Antimicrobial Pharmacodynamics in Theory and Clinical Practice - C. Nightingale, et al., (M-D, 2002)...

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Marcel Dekker Inc New York bull BaselTM

edited by

Charles H NightingaleHartford Hospital

Hartford Connecticut

Antimicrobial Pharmacodynamics

in Theory and Clinical Practice

Takeo MurakawaFujisawa Pharmaceutical Company Ltd

Osaka Japan

Paul G AmbroseCognigen Corporation

Buffalo New York

Copyright copy 2001 by Marcel Dekker Inc All Rights Reserved

ISBN 0-8247-0561-0

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Current printing (last digit)10 9 8 7 6 5 4 3 2 1

PRINTED IN THE UNITED STATES OF AMERICA

INFECTIOUS DISEASE AND THERAPY

Series Editor

Burke A Cunha

Winthrop-University HospitalMineola and

State University of New York School of MedicineStony Brook New York

1 Parasitic Infections in the Compromised Host edited by Peter DWalzer and Robert M Genta

2 Nucleic Acid and Monoclonal Antibody Probes Applications inDiagnostic Methodology edited by Bala Swaminathan and GyanPrakash

3 Opportunistic Infections in Patients with the Acquired Immuno-deficiency Syndrome edited by Gifford Leoung and John Mills

4 Acyclovir Therapy for Herpesvirus Infections edited by David ABaker

5 The New Generation of Quinolones edited by Clifford Siporin Carl LHeifetz and John M Domagala

6 Methicillin-Resistant Staphylococcus aureus Clinical Managementand Laboratory Aspects edited by Mary T Cafferkey

7 Hepatitis B Vaccines in Clinical Practice edited by Ronald W Ellis8 The New Macrolides Azalides and Streptogramins Pharmacology

and Clinical Applications edited by Harold C Neu Lowell S Youngand Stephen H Zinner

9 Antimicrobial Therapy in the Elderly Patient edited by Thomas TYoshikawa and Dean C Norman

10 Viral Infections of the Gastrointestinal Tract Second Edition Revisedand Expanded edited by Albert Z Kapikian

11 Development and Clinical Uses of Haemophilus b Conjugate Vac-cines edited by Ronald W Ellis and Dan M Granoff

12 Pseudomonas aeruginosa Infections and Treatment edited byAldona L Baltch and Raymond P Smith

13 Herpesvirus Infections edited by Ronald Glaser and James F Jones14 Chronic Fatigue Syndrome edited by Stephen E Straus15 Immunotherapy of Infections edited by K Noel Masihi16 Diagnosis and Management of Bone Infections edited by Luis E

Jauregui17 Drug Transport in Antimicrobial and Anticancer Chemotherapy

edited by Nafsika H Georgopapadakou

18 New Macrolides Azalides and Streptogramins in Clinical Practiceedited by Harold C Neu Lowell S Young Stephen H Zinner andJacques F Acar

19 Novel Therapeutic Strategies in the Treatment of Sepsis edited byDavid C Morrison and John L Ryan

20 Catheter-Related Infections edited by Harald Seifert Bernd Jansenand Barry M Farr

21 Expanding Indications for the New Macrolides Azalides andStreptogramins edited by Stephen H Zinner Lowell S YoungJacques F Acar and Harold C Neu

22 Infectious Diseases in Critical Care Medicine edited by Burke ACunha

23 New Considerations for Macrolides Azalides Streptogramins andKetolides edited by Stephen H Zinner Lowell S Young Jacques FAcar and Carmen Ortiz-Neu

24 Tickborne Infectious Diseases Diagnosis and Management editedby Burke A Cunha

25 Protease Inhibitors in AIDS Therapy edited by Richard C Ogdenand Charles W Flexner

26 Laboratory Diagnosis of Bacterial Infections edited by Nevio Cimolai27 Chemokine Receptors and AIDS edited by Thomas R OrsquoBrien28 Antimicrobial Pharmacodynamics in Theory and Clinical Practice

edited by Charles H Nightingale Takeo Murakawa and Paul G Am-brose

29 Pediatric Anaerobic Infections Diagnosis and Management ThirdEdition Revised and Expanded Itzhak Brook

Additional Volumes in Production

Preface

To use antibiotics appropriately the clinician needs to understand fundamentalpharmacodynamic concepts These concepts are essential for they form the verybasis for therapeutic strategies that maximize clinical benefit while minimizingtoxicity to the patient The objectives of this book are first to review the constel-lation of scientific and medical literature concerning antibiotics and pharmacody-namics The relevance of this complex information is then synthesized into aneasy-to-understand discussion of concept and theory Finally the reader is shownhow to apply these theories and concepts with specific examples to the clinicalpractice of medicine and pharmacy In other words this book takes the readerfrom the test tube through the animal and human volunteer laboratory to thepatientrsquos bedside

The book includes a thorough discussion of the pharmacodynamics of allmajor classes of the antimicrobial armamentarium These include penicillinscephalosporins cephamycins carbapenems monobactams aminoglycosidesquinolones macrolides antifungals antivirals and others Additionally a phar-macodynamic discussion of new classes of antimicrobial agents that are uponthe horizon such as the ketolide antibiotics is included

This book is unique in that no other text of its kind currently exists Theinformation that this book provides integrates medical microbiology clinical in-fectious diseases and pharmacokinetics This book pulls together in one text theessential elements of these disciplines and does so in a very understandable andpractical manner

The infectious disease physicians and pharmacists we selected as contribu-tors are eminently qualified and are recognized experts in their field Moreoverthese authors were chosen on the basis of their ability to convey their perspective

iii

iv Preface

and expertise lucidly which makes them ideal teachers They all agreed that therewas a need for such a book and were excited about joining in this venture

This book will find an audience in a large array of healthcare disciplinesincluding college educators medical pharmacy and microbiology students in-fectious disease physicians and pharmacy specialists medical house staff clinicaland staff pharmacists clinical microbiologists and other healthcare decisionmakers

Charles H NightingaleTakeo MurakawaPaul G Ambrose

Contents

Preface iiiContributors vii

1 Pharmacodynamics of Antimicrobials General Concepts andApplications 1William A Craig

2 Microbiology and Pharmacokinetics 23Charles H Nightingale and Takeo Murakawa

3 In Vitro Antibiotic Pharmacodynamic Models 41Michael J Rybak George P Allen and Ellie Hershberger

4 Animal Models of Infection for the Study of AntibioticPharmacodynamics 67Michael N Dudley and David Griffith

5 β-Lactam Pharmacodynamics 99JoCarol J McNabb and Khanh Q Bui

6 Aminoglycoside Pharmacodynamics 125Myo-Kyoung Kim and David P Nicolau

7 Pharmacodynamics of Quinolones 155Robert C Owens Jr and Paul G Ambrose

v

vi Preface

8 Glycopeptide Pharmacodynamics 177Gigi H Ross David H Wright John C Rotschafer andKhalid H Ibrahim

9 Macrolide Azalide and Ketolide Pharmacodynamics 205Charles H Nightingale and Holly M Mattoes

10 Metronidazole Clindamycin and StreptograminPharmacodynamics 221Kenneth Lamp Melinda K Lacy and Collin Freeman

11 Tetracycline Pharmacodynamics 247Burke A Cunha and Holly M Mattoes

12 Pharmacodynamics of Antivirals 259George L Drusano Sandra L Preston and Peter J Piliero

13 Antifungal Pharmacodynamics 285Michael E Klepser and Russell E Lewis

14 Human Pharmacodynamics of Anti-Infectives Determinationfrom Clinical Trial Data 303George L Drusano

15 Antibacterial Resistance 327Philip D Lister

16 Basic Pharmacoeconomics 367Mark A Richerson and Eugene Moore

17 Utilizing Pharmacodynamics and Pharmacoeconomics inClinical and Formulary Decision Making 385Paul G Ambrose Annette Zoe-Powers Rene RussoDavid T Jones and Robert C Owens Jr

Index 409

Contributors

George P Allen PharmD Postdoctoral Fellow Pharmacy Practice WayneState University and Detroit Receiving Hospital and University Health CenterDetroit Michigan

Paul G Ambrose PharmD Director Infectious Disease Research CognigenCorporation Buffalo New York

Khanh Q Bui PharmD Bristol-Myers Squibb Company Chicago Illinois

William A Craig MD Professor of Medicine and Therapeutics Departmentof Infectious Diseases University of Wisconsin and William S Middleton Me-morial Veterans Hospital Madison Wisconsin

Burke A Cunha MD Chief Infectious Disease Division Winthrop-University Hospital Mineola and Professor of Medicine State University ofNew York School of Medicine Stony Brook New York

George L Drusano MD Professor Department of Medicine and Pharmacol-ogy Clinical Research Institute Albany Medical College Albany New York

vii

viii Contributors

Michael N Dudley PharmD Vice President Department of Pharmacologyand Microbiology Microcide Pharmaceuticals Inc Mountain View California

Collin Freeman PharmD Clinical Science Specialist Department of Scien-tific Affairs Bayer Corporation West Haven Connecticut

David Griffith BS Research Scientist Department of Pharmacology Micro-cide Pharmaceuticals Inc Mountain View California

Ellie Hershberger PharmD Director Infectious Disease Laboratory Re-search Institute William Beaumont Research Institute Royal Oak Michigan

Khalid H Ibrahim PharmD Infectious Diseases Research Fellow Depart-ment of Experimental and Clinical Pharmacology University of Minnesota Min-neapolis Minnesota

David T Jones PharmD MD Department of Cardiology and Internal Med-icine University of California at Davis Sacramento California

Myo-Kyoung Kim PharmD Infectious Disease Fellow Department of Phar-macy Research Hartford Hospital Hartford Connecticut

Michael E Klepser PharmD Associate Professor College of PharmacyUniversity of Iowa Iowa City Iowa

Melinda K Lacy PharmD Assistant Professor Department of PharmacyPractice School of Pharmacy University of Kansas Medical Center Kansas CityKansas

Kenneth Lamp PharmD Medical Science Manager Department of UnitedStates Medicines Bristol-Myers Squibb Plainsboro New Jersey

Russell E Lewis PharmD Assistant Professor Department of Clinical Sci-ences and Administration University of Houston College of Pharmacy HoustonTexas

Philip D Lister PhD Associate Professor Department of Medical Microbiol-ogy and Immunology Creighton University School of Medicine Omaha Ne-braska

Holly M Mattoes PharmD Scientific Communications Manager DesignWriteIncorporated Princeton New Jersey

Contributors ix

JoCarol J McNabb PharmD Assistant Professor College of PharmacyUniversity of Nebraska Medical Center Omaha Nebraska

Eugene Moore PharmD Coordinator Army Ambulatory Care PharmacistProgram Department of Defense Pharmacoeconomic Center Fort Sam HoustonTexas

Takeo Murakawa PhD Supervisor International Development and Regula-tory Affairs Development Division Fujisawa Pharmaceutical Company LtdOsaka and Lecturer (Part) Graduate School of Pharmaceutical Sciences KyotoUniversity Kyoto and Graduate School of Pharmaceutical Sciences TokushimaUniversity Tokushima Japan

David P Nicolau PharmD Division of Infectious Disease Pharmacy Re-search Hartford Hospital Hartford Connecticut

Charles H Nightingale PhD Vice-President for Research and Director ofthe Institute for International Healthcare Studies Hartford Hospital HartfordConnecticut

Robert C Owens Jr PharmD Clinical Specialist Infectious DiseasesMaine Medical Center Portland Maine and University of Vermont College ofMedicine Burlington Vermont

Peter J Piliero MD Assistant Professor Department of Medicine AlbanyMedical College Albany New York

Sandra L Preston PharmD Assistant Professor of Medicine Clinical Re-search Initiative Albany Medical College Albany New York

Mark A Richerson PharmD MS Commander Medical Service CorpsUnited States Navy and Department of Defense Pharmacoeconomic Center FortSam Houston Texas

Gigi H Ross PharmD Infectious Diseases Scientific Liaison Clinical Af-fairs Ortho-McNeil Pharmaceutical Raritan New Jersey

John C Rotschafer PharmD FCCP Professor Department of Experi-mental and Clinical Pharmacology College of Pharmacy University of Minne-sota Minneapolis Minnesota

Rene Russo PharmD Postdoctoral Fellow College of Pharmacy RutgersUniversity Piscataway New Jersey

x Contributors

Michael J Rybak PharmD FCCP BCPS Professor of Pharmacy andMedicine and Director Anti-Infective Research Laboratory Department of Phar-macy Practice Wayne State University and Detroit Receiving Hospital and Uni-versity Health Center Detroit Michigan

David H Wright PharmD Manager Professional Education and ScientificAffairs Ortho-McNeil Pharmaceutical Raritan New Jersey

Annette Zoe-Powers PharmD Director Department of Infectious DiseasesBristol-Myers Squibb Company Plainsboro New Jersey

1

Pharmacodynamics of AntimicrobialsGeneral Concepts and Applications

William A Craig

University of Wisconsin and William S Middleton Memorial Veterans HospitalMadison Wisconsin

1 INTRODUCTION

lsquolsquoPharmacodynamicsrsquorsquo is the term used to reflect the relationship between mea-surements of drug exposure in serum tissues and body fluids and the pharmaco-logical and toxicological effects of drugs With antimicrobials pharmacodynam-ics is focused on the relationship between concentrations and the antimicrobialeffect Studies in the past have focused on pharmacokinetics and descriptions ofthe time course of antimicrobials in serum tissues and body fluids Much lessemphasis has been placed on the time course of antimicrobial activity Studiesover the past 20 years have demonstrated marked differences in the time courseof antimicrobial activity among antibacterials and antifungals [15208488] Fur-thermore the pattern of antimicrobial activity over time is an important determi-nant of optimal dosage regimens [21] This chapter focuses on general conceptsand the application of pharmacodynamics to antimicrobial therapy

1

2 Craig

2 MEASUREMENTS OF ANTIMICROBIAL ACTIVITY

21 Minimum Inhibitory and Minimum Bactericidal

Concentrations

The minimum inhibitory and minimum bactericidal concentrations (MIC andMBC) have been the major parameters used to measure the in vitro activity ofantimicrobials against various pathogens Although the MIC and MBC are excel-lent predictors of the potency of an antimicrobial against the infecting organismthey provide essentially no information on the time course of antimicrobial activ-ity For example the MBC provides minimal information on the rate of bacteri-cidal and fungicidal activity and on whether killing can be increased by higherdrug concentrations In addition the MIC provides no information on growthinhibitory effects that may persist after antimicrobial exposure These persistenteffects are due to three different phenomena the postantibiotic effect (PAE)the postantibiotic sub-MIC effect (PAE-SME) and the postantibiotic leukocyteenhancement (PALE) [176263] The effects of increasing concentrations on thebactericidal and fungicidal activity of antimicrobials combined with the magni-tude of persistent effects give a much better description of the time course ofantimicrobial activity than that provided by the MIC and MBC

22 Killing Activity

Antimicrobials exhibit two primary patterns of microbial killing The first patternis characterized by concentration-dependent killing over a wide range of concen-trations With this pattern higher drug concentrations result in a greater rate andextent of microbial killing This pattern is observed with the aminoglycosidesfluoroquinolones ketolides metronidazole and amphotericin B [9192028] Thesecond pattern is characterized by minimal concentration-dependent killing Withthis pattern saturation of the killing rate occurs at low multiples of the MICusually around four to five times the MIC Drug concentrations above these val-ues do not kill microbes faster or more extensively This pattern is also calledtime-dependent killing because the extent of microbial killing is primarily depen-dent on the duration of exposure This pattern is observed with β-lactam antibiot-ics macrolides clindamycin glycopeptides tetracyclines trimethoprim linezo-lid and flucytosine [48152029]

The different patterns of bacterial killing are illustrated in Fig 1 by showingthe effect of increasing drug concentrations on the in vitro antimicrobial activityof tobramycin ciprofloxacin and ticarcillin against a standard strain of Pseudo-monas aeruginosa [20] Increasing concentrations of tobramycin and ciprofloxa-cin produced more rapid and extensive bacterial killing as exhibited by thesteeper slopes of the killing curves With ticarcillin there was a change in slopeas the concentration was increased from one to four times the MIC However

Antimicrobial Pharmacodynamics 3

FIGURE 1 Time-kill curves of Pseudomonas aeruginosa ATCC 27853 with ex-posure to tobramycin ciprofloxacin and ticarcillin at concentrations fromone-fourth to 64 times the MIC (From Ref 20)

higher concentrations did not alter the slope The slight reduction in bacterialnumbers at the higher doses is due to an earlier onset of bacterial killing From2 h on ticarcillin concentrations from 4 to 64 times the MIC produced the samerates of killing

23 Persistent Effects

lsquolsquoPostantibiotic effectrsquorsquo is the term used to describe the persistent suppression ofbacterial growth following antimicrobial exposure [151863] If reflects the timeit takes for an organism to recover from the effects of exposure to an antimicrobialand resume normal growth This phenomenon was first observed in the 1940s inearly studies with penicillin against staphylococci and streptococci [1238] Laterstudies starting in the 1970s extended this phenomenon to newer drugs and togram-negative organisms The postantibiotic effect is demonstrated in vitro byfollowing bacterial growth kinetics after drug removal

Moderate to prolonged in vitro postantibiotic effects are observed for allantibacterials with susceptible gram-positive bacteria such as staphylococci andstreptococci [10] Moderate to prolonged in vitro PAEs are also observed withgram-negative bacilli for drugs that are inhibitors of protein or nucleic acid syn-thesis In contrast short or no postantibiotic effects are observed for β-lactamantibiotics with gram-negative bacilli The only exception has been for carbapen-ems which exhibit moderate PAEs primarily with strains of Pseudomonas aeru-

4 Craig

ginosa [1647] In vitro postantifungal effects (PAFEs) have been observed withvarious yeasts following exposure to amphotericin B and flucytosine but not totriazoles such as fluconazole [3984]

The postantibiotic sub-MIC effect demonstrates the additional effect sub-MIC concentrations can have on the in vitro PAE For example exposure ofstreptococci in the PAE phase to macrolides at drug concentrations of one-tenthand three-tenths of the MIC increased the duration of the postantibiotic effectby about 50 and 100 respectively [1871] The PAE phase can also makestreptococci hypersensitive to the killing effects of penicillin [17] The durationof the postantibiotic sub-MIC effects reported in the literature includes the dura-tion of the PAE plus the enhanced duration due to sub-MIC concentrations Mor-phological changes such as filaments can also be produced by sub-MIC concen-trations [60]

Postantibiotic leukocyte enhancement describes the effects of leukocyteson bacteria during the postantibiotic phase Studies have demonstrated that suchbacteria are more susceptible to intracellular killing or phagocytosis by leuko-cytes [1862] This phenomenon can also prolong the duration of the in vitropostantibiotic effect Antimicrobials that produce the longest postantibiotic ef-fects tend to exhibit the most prolonged effects when exposed to leukocytes

The postantibiotic effect has also been demonstrated in vivo in a varietyof animal infection models [1822] The in vivo phenomenon is actually a combi-nation of the in vitro PAE and sub-MIC effects from gradually falling drug con-centrations The largest number of studies have used the neutropenic mouse thighinfection model [88] When performed in non-neutropenic mice the in vivo PAEwould also include any postantibiotic leukocyte enhancement effects

There are several important differences between the in vivo and in vitroPAEs In most cases in vivo PAEs are longer than in vitro PAEs most likelybecause of the additive effect of sub-MIC concentrations Simulation of humanpharmacokinetics can further enhance the duration of the in vivo PAE by a similarmechanism Prolongation of sub-MIC concentrations of amikacin by simulatingthe human drug half-life (2 h) extended the duration of in vivo PAEs by 40ndash100over values observed with a dose producing the same area under the concentrationversus time curve (AUC) but eliminated with a murine half-life of 20 min [27]In vivo PAEs with some drugs are further prolonged by the presence of leuko-cytes In general the presence of neutrophils tends to double the duration of thein vivo postantibiotic effect for aminoglycosides and fluoroquinolones with gram-negative bacilli [1827] However leukocytes have no major effect on the mini-mal in vivo PAEs observed for β-lactams with gram-negative bacilli

There are also some differences between the in vitro and in vivo PAEs thatquestion the value of measuring the in vitro PAE First the duration of the invitro PAE is not predictive of the duration of the in vivo PAE [40] Secondprolonged PAEs for penicillin and cephalosporins with streptococci are observed


in vitro but not in vivo [228088] Third in vitro studies that suggest that the PAEof aminoglycosides decreases and disappears over a prolonged dosing interval orwith repeated doses have not been confirmed in vivo [3564] Fourth fluconazoleexhibits a postantifungal effect in vivo but not in vitro [384]

3 PATTERNS OF ANTIMICROBIAL ACTIVITY

The pharmacodynamic characteristics described above suggest that the timecourse of antimicrobial activity can vary markedly for different antibacterial andantifungal agents As shown in Table 1 these drugs exhibit three major patternsof antimicrobial activity The first pattern in characterized by concentration-dependent killing and moderate to prolonged persistent effects Higher concentra-tions would kill organisms more rapidly and more extensively than lower levelsThe prolonged persistent effects would allow for infrequent administration oflarge doses This pattern is observed with aminoglycosides fluoroquinolonesdaptomycin ketolides metronidazole and amphotericin B The goal of a dosingregimen for these drugs would be to maximize concentrations The peak leveland the AUC should be the pharmacokinetic parameters that would determinein vivo efficacy

The second pattern is characterized by time-dependent killing and minimalto moderate persistent effects High drug levels would not kill organisms betterthan lower concentrations Furthermore organism regrowth would start very soonafter serum levels fell below the MIC This pattern is observed with β-lactamsmacrolides clindamycin oxazolidinones and flucytosine The goal of a dosingregimen for these drugs would be to optimize the duration of exposure Theduration of time that serum levels exceed some minimal value such as the MICshould be the major pharmacokinetic parameter determining the in vivo efficacyof these drugs

The third pattern is also characterized by time-dependent killing but theduration of the persistent effects is much prolonged This can prevent any re-growth during the dosing interval This pattern is observed with azithromycintetracyclines quinupristin-dalfopristin glycopeptides and fluconazole The goalof a dosing regimen is to optimize the amount of drug administered to ensurethat killing occurs for part of the time and there is no regrowth during the dosinginterval The AUC should be the primary pharmacokinetic parameter that woulddetermine in vivo efficacy

4 PHARMACOKINETICPHARMACODYNAMIC

PARAMETERS

By using the MIC as a measure of the potency of drugndashorganism interactionsthe pharmacokinetic parameters determining efficacy can be converted to

6 Craig

TA

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pharmacokineticpharmacodynamic (PKPD) parameters [21] Serum (orplasma) concentrations are used for determining the pharmacokinetic indicesBecause most infections occur in tissues and the common bacterial pathogensare extracellular organisms interstitial fluid concentrations at the site of infectionshould be the primary determinants of efficacy Serum levels are much betterpredictors of interstitial fluid levels than tissue homogenate concentrations Be-cause tissue homogenates mix the interstitial intracellular and vascular compart-ments together they tend to underestimate or overestimate the interstitial fluidconcentration depending on the ability of the drug to accumulate intracellularly[74]

Identification of the primary PKPD parameter that determines efficacy iscomplicated by the high degree of interdependence among the various parame-ters For example a larger dose produces a higher peakMIC ratio a higher AUCMIC ratio and a longer duration of time above MIC If the higher dose producesa better therapeutic effect than a lower dose it is difficult to determine whichPKPD parameter is of major importance because all three increased Howevercomparing the effects of dosage regimens that include different dosing intervalscan reduce much of the interdependence among PKPD parameters Such studiesare often referred to as dose-fractionation studies [3758] For example dividingseveral total doses into 1 2 4 8 and 24 doses administered at 24 12 6 3 and1 h intervals respectively can allow one to identify which PKPD parameter ismost important for in vivo efficacy

Several investigators have used this study design in animal infection modelsto correlate specific PKPD parameters with efficacy for various antimicrobialsagainst gram-positive cocci gram-negative bacilli and Candida species[3492829576187] This is demonstrated graphically in Fig 2 for ceftazidimeagainst Klebsiella pneumoniae and in Fig 3 for temafloxacin against Streptococ-cus pneumoniae In these studies pairs of mice were treated with multiple dosageregimens that varied both the dose and the dosing interval The number of colony-forming units (CFUs) remaining in the thigh after 24 h of therapy was plottedagainst the peakMIC and 24 h AUCMIC ratios and the percentage of time thatserum levels exceeded the MIC that was calculated for each dosage regimen frompharmacokinetic indices As shown in Fig 2 there was a very poor relationshipbetween CFUsthigh and the peakMIC and 24 h AUCMIC ratios On the otherhand an excellent correlation was observed between the number of bacteria inthe thighs and the percentage of time that serum levels exceeded the MIC How-ever the best correlation in Fig 3 was observed with the 24 h AUCMIC ratiofollowed by the peakMIC ratio

The specific PKPD parameters correlating with efficacy in animal infec-tion models for different antibacterials and antifungals are listed in Table 2 Asexpected time above MIC has consistently been the only PKPD parameter corre-lating with the therapeutic efficacy of β-lactam antibiotics Time above MIC is

8 Craig

FIGURE 2 Relationship between three pharmacodynamic parameters (peakMIC ratio 24 h AUCMIC ratio and percentage of time that serum levels ex-ceed the MIC) and the number of K pneumoniae ATCC 53816 in the thighsof neutropenic mice after 24 h of therapy with ceftazidime Each point repre-sents data for one mouse The dotted line reflects the number of bacteria atthe beginning of therapy

FIGURE 3 Relationship between three pharmacodynamic parameters (peakMIC ratio 24 h AUCMIC ratio and percentage of time that serum levels ex-ceed the MIC) and the number of S pneumoniae ATCC 10813 in the thighsof neutropenic mice after 24 h of therapy with temafloxacin Each point repre-sents data for one mouse The dotted line reflects the number of bacteria atthe beginning of therapy


TABLE 2 PKPD Parameters Determining Efficacy for DifferentAntimicrobials

PKPD parameter Antimicrobial

Time above MIC Penicillins cephalosporins aztreonam carbapenemstribactams macrolides clindamycin oxazolidinonesflucytosine

PeakMIC ratio Aminoglycosides fluoroquinolones daptomycin van-comycin teicoplanin amphotericin B

AUCMIC ratio Aminoglycosides fluoroquinolones daptomycin van-comycin ketolides quinupristin-dalfopristin tetracy-clines fluconazole

also the parameter correlating with efficacy of the macrolides clindamycin oxa-zolidinones and flucytosine

The AUCMIC and peakMIC ratios have been the PKPD parameters thatcorrelate with efficacy for aminoglycosides and fluoroquinolones Most studieshave shown slightly better correlation with the AUCMIC ratio than with thepeakMIC ratio PeakMIC ratios appear to be more important in infections wherethe emergence of resistant subpopulations is a significant risk and for drugs thatact on the cell membrane such as daptomycin and amphotericin B [93776]

Although vancomycin tetracyclines azithromycin and quinupristin-dalfopristin do not exhibit concentration-dependent killing the AUCMIC ratiohas been the major PKPD parameter correlating with therapeutic efficacy ofthese drugs in neutropenic animals [2129] A study in normal mice with vanco-mycin and teicoplanin against a strain of S pneumoniae that used mortality asan endpoint demonstrated that the peakMIC ratio was the most important param-eter [55]

5 MAGNITUDE OF PKPD PARAMETER REQUIRED

FOR EFFICACY

Because PKPD parameters can correct for differences in a drugrsquos pharmacoki-netics and intrinsic antimicrobial activity one would expect that the magnitude ofthe PKPD parameters required for efficacy would be similar in different animalspecies Thus results from studies in animal infection models could be predictiveof the activity of drugs in humans This would be especially helpful in designingdosage regimens for both old and new antibacterials in situations where it isdifficult to obtain sufficient clinical data such as with newly emerging resistantorganisms Studies in animals would also allow one to determine if the magnitudeof the PKPD parameter required for efficacy was similar for (1) different drugs

10 Craig

within the same antimicrobial class (2) different dosing regimens (3) differentpathogens and (4) different sites of infection

51 Animal Infection Models

The largest number of studies addressing the magnitude of the PKPD parameterswith various drugs dosing regimens pathogens sites of infection and animalspecies have been performed with β-lactams and fluoroquinolones Time aboveMIC is the PKPD parameter that correlates with the therapeutic efficacy of thevarious β-lactam antibiotics Studies in animal infection models demonstrate thatantibiotic concentrations do not need to exceed the MIC for 100 of the dosinginterval to obtain a significant antibacterial effect [2326285787] In fact an invivo bacteriostatic effect is observed when serum levels exceed the MIC for about30ndash40 of the dosing interval Very similar percentages for time above MIChave been observed in murine thigh and lung infection models for various dosingintervals from 1 to 24 h and with several broad-spectrum cephalosporins againstboth gram-negative bacilli and gram-negative streptococci provided unbounddrug levels were used for highly protein bound cephalosporins such as ceftri-axone

If one uses survival after several days of therapy as the endpoint for efficacyof β-lactams in animal infection models then slightly higher percentages of timeabove MIC are necessary [23070] Figure 4 illustrates the relationship betweentime above MIC and mortality for animals infected with S pneumoniae that weretreated for several days with penicillins or cephalosporins Several studies in-

FIGURE 4 Relationship between the percentage of time serum levels of β-lactams exceed the MIC and survival in animal models infected with S pneu-moniae (Data from Refs 5 30 and 70)


cluded penicillin-intermediate and penicillin-resistant strains The mortality wasclose to 100 if serum levels were above the MIC for 20 or less of the dosinginterval As soon as the percentage of time above MIC reached 40ndash50 or highersurvival was in the order of 90ndash100

The PKPD parameter that best correlates with the efficacy of the fluoro-quinolones is the 24 h AUCMIC ratio [6728] The magnitude of this PKPDparameter required to produce a bacteriostatic effect in animal infection modelsvaried for most organisms from 25 to 50 [2841] These values are equivalent toaveraging one to two times the MIC over a 24 h period [ie (1ndash2) 24 24ndash48] This magnitude was independent of the dosing interval the site of infectionand the fluoroquinolone used provided unbound drug concentrations were usedfor moderate to highly protein bound drugs such as gemifloxacin [7]

The relationship between the 24 h AUCMIC values and outcome for flu-oroquinolones as reported in the literature from studies that treated animals forat least 2 days reported survival results at the end of therapy and provided phar-macokinetic data is illustrated in Fig 5 [28] The infections in these studies in-cluded pneumonia peritonitis and sepsis produced by gram-negative bacilli anda few gram-positive cocci in immunosuppressed mice rats and guinea pigs Ingeneral 24 h AUCMIC ratios less than 30 were associated with greater than50 mortality whereas AUCMIC values of 100 or greater were associated with

FIGURE 5 Relationship between 24 h AUCMIC for fluoroquinolones and sur-vival in immunosuppressed animals infected with gram-negative bacilli anda few gram-positive cocci (From Ref 28)

12 Craig

almost no mortality A value of 100 is equivalent to having serum concentrationsaverage about four times the MIC over a 24 h period (ie 4 times 24 96)

Differences in the magnitude of the PKPD parameter are observed withdifferent classes of β-lactams and with some organisms with both β-lactams andfluoroquniolones For the same types of organisms the percentages for timeabove MIC for a bacteriostatic effect were slightly lower for penicillins than forcephalosporins and even lower for carbapenems [26] These differences are dueto the rate of killing which is fastest with the carbapenems and slowest with thecephalosporins In addition the percentage for time above MIC required for effi-cacy with staphylococci was less than observed with gram-negative bacilli andstreptococci [23] This difference is due to the prolonged in vivo PAEs observedfor β-lactams with staphylococci but not with gram-negative bacilli and strepto-cocci In non-neutropenic mice the magnitude of the 24 h AUCMIC for fluoro-quinolones required for efficacy was about threefold lower for S pneumoniaethan for Klebsiella pneumoniae [728] In vitro models have also demonstrateda lower AUCMIC value for strains of S pneumoniae [5659]

52 Drug Combinations

Very little is known about determining the magnitude of PKPD parameters whendrugs are used in combination Some investigators have suggested that one canadd the magnitude of the 24 h AUCMIC ratios for each of the drugs to estimatethe pharmacodynamic activity of the combination [82] However a study in neu-tropenic mice infected with Pseudomonas aeruginosa demonstrated that the mag-nitudes of the PKPD parameters required when a β-lactam aminoglycoside orfluoroquinolone is used alone are also important in predicting the efficacy ofthese drugs used in combination [66] Thus adding the 24 h AUCMIC ratio ofβ-lactams to that of aminoglycosides or fluoroquinolones has a poor predictivevalue for their activity in combination Instead one must add the effect producedby the percentage of time above MIC for β-lactams to the effect resulting fromthe 24 h AUCMIC ratio of aminoglycosides and fluoroquinolones to accuratelypredict the activity of these combinations Adding the 24 h AUCMIC ratios isappropriate for aminoglycoside-fluoroquinolone combinations because that is theimportant PKPD parameter for both drugs

53 Human Infections

Bacteriological cure in patients with acute otitis media and acute maxillary sinus-itis provides a sensitive model for determining the relationship between outcomeand time above MIC for multiple β-lactam antibiotics A variety of clinical trialshave included pretherapy and repeat sinus puncture or tympanocentesis of middleear fluid after 2ndash7 days of therapy to determine whether the initial organismisolated had been eradicted [25323348547779] Figure 6 demonstrates the


relationship between time above MIC and the bacteriological cure rate formany β-lactams against S pneumoniae and Haemophilus influenzae in patientswith these two infections Several of the recent studies have included penicillin-intermediate and penicillin-resistant strains In general percentages for timeabove MIC greater than 40 were required to achieve an 85ndash100 bacteriologi-cal cure rate for both organisms including resistant pneumococci

Commonly used parenteral doses of ceftriaxone cefotaxime penicillin Gand ampicillin provide free-drug concentrations above the MIC90 for penicillin-intermediate strains of S pneumoniae for at least 40ndash50 of the dosing intervalA variety of clinical trials in severe pneumococcal pneumonia including bacter-emic cases have demonstrated that these β-lactams are as effective against theseorganisms as against fully susceptible strains [435072] Thus the magnitude ofthe PKPD parameter determining efficacy for β-lactams against pneumococciis very similar in animal infection models and in human infections such as pneu-monia sinusitis and otitis media

As illustrated in Fig 5 high survival in animal infection models treatedwith fluoroquinolones was observed with 24 h AUCMIC values of 100 or higherVery similar values were observed in two clinical trials Forrest et al [45] found

FIGURE 6 Relationship between time above MIC and bacteriological cure forvarious β-lactams against penicillin-susceptible (PSSP) penicillin-intermedi-ate (PISP) and penicillin-resistant (PRSP) S pneumoniae and H influenzaein patients with acute otitis media and acute maxillary sinusitis (Data fromRefs 25 32 and 33)

14 Craig

that a 24 h AUCMIC value of 125 or higher was associated with satisfactoryoutcome in seriously ill patients treated with intravenous ciprofloxacin Lowervalues resulted in clinical and microbiological cure rates of less than 50 An-other study in patients with a variety of bacterial infections treated with levoflox-acin found that a peakMIC ratio of 12 or higher and a 24 h AUCMIC ratio of100 or higher resulted in a statistically improved outcome [73] These studiesfurther demonstrate that the magnitude of the PKPD parameter in animal infec-tion models can be predictive of the magnitude of the parameter required foreffective therapy in humans

54 Prevention of Resistance

A peakMIC ratio of 8ndash10 has been shown both in vitro and in vivo to preventthe emergence of resistant mutants during therapy with aminoglycosides and flu-oroquinolones [1331] A 24 h AUCMIC ratio greater than 100 has also beenassociated with a significantly reduced risk for the emergence of resistance duringtherapy [82] The conclusion of this study was dependent almost entirely on theresults with ciprofloxacin in patients with gram-negative bacillary infections pri-marily those due to P aeruginosa A 24 h AUCMIC ratio greater than 100 didnot reduce the risk for the emergence of resistant organisms in patients treatedwith cephalosporins for infections due to gram-negative bacilli producing type1 β-lactamase Much more data are needed on the relationship between PKPDparameters and the development of resistance

6 APPLICATIONS OF PHARMACODYNAMICS

Knowledge of the pharmacodynamics of antimicrobials have proven useful for(1) establishing newer optimal dosing regimens for established drugs (2) devel-oping new antimicrobials or new formulations (3) establishing susceptibilitybreakpoints and (4) formulating guidelines for empirical therapy of infections

61 New Dosage Regimens

Administration of β-lactams by continuous infusion enhances their ability tomaintain serum levels above the MIC Despite many potential advantages of con-tinuous infusion only a few clinical trials have documented the success of thistype of dosage regimen [1424] Recent clinical trials have been designed to deter-mine if continuous infusion will (1) allow for the use of daily dosages of druglower than those required for intermittent administration or (2) improve efficacyagainst bacteria with reduced susceptibility For example equivalent outcomeshave been observed with 3ndash4 gday of ceftazidime by continuous infusion com-pared with 6 gday administered intermittently [5169] Initial results with con-tinuous infusion of large doses of ampicillin have demonstrated success for


moderately ampicillin-resistant strains (ampicillin MIC 32ndash64 mgL) ofvancomycin-resistant Enterococcus faecium [21]

The peakMIC ratio appears to be the major PKPD parameter determiningthe clinical efficacy of aminoglycosides High rates of clinical success in severegram-negative bacillary infections and rapid resolution of fever and leukocytosisin gram-negative bacillary nosocomial pneumonia require a peakMIC ratio of8ndash10 [5365] The once-daily dosage regimen for aminoglycosides was designedto enhance peak serum levels In addition once-daily dosing has the potential todecrease the nephro- and ototoxicity associated with these drugs [85] Uptake ofaminoglycosides into renal tubular cells and middle ear endolymph is more effi-cient with low sustained concentrations than with high intermittent levels[348386]

Most meta-analyses of clinical trials have demonstrated a small but signifi-cant increase in clinical outcome with once-daily dosing and a trend toward de-creased nephrotoxicity [1101144464967] Studies have also demonstratedthat the onset of nephrotoxicity occurs several days later when the drug is admin-istered once daily than when multiple-daily dosage regimens are followed[527581] Nevertheless once-daily dosing may not be ideal for all indicationsStudies in experimental enterococcal endocarditis have shown a greater reduc-tion in bacterial vegetation titers when the aminoglycoside is administered bymultiple-dosing regimens than by once-daily administration [4285]

62 New Antimicrobials and Formulations

Identification of the PKPD parameter and its magnitude required for efficacyhas proven useful for selecting the dosage regimen for phase III clinical trials ofnew antimicrobials For example the dosage regimen for linezolid (600 mg twicedaily) was designed to produce serum levels above 8 mgL for more than 50of the dosing interval [8] New extended release formulations of cefaclor andclarithromycin and the 141 amoxicillin-clavulanate formulation were all de-signed to enhance the duration of time serum levels exceed the MIC

63 Susceptibility Breakpoint Determinations

The Subcommittee on Antimicrobial Susceptibility Testing of the National Com-mittee for Clinical Laboratory Standards (NCCLS) recently incorporated pharma-codynamics as one of the factors to consider when establishing susceptibilitybreakpoints [68] For example pharmacodynamic breakpoints for β-lactam anti-biotics would be defined as the highest MIC that serum concentrations followingstandard dosage would exceed for at least 40 of the dosing interval Table 3compares the old and new susceptibility breakpoints of several oral β-lactamsfor S pneumoniae with the pharmacodynamic breakpoints predicted from serumconcentrations in children and adults The two values for cefaclor reflect the

16 Craig

TABLE 3 Pharmacodynamic and New and Old NCCLS SusceptibilityBreakpoints for Oral β-Lactams with Streptococcus pneumoniae

PharmacodynamicOld NCCLS (T MIC 40) New NCCLS

Drug breakpoint (mgL) breakpoint (mgL) breakpoint (mgL)

Amoxicillin 05 2 2Cefaclor mdash 05ndash1 1Cefuroxime 05 1 1Cefprozil mdash 1ndash2 2Cefpodoxime mdash 05 05

difference in serum levels between standard doses and the extended-release for-mulation and those for cefprozil reflect the difference in serum levels from chil-dren and adults The NCCLS breakpoints are identical to the pharmacodynamicbreakpoints

64 Guidelines for Empirical Therapy

Because the magnitude of PKPD parameters determined in animal infectionmodels can be predictive of antimicrobial efficacy in human infections it is easyto understand why pharmacodynamics is being used more and more in establish-ing guidelines for empirical therapy Recently published guidelines for otitis me-dia acute bacterial rhinosinusitis and community-acquired pneumonia have usedthe ability of antimicrobials to reach the magnitude of PKPD parameters requiredfor efficacy for both susceptible pathogens and those with decreased susceptibilityto rank or select antimicrobials for empirical therapy of these respiratory infec-tions [365078]

7 SUMMARY

Studies over the past 20 years have demonstrated that antibacterials and antifun-gals can vary markedly in their time course of antimicrobial activity Three differ-ent patterns of antimicrobial activity are observed Specific PKPD parameterssuch as the peakMIC and AUCMIC ratios and the time above MIC have alsobeen shown to be major determinants of in vivo efficacy The magnitude of thePKPD parameters required for efficacy are relatively similar in animal infectionmodels and human infections and are largely independent of the dosing intervalthe site of infection the drug used within each antimicrobial class and the typeof infecting pathogen However additional studies are needed to extend currentobservations to other antimicrobials and organisms and to correlate PKPD pa-


rameters with therapeutic efficacy in a variety of animal infection models andhuman infections Pharmacodynamics has many applications including use forestablishing optimal dosing regimens for old drugs for developing new antimi-crobials and formulations for setting susceptibility breakpoints and for providingguidelines for empirical therapy

REFERENCES

1 Ali MZ Goetz MB A meta-analysis of the relative efficacy and toxicity of singledaily dosing versus multiple daily dosing of aminoglycosides Clin Infect Dis 199724796ndash809

2 Andes D Craig WA In vivo activities of amoxicillin and amoxicillin-clavulanateagainst Streptococcus pneumonia Application to breakpoint determinations Anti-microb Agents Chemother 1998 422375ndash2379

3 Andes D van Ogtrop M Characterization and quantitation of the pharmacodynamicsof fluconazole in a neutropenic murine disseminated candidiasis infection modelAntimicrob Agents Chemother 1999 432116ndash2120

4 Andes D van Ogtrop ML In vivo characterization of the pharmacodynamics offlucytosine in a neutropenic murine disseminated candidiasis model AntimicrobAgents Chemother 2000 44938ndash942

5 Andes DR Craig WA Pharmacokinetics and pharmacodynamics of antibiotics inmeningitis Infect Dis Clinics N Am 1999 13595ndash618

6 Andes DR Craig WA Pharmacodynamics of fluoroquinolones in experimentalmodels of endocarditis Clin Infect Dis 1998 2747ndash50

7 Andes D Craig WA Pharmacodynamics of gemifloxacin against quinolone-resistantstrains of Streptococcus pneumoniae with known resistance mechanisms Abstractsof the 39th Interscience Conference on Antimicrobial Agents and ChemotherapyAm Soc Microbiol Washington DC 1999

8 Andes D Van Ogtrop M Craig WA Pharmacodynamic activity of a new oxazolidi-none in an animal infection model Abstracts of the 38th Interscience Conference onAntimicrobial Agents and Chemotherapy Am Soc Microbiol Washington DC 1998

9 Andes D In-vivo pharmacodynamics of amphotericin B against Candida albicansAbstracts of the 39th Interscience Conference on Antimicrobial Agents and Chemo-therapy Am Soc Microbiol Washington DC 1999

10 Bailey TC Little JR Littenberg B Reichley RM Dunagan WC A meta-analysisof extended-interval dosing versus multiple daily dosing of aminoglycosides ClinInfect Dis 1997 24786ndash795

11 Barza M Ioannidis JPA Cappelleri JC Lau J Single or multiple daily doses ofaminoglycosides A meta-analysis Br Med J 1996 312338ndash345

12 Bigger JW The bactericidal action of penicillin on Staphylococcus pyogenes IrishJ Med Sci 1994 227533ndash568

13 Blaser J Stone BB Groner MC et al Comparative study with enoxacin and netil-micin in a pharmacodynamic model to determine importance of ratio of antibioticpeak concentration to MIC for bactericidal activity and emergence of resistanceAntimicrob Agents Chemother 1987 311054ndash1060

18 Craig

14 Bodey GP Ketchel SJ Rodriguez N A randomized study of carbenicillin plus cefa-mandole or tobramycin in the treatment of febrile episodes in cancer patients AmJ Med 1979 67608ndash616

15 Bundtzen RW Gerber AU Cohn DL Craig WA Postantibiotic suppression of bac-terial growth Rev Infect Dis 1981 328ndash37

16 Bustamante CL Wharton RC Wade JC In vitro activity of ciprofloxacin in combi-nation with ceftazidime aztreonam and azlocillin against multiresistant isolates ofPseudomonas aeruginosa Antimicrob Agents Chemother 1990 341814ndash1815

17 Cars O Odenholt-Tornqvist I The post-antibiotic sub-MIC effict in vitro and invivo J Antimicrob Chemother 1993 31(suppl D)159ndash166

18 Craig WA Gudmundsson S Postantibiotc effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams and Wilkins Baltimore MD 1996296ndash329

19 Craig WA Andes D Differences in the in vivo pharmacodynamics of telithromycinand azithromycin against Streptococcus pneumoniae Abstracts of 40th InterscienceConference on Antimicrobial Agents and Chemotherapy Am Soc Microbiol Wash-ington DC 2000

20 Craig WA Ebert SC Killing and regrowth of bacteria in vitro A review Scand JInfect Dis 1991 suppl 7463ndash70

21 Craig WA Pharmacokineticpharmacodynamics parameters Rationale for antibac-terial dosing of mice and men Clin Infect Dis 1997 261ndash12

22 Craig WA Postantibiotic effects in experimental infection models Relationship toin vitro phenomena and to treatment of infections in man J Antimicrob Chemother1993 31149ndash158

23 Craig WA Interrelationship between pharmacokinetics and pharmacodynamics indetermining dosage regimens for broad-spectrum cephalosporins Diagn MicrobiolInfect Dis 1995 211ndash8

24 Craig WA Ebert SC Continuous infusion of β-lactam antibiotics AntimicrobAgents Chemother 1992 362577ndash2583

25 Craig WA Andes D Pharmacokinetics and pharmacodynamics of antibiotics in oti-tis media Pediatr Infect Dis J 1996 15255ndash259

26 Craig WA Ebert S Watanabe Y Differences in time above MIC required for effi-cacy of beta-lactams in animal infection models Abstracts of the 33rd InterscienceConference on Antimicrobial Agents and Chemotherapy Am Soc Microbiol Wash-ington DC 1993

27 Craig WA Redington J Ebert SC Pharmacodynamics of amikacin in-vitro and inmouse thigh and lung infections J Antimicrob Chemother 1991 27(suppl C)29ndash40

28 Craig WA Dalhoff A Pharmacodynamics of fluoroquinolones in experimental ani-mals In J Kuhlman A Dalhoff HJ Zeiler eds Handbook of Experimental Pharma-cology Vol 127 Quinolone Antibacterials pp 207ndash232

29 Craig WA Postantibiotic effects and the dosing of macrolides azalides and strepto-gramins In SH Zinner LS Young JF Acar HC Neu eds Expanding Indicationsfor the New Macrolides Azalides and Streptogramins Marcel Dekker New York199727ndash38

30 Craig WA Antimicrobial resistance issues of the future Diagn Microbiol Infect Dis1996 25213ndash217


31 Craig WA Does the dose matter Clin Infect Dis 2000 in press32 Dagan R Abramason O Leibovitz E Greenberg D Lang R Goshen S Yagupsky

P Leiberman A Fliss DM Bacteriologic response to oral cephalosporins Are estab-lished susceptibility breakpoints appropriate in the case of acute otitis media J InfectDis 1997 1761253ndash1259

33 Dagan R Leibovitz E Fliss DM Leiberman A Jacobs MR Craig W Yagupsky PBacteriologic efficacies of oral azithromycin and oral cefaclor in treatment of acuteotitis media in infants and young children Antimicrob Agents Chemother 2000 4443ndash50

34 De Broe ME Verbist L Verpooten GA Influence of dosage schedule on renal corti-cal accumulation of amikacin and tobramycin in man J Antimicrob Chemother1991 27(suppl C)41ndash47

35 Den Hollander JG Mouton JW van Goor MP et al Alteration of postantibioticeffect during one dosing interval of tobramycin simulated in an in vitro pharmacoki-netic model Antimicrob Agents Chemother 1996 40784ndash786

36 Dowell SF Butler JC Giebink GS Jacobs MR Jernigan D Musher DM RakowskyA Schwartz B Acute otitis media Management and surveillance in an era of pneu-mococcal resistancemdashA report from the drug-resistant Streptococcus pneumoniaetherapeutic working group Pediatr Infect Dis J 1999 181ndash9

37 Drusano GL Johnson DE Rosen M Pharmacodynamics of a fluoroquinolone anti-microbial agent in a neutropenic rat model of Pseudomonas sepsis AntimicrobAgents Chemother 1993 37483ndash490

38 Eagle H Musselman AD The slow recovery of bacteria from the toxic effects ofpenicillin J Bacteriol 1949 58475ndash490

39 Ernst EJ Klepser ME Pfaller MA Postantifungal effects of echinocandin azole andpolyene antifungal agents against Candida albicans and Cryptococcus neoformansAntimicrob Agents Chemother 2000 441108ndash1111

40 Fantin B Ebert S Leggett J et al Factors affecting the duration of in-vivo postanti-biotic effect for aminoglycosides against gram-negative bacilli J Antimicrob Che-mother 1990 27829ndash836

41 Fantin B Leggett J Ebert S Craig WA Correlation between in vitro and in vivoactivity of antimicrobial agents against gram-negative bacilli in a murine infectionmodel Antimicrob Agents Chemother 1991 351413ndash1422

42 Fantin B Carbon C Importance of the aminoglycoside dosing regimen in thepenicillin-netilimicin combination for treatment of Enterococcus faecalisndashinducedexperimental endocarditis Antimicrob Agents Chemother 1990 342387ndash2389

43 Feikin DR Schuchat A Kolczak M Barrett NL Harrison LH Lefkowitz LMcGreer A Farley MM Vugia DJ Lexau C Stefonek KR Patterson JE JorgensenJH Mortality from invasive pneumococcal pneumonia in the era of antibiotic resis-tance 1995ndash1997 Am J Public Health 2000 90223ndash229

44 Ferriols-Lisart R Alos-Alminana M 1996 Effectiveness and safety of once-dailyaminoglycosides A meta-analysis Am J Health Syst Pharmacy 1996 531141ndash1150

45 Forrest A Nix DE Ballow CH Goss TF Birmingham MC Schentag JJ Pharmaco-dynamics of intravenous ciprofloxacin in seriously ill patients Antimicrob AgentsChemother 1993 371073ndash1081

20 Craig

46 Galloe AM Gaudal N Christensen HR Kampmann JP Aminoglycosides Singleor multiple daily dosing A meta analysis on efficacy and safety Eur J Clin Pharma-col 1995 4839ndash43

47 Gudmundsson S Vogelman B Craig WA The in vivo postantibiotic effect of imi-penem and other new antimicrobials J Antimicrob Chemother 1986 18(suppl E)67ndash73

48 Gwaltney JM Savolainen S Rivas P Schenk P Scheld WM Sydnor A KeyserlingC Leigh A Tack KJ Comparative effectiveness and safety of cefdinir and amoxicil-lin-clavulanate in treatment of acute community-acquired bacterial sinusitis Antimi-crob Agents Chemother 1997 411517ndash1520

49 Hatala R Dinh T Cook DJ Once-daily aminoglycoside dosing in immunocompetentadults A meta-analysis Ann Intern Med 1996 124717ndash725

50 Heffelfinger JD Dowell SF Jorgensen JH et al Management of community-acquired pneumonia in the era of pneumococcal resistance Arch Intern Med 20001601399ndash1408

51 Houlihan HH Mercier RC McKinnon PS et al Continuous infusion versus inter-mittent administration of ceftazidime in critically ill patients with gram-negativeinfection Abstracts of the 37th Interscience Conference on Antimicrobial Agentsand Chemotherapy Am Soc Microbiol Washington DC 1997

52 Int Antimicrob Therap Coop Group of EORTC Efficacy and toxicity of single dailydoses of amikacin and ceftriaxone versus multiple daily doses of amikacin and cef-tazidime for infection in patients with cancer and granulocytopenia Ann Intern Med1993 119584

53 Kashuba AD Nafziger AN Drusano GL Bertino JS Optimizing aminoglycosidetherapy for nosocomial pneumonia caused by gram-negative bacteria AntimicrobAgents Chemother 1999 43623ndash629

54 Klein JO Microbiologic efficacy of antibacterial drugs for acute otitis media PediatrInfect Dis 1993 J 12973ndash975

55 Knudsen JD Fuursted K Raber S Espersen F Fridmodt-Moller N Pharmacody-namics of glycopeptides in the mouse peritonitis model of Streptococcus pneumon-iae and Staphylococcus aureus infection Antimicrob Agents Chemother 2000 441247ndash1254

56 Lacy MK Lu W Xu X Tessier PR Nicolau DP Quintiliani R Nightingale CHPharmacodynamic comparisons of levofloxacin ciprofloxacin and ampicillinagainst Streptococcus pneumoniae in an in vitro model of infection AntimicrobAgents Chemother 1999 43672ndash677

57 Leggett JE Fantin B Ebert S Totsuka K Vogelman B Calame W Mattie H CraigWA Comparative antibiotic dose-effect relations at several dosing intervals in mu-rine pneumonitis and thigh-infection models J Infect Dis 1989 159281ndash292

58 Leggett JE Ebert S Fantin B Craig WA Comparative dose-effect relations at sev-eral dosing intervals for beta-lactam aminoglycoside and quinolone antibioticsagainst gram-negative bacilli in murine thigh-infection and pneumonitis modelsScand J Infect Dis 1991 (suppl 74)179ndash184

59 Lister PD Sanders CC Pharmacodynamics of levofloxacin and ciprofloxacin againstStreptococcus pneumoniae J Antimicrob Chemother 1999 4379ndash86

60 Lorian V Effect of low antibiotic concentrations on bacteria Effects on ultrastruc-


ture virulence and susceptibility to immunodefenses In V Lorian ed Antibioticsin Laboratory Medicine 4th ed Williams and Wilkins Baltimore 1991493ndash555

61 Louie A Drusano GL Banerjee P Liu QF Liu W Kaw P Shayegani M TaberH Miller MH Pharmacodynamics of fluconazole in a murine model of systemiccandidiasis Antimicrob Agents Chemother 1998 421105ndash1109

62 McDonald PJ Wetherall BL Pruul H Postantibiotic leukocyte enhancement In-creased susceptibility of bacteria pretreated with antibiotics to activity of leukocytesRev Infect Dis 1981 338ndash44

63 McDonald PJ Craig WA Kunin CM Persistent effect of antibiotics on Staphylococ-cus aureus after exposure for limited periods of time J Infect Dis 1977 135217ndash223

64 McGrath BJ Marchbanks CR Gilbert D Dudley MN In vitro postantibiotic effectfollowing repeated exposure to imipenem temofloxacin and tobramycin Antimi-crob Agents Chemother 1993 371723ndash1725

65 Moore RD Lietman PS Smith CR Clinical response to aminoglycoside therapyImportance of the ratio of peak concentration to minimal inhibitory concentrationJ Infect Dis 1987 15593ndash99

66 Mouton JW van Ogtrop ML Andes D Craig WA Use of pharmacodynamic indicesto predict efficacy of combination therapy in vivo Antimicrob Agents Chemother1999 432473ndash2478

67 Munckhof WJ Grayson JL Turnidge JD A meta-analysis of studies on the safetyand efficacy of aminoglycosides given either once daily or as divided doses J Anti-microb Chemother 1996 37645ndash663

68 National Committee for Clinical Laboratory Standards Development of In-VitroSusceptibility Testing Criteria and Quality Control Parameters Approved Guideline2nd ed Document M23ndashA2 January 2000

69 Nenko AS Cappelletty DM Kruse JA et al Continuous infusion versus intermittentadministration of ceftazidime in critically ill patients with suspected gram-negativeinfection Abstracts of the 35th Interscience Conference on Antimicrobial Agentsand Chemotherapy Am Soc Microbiol Washington DC 1995

70 Nicolau DP Onyeji CO Zhong M Tessier PR Banevicius MA Nightingale CHPharmacodynamic assessment of cefprozil against Streptococcus pneumoniae Im-plications for breakpoint determinations Antimicrob Agents Chemother 2000 441291ndash1295

71 Odenholt-Tornqvist I Lowdin E Cars O Postantibiotic sub-MIC effects of vanco-mycin roxithromycin sparfloxacin and amikacin Antimicrob Agents Chemother1992 361852ndash1858

72 Pallares R Linares J Vadillo M Cabellos C Manresa R Viladrich PF Martin RGudiol F Resistance to penicillin and cephalosporin and mortality from severe pneu-mococcal pneumonia in Barcelona Spain N Engl J Med 1995 333474ndash480

73 Preston SL Drusano GL Berman AL Fowler CL Chow AT Dornseif B ReichiV Natarajan J Corrado M Pharmacodynamics of levofloxacin A new paradigmfor early clinical trials J Am Med Assoc 1998 279125ndash129

74 Redington J Ebert SC Craig WA Role of antimicrobial pharmacokinetics and phar-macodynamics in surgical prophylaxis Rev Infect Dis 1991 13(suppl 10)S790ndashS799

22 Craig

75 Rybak MJ Abate BJ Kang SL Ruffing MJ Lerner SA Drusano GL Prospectiveevaluation of the effect of an aminoglycoside dosing regimen on rates of observednephrotoxicity and ototoxicity Antimicrob Agents Chemother 1999 431549ndash1555

76 Safdar N Andes D Craig WA In-vivo pharmacodynamic characterization of dapto-mycin Abstracts of 37th Infectious Diseases Society of America Washington DC1999

77 Scheld WM Sydnor A Farr B Gratz JC Gwaltney JM Comparison of cyclacillinand amoxicillin for therapy for acute maxillary sinusitis Antimicrob Agents Chemo-ther 1986 30350ndash353

78 Sinus and Allergy Health Partnership Antimicrobial treatment guidelines for acutebacterial rhinosinusitis Otolaryngol Head Neck Surg 2000 123(suppl 1)1ndash32

79 Sydnor A Gwaltney JM Cocchetto DM Scheld WM Comparative evaluation ofcefuroxime axetil and cefaclor for treatment of acute bacterial maxillary sinusitisArch Otolaryngol Head Neck Surg 1989 1151430ndash1433

80 Tauber MG Zak O Scheld WM Hengstler B Sande MA The postantibiotic effectin the treatment of experimental meningitis caused by Streptococcus pneumoniaein rabbits J Infect Dis 1984 149575ndash583

81 Ter Braak EW De Vries PJ Bouter KP Van der Vegt SG Dorrestein GC NorthierJW Van Dijk A Verkooyen RP Verbrugh HA Once-daily dosing regimen foraminoglycoside plus β-lactam combination therapy of serious bacterial infectionsComparative trial with netilmicin plus ceftriaxone Am J Med 1990 8958ndash66

82 Thomas JK Forrest A Bhavnani SM Hyatt JM Cheng A Ballow CH Schentag JJPharmacodynamic evaluation of factors associated with the development of bacterialresistance in acutely ill patients during therapy Antimicrob Agents Chemother 199842521ndash527

83 Tran BH Deffrennes D Aminoglycoside ototoxicity Influence of dosage regimenon drug uptake and correlation between membrane binding and some clinical fea-tures Acta Otolaryngol (Stockholm) 1988 105511ndash515

84 Turnidge JD Gudmundsson S Bogelman B Craig WA The postantibiotic effectof antifungal agents against common pathogenic yeast J Antimicrob Chemother1994 3483ndash92

85 Urban A Craig WA Daily dosing of aminoglycosides Curr Clin Top Infect Dis1997 17236ndash255

86 Verpooten GA Giuliano RA Verbist L Estermans G De Broe ME Once-dailydosing decreases renal accumulation of gentamicin and netilmicin Clin PharmacolTher 1989 4522ndash27

87 Vogelman B Gudmundsson S Leggett J Turnidge J Ebert S Craig WA Correla-tion of antimicrobial pharmacokinetic parameters with therapeutic efficacy in ananimal model J Infect Dis 1988 158831ndash847

88 Vogelman B Gudmundsson S Turnidge J Craig WA The in vivo postantibioticeffect in a thigh infection in neutropenic mice J Infect Dis 1988 157287ndash298

2

Microbiology and Pharmacokinetics

Charles H Nightingale

Hartford Hospital Hartford Connecticut

Takeo Murakawa

Fujisawa Pharmaceutical Company Ltd Osaka Kyoto University Kyoto andTokushima University Tokushima Japan

1 INTRODUCTION

Pharmacodynamics as applied to antimicrobial agents is simply the indexing ofmicrobiological data to the drugrsquos concentration in the body (pharmacokinetics)Antimicrobial agents are different from most therapeutic drugs in their mecha-nism of action The major difference between the agents and other drugs involvesthe fact that the target of antimicrobial agents is not the human body but microor-ganisms that live in it A strong understanding of microbiology and pharmacoki-netics under dynamic in vivo conditions is needed to fully understand the topicof microbial pharmacodynamics However it is beyond the scope of this chapterto present such material in detailed survey form Other chapters of this text willdiscuss specific drug classes and illustrate the important pharmacokinetic andmicrobiological properties of those drugs We prefer to discuss the importantaspects of microbiology and pharmacokinetics as they apply to antimicrobialsand the treatment of infections We refer the reader to other chapters of this

23

24 Nightingale and Murakawa

textbook as well as to standard texts [12] and primary articles on microbiologyand pharmacokinetics for a more basic explanation of these topics

2 MICROBIOLOGY

21 Effect of Antimicrobial Agents on Microorganisms

To understand this topic one must first have a concept of what is meant by theterm antimicrobial agent In this chapter this term refers to a chemical entity (asubstance of particular chemical structure) that has the ability to bind to someimportant site in a microorganism and cause microbial death This important siteis a place in the microorganism where the organism undergoes some biochemicalreaction that is part of its life cycle The binding substance (which we call anantimicrobial agent) blocks or interferes with the biochemical reaction and thusprevents the organism from completely fulfilling those acts necessary to supportits life cycle The results of this lsquolsquointerferencersquorsquo by a chemical (natural or syn-thetic) that we call an antimicrobial agent usually results in cell death if the agentis in high enough concentration for a long enough period of time Assuming thatthis description is fundamentally correct it follows then that three events mustoccur in order for the antimicrobial agent to eradicate the microorganism

1 The antimicrobial agent must reach the action sites and bind to thebinding sites in the microorganism The binding site can be a penicillin-binding protein a DNA gyrase a topoisomerase or a ribosome or anyother point of attachment that interferes with the biochemical reactionsin the microorganism The actual site varies depending upon the chemi-cal structure of the class of the agent and also varies somewhat withinthe same agent class This ability to bind to an active site can be thoughtof as being roughly descriptive (although not completely) of what wewould commonly call the microbiological activity of the antimicrobialagents

2 The antimicrobial agent must occupy a sufficient number of activesites The occupation of a sufficient number of sites is an interestingtopic and is central to the definition of pharmacodynamics The mecha-nism of action of the antimicrobial agent does not have to be fullyelucidated for the purpose of this discussion First it must be recognizedthat the number of active sites that must be occupied is largely un-known Obviously the number is greater than one because we knowat least intuitively that there are many active sites involving manymolecules of active proteins such as enzymes or DNA and we mustdescribe them very qualitatively One term that qualitatively describesthe number of sites is lsquolsquosufficientrsquorsquo The drug must diffuse usually (butnot always) on the basis of the drugrsquos concentration gradient to the

Microbiology and Pharmacokinetics 25

metabolically active site of the microorganism Because concentrationis important one might speculate upon the issue of identifying theimportant place where the drug concentration is favorable to its bindingto active sites Intuitively it is the drug concentration immediately sur-rounding the active site that ultimately controls this process But thatconcentration is in the bacterial cell and is largely unknown

3 The antimicrobial agent must reside on the active site for a period oftime Because antibiotics are not contact poisons but agents that inter-fere with a biochemical process essential for the bacterial life cycle adefinite time must evolve in order for the agent to elicit the desiredresponse The length of time at the active site is related to the strengthof binding to that site

When these three conditions exist the antimicrobial agents will have the desiredeffect on the bacteria

22 Parameters for Microbiological Activity of

Antimicrobial Agents

The killing properties of antimicrobial agents are important for pharmacodynamicconsiderations As described above the killing properties of antimicrobial agentsare dependent upon the antibiotic characteristics such as number of binding sitesand the binding or interfering properties at the active sites ie the drug concen-tration and length of time required to lead to microbial death It is difficult tocompletely characterize these properties However the overall results may besufficient from a practical perspectivemdashthe killing concentration surrounding thebacterial cell should be a good surrogate for the actual drug concentration at theactive site

The following parameters of antimicrobial activity are of common or practi-cal use

1 The minimal inhibitory concentration (MIC) is the lowest drug concen-tration without visible growth for 16ndash20 h at 35degC in the medium

2 The minimal bactericidal concentration (MBC) is the lowest concentra-tion required to kill 999 of the bacterial cells of the inoculum after16ndash20 h exposure to the drug at 35degC

For pharmacodynamic considerations it is important to understand that (1) theseparameters are determined under in vitro conditions and that (2) we cannot useonly one cell of a microorganism for the testsmdashfor example a million cells areused as a unit

Susceptibility of cells is not homogeneous ie each cell of a million cellpopulation has a different MIC or MBC and the total bacterial population showsan MIC distribution that is different for each organismndashdrug combination


23 Kinetic Killing Properties

The killing rate is an important parameter and will vary with the kind of microor-ganism and the strain of the organism as well as the type of antimicrobial agentThe killing properties of antimicrobial agents have two basic forms of relation-ships with drug concentration concentration dependence and time-dependenceIt should be noted that the issues of concentration-dependent and time-dependentbacterial killing are an abbreviated way of describing an antibioticndashbacterial in-teraction This is shown in Fig 1 which illustrates the usual in vitro experimentsto determine whether a drug is static or cidal If we grow bacteria in broth in theabsence of antibiotic the control curve results If we add a small quantity ofdrug the rate of growth still exceeds the rate of death and the organism continuesto grow Obviously that small amount of drug did not have much of an effecton the bacterial life cycle If we add more drug the rate of growth and deathmight be equal and we say that the drug exhibits static properties If we addeven more drug the rate of killing might exceed the rate of living and the drugis now termed a cidal agent If we continue to add increasing amounts of drugthe rate of killing relative to the rate of living continues to increase and if we lookat just this concentration range we conclude that the drug exhibits concentration-dependent killing If we add even more drug we eventually reach a point whereadditional drug does not increase the rate of killing any further and if we justlook at its behavior in that concentration range we say that the drug exhibits time-dependent killing properties In the example illustrated in Fig 1 the same drugexhibited no effect static behavior cidal behavior and concentration-dependentand time-dependent cidal behavior In essence the same drug can have all of

FIGURE 1 Relationship between net effect on bacterial growth and death andtime of exposure to antibiotic at various fixed concentrations


these properties depending on the drugrsquos concentration in relation to its MICTherefore when we say that a drug is a concentration- or time-dependent killerwe seem to be speaking in incomplete sentences A more complete descriptionwould be to add the phrase lsquolsquounder the concentrations achieved after clinicaldosingrsquorsquo The importance of this phenomenon is that although investigators maystudy the properties of a drug and classify it as static or cidal this is arbitrarybecause the same experiment under different conditions would result in a differ-ent classification This is why for the tetracyclines the official US packageinsert has phrases that indicate that these agents are static but exhibit cidal proper-ties against some bacteria

It is important to also realize that the killing property of a drug is relatedto its rate of growth If antibiotics interfere with certain biochemical reactionsin the bacteria that are normally part of their life cycle then the drug needs tobe present bound to the active site at the time the biochemical reaction is tooccur As an example penicillins or cephalosporins show bactericidal activitywhen cell growth is maximal but not for resting cells because the drugs inhibitthe synthesis of the bacterial cell wall and the bacteria must be actively makingcell walls for the antibiotic to have its effect

Sometimes the killing curve shows a reduction in the number of viablecells of an organism even at concentrations lower than the MIC (lsquolsquosubinhibitoryconcentrationsrsquorsquo) This means that some cells in the population are more sensitiveto the drug than the MIC reported for the entire population would indicate Insuch a case the MIC may change as a function of inoculum size

A postantibiotic effect (PAE) is observed with cephalosporins againstgram-negative bacteria When the bacteria are exposed to antibiotics at severaltimes the MIC for a certain period of time and then the antibiotic is removedslow bacterial growth is observed for a certain period of time Possible reasonsfor this phenomenon are that the drug continues to bind to the active sites of thebacteria or that the antibiotics remain in the cell even after the antibiotic hasdisappeared from outside the cell or there may be some methodological problemassociated with the removal of the drug from the test system

The MIC MBC and killing kinetics properties such as concentration-de-pendent and time-dependent properties are commonly used as parameters formicrobiological activity of antimicrobial agents and therefore are of major impor-tance However it is important to understand that these definitions and most dataare obtained in vitro not at the actual sites of infection in the body It is difficultor impossible to obtain the MIC in body fluids and in addition the reported datahave a large amount of variability associated with them due to the twofold dilu-tional techniques commonly used in the field of microbiology This represents100 variability in the reported MIC value As a guide the MIC values are veryuseful but to consider them to be absolute infallible numbers is misleading andconfusing


3 PHARMACOKINETICS

Parameters for the pharmacokinetic properties of antimicrobial agents are thesame as for those of other therapeutic drugs There is no particular issue in phar-macokinetic considerations with antimicrobial agents except that the target siteis located in the infection site where microorganisms are living The infectionsites are distributed throughout the body and an effective concentration of anantimicrobial agent is required to eradicate microorganisms in those infectionsites where they are living

31 Pharmacokinetic Parameters for

Pharmacodynamic Consideration

We do not administer a drug directly into microorganisms but into a humanbody It must be driven to the place where the organism lives by the drug concen-tration in blood Most organisms live in some interstitial fluid (IF) in the bodyalthough some reside inside mammalian cells Assuming we are considering or-ganisms that live in IF it is the drug concentration in this fluid that is importantFortunately drug transfer from the bloodstream into the IF is rather rapid dueto the leakiness of the capillary bed membrane and blood (serum or plasma)concentrations control the IF drug concentration which controls or influencesthe drug concentration around the bacterial active sites In this way we lsquolsquobackintorsquorsquo the realization that drug concentrations in the serum (lsquolsquoserumrsquorsquo will beused in this chapter to mean plasma blood or serum) are important for a sufficientnumber of active sites to be occupied by the drug However it is not true forevery injection site This forms the basis for modern pharmacodynamic theorywhich allows one to index microbiological activity to the serum concentrationof an antibiotic The concentration of the drug in the serum is a major functionof the drugrsquos pharmacokineticsThe pharmacokinetic parameters related to microbiological activity of an antibi-otic are

Peak drug concentration Cp

Half life T12

Area under the drug concentration curve AUCTime period during which drug concentrations exceed MIC or MBC

32 Difference of Pharmacokinetic Parameters in Serum

and Tissues or Fluids

As mentioned above pharmacokinetic parameters should be those based on theantibiotic concentration at the infection site However determination of the drugconcentration at various infection sites is difficult in practice A drug is distributedor penetrated into tissue or tissue fluids of infection sites via the bloodstream


When the drug concentrations at the infection site and in the bloodstream areproportional it is possible to use the serum or blood concentration to index tomicrobiological activity If there is a big difference between the kinetic propertiesin the blood and those at the site of infection (tissuesfluids) then the drug con-centration at the site of infection should be used to index to microbiologicalactivity Serumndashtissue protein binding drug distribution penetration rate anddrug metabolism at these sites will be factors in effecting differences in bloodand infection site drug kinetics

4 CLINICAL ASPECTS

As mentioned before there are major differences between antimicrobial agentsand other therapeutic drugs because the target of antimicrobial agents is not thehuman body but microorganisms that cause infectious diseases The human bodyitself has host defense mechanisms to kill invasive microorganisms Infectiousdiseases are caused by the bacteria creating through the large number of themgrowing in the body a condition that exceeds the bodyrsquos host defense capabilityto recognize and eradicate the invasive microorganism Therefore eradication ofmicroorganisms from the body or infection sites depends upon a combination ofactivities partly provided by the body and partly provided by an antibacterialdrug If host defense activity is normal and the infecting organisms are not verypathogenic clinical efficacy will result even when the activity of the antibioticis modest However when the host defense mechanism is weak or impaired asis the case with immunocompromised patients the antimicrobial agents itselfmust kill and eradicate the infecting microorganisms In such a situation thedosing and activity of the antibiotic must be maximized

5 PHARMACODYNAMICS

51 Pharmacodynamic Parameters

The drug concentration in the serum and the time that it remains there are relatedto the occupation of a sufficient number of binding sites in the bacteria and thelength of time those sites are occupied To produce a pharmacodynamic parame-ter it is only necessary to index a measure of microbiological activity such as theminimum inhibitory concentration (MIC) or minimum bactericidal concentration(MBC) to the area under the curve (AUC) In this chapter we will use the termMIC however the MBC or any other measure of microbiological activity couldalso be used It is interesting that the mathematical product of drug concentrationin the serum (Cp) multiplied by time is termed the drugrsquos area under the serumconcentrationndashtime curve (AUC) A schematic representation of the AUC in-


dexed to the MIC of bacteria is shown in Fig 2 This pharmacodynamic parame-ter AUCMIC is an important and fundamental parameter that relates microbio-logical activity to serum concentration One simple way of thinking about theAUCMIC ratio is that the AUC represents the concentration of drug and timeof exposure that presents itself to the bacteria over and above the minimumneeded to do the job (MIC) The higher this ratio (AUCMIC) the better is thechance of the antibiotic being successful It should also be noted that in natureantibiotics eradicate bacteria (fulfill the three requirements described earlier) byan interaction of microbiological activity and pharmacokinetics not just one ofthe two Decisions related to drug selection should therefore take both of theseproperties into consideration Unfortunately most clinicians because of theirtraining will choose antibiotics on the basis of microbiological activity aloneWe believe that this may result in poor decisions The reason for this belief isthat one can choose the most active antibiotic in the world (lowest MIC againstthe target organism) but if it does not go to where the bacteria reside it will notkill anything The reverse is also true Good pharmacokinetics with very poorantimicrobial activity will also not result in a satisfactory outcome Both needto be considered simultaneously hence the importance of pharmacodynamics

Under certain conditions the AUCMIC [(Cp time)MIC] ratio can besimplified These simplifying assumptions are based upon whether the drug isclassified as a concentration-dependent or time-dependent bacterial killer Thisdetermination is made from in vitro experiments such as those for which data areshown in Fig 3 For the aminoglycoside we can see that as the drug concentrationincreases the rate of killing increases and hence this drug is considered to be aconcentration-dependent killer If one makes the concentration high enough therate of killing is so fast that the contribution of drug exposure to the entire processis insignificant and can be ignored The ratio AUCMIC [(Cp time)MIC] sim-

FIGURE 2 Pharmacodynamic relationship AUCMIC


FIGURE 3 Concentration-dependent vs independent bacterial killing (FromRef 21)

plifies to CpMIC For such a drug the question arises regarding how high the drugconcentration must be in relation to the MIC in order to make this simplifyingassumption The answer is illustrated in Fig 4 where it can be seen that whenone reaches a CpMIC ratio of 10ndash12 1 the simplifying assumption seems tohold Several other investigators have demonstrated the same phenomenon [34]Achieving a CpMIC ratio of 10 or greater forms the basis for high and infrequentdosing of aminoglycosides [5ndash7] If such a ratio is achieved bacterial killing isat a maximum If it is not achieved then the drug can still be quite effectivehowever the simplifying assumption cannot be made and the entire AUC mustbe applied to the ratio A good dosing strategy for the clinical use of such drugsis to use the highest possible dose that achieves this ratio and does not causetoxicity

FIGURE 4 Clinical response vs CpMIC (from Ref 7)


Examination of Fig 3 also reveals that other agents such as the β-lactamsexhibit different bacterial killing attributes It can be seen that once these drugsachieve concentrations of 2ndash4 times the MIC further increases in concentrationdo not yield greater rates of bacterial killing Bacterial killing is at a maximumunder these conditions the contribution of drug concentration to the entire killingprocess is minimal and can be ignored The AUCMIC ratio simplifies to drugexposure of the bacteria and is expressed pharmacodynamically as the time theserum concentrations remain above the MIC of the bacteria (T MIC) For drugsthat display such behavior the clinical dosing strategy is to maximize the T MIC While the minimal T MIC varies with different drugndashbug combinationsit appears from animal [8ndash11] and clinical [12] data that an acceptable workingvalue of T MIC of about 50 of the dosing interval is associated with satisfac-tory outcomes This number is appropriate for patients with intact or functioningimmune systems For immunocompromised patients being 4ndash5 times the MICis related to better outcomes and is roughly equivalent to being above the MICfor the entire dosing interval [13] The working number of 50 T MIC or100 T MIC for immunocompromised patients is just a good approximationof what is necessary to achieve satisfactory clinical response This will vary de-pending upon different conditions such as host factors and drugndashbacteria interac-tions

It is important to also realize that the pharmacodynamic classification ofhow a drug interacts with bacteria is directly related to the bacterial rate ofgrowth If antibiotics interfere with certain biochemical reactions in the bacteriathat are normally part of their life cycle then the drug needs to be present boundto the active site at the time the biochemical reaction would normally occurAlthough this is intuitively obvious what seems to be overlooked is that thepharmacodynamic parameter that correlates best with bacterial eradication willchange with the growth properties of the bacteria A good example of this is arecently published study by Lutsar et al [14] who studied gatifloxacin pharmaco-dynamics in the cerebral spinal fluid (CSF) of rabbits Because gatifloxacin is aquinolone it is expected to be a concentration-dependent bactericidal agent Assuch one would expect that the pharmacodynamic parameters AUCMIC or CpMIC might predominate Whereas these investigators found that AUCcsfMIC didcorrelate with outcomes when the AUCcsf was held constant it was the T MICthat correlated with good outcome not CpcsfMIC This may at first seem counter-intuitive but it can be explained on the basis of the rate of growth of bacteriain CSF If the rate of growth is relatively slow the antibiotic will act only if itis present on the active site when that site is involved in the necessary biochemicalreaction that is part of the bacteriarsquos life cycle When one considers this it is notsurprising that a relationship exists with T MIC rather than high and transitorypeak concentrations


Do bacteria grow more slowly in CSF We believe they do because rapidlygrowing bacteria in the CSF will probably kill the host before therapy can beinitiated We hypothesize that the CSF is a much poorer growth medium forbacteria than either broth or other sites in the body and this slower growth allowstherapy to be initiated but also changes the pharmacodynamic parameter thatcorrelates with effective bacterial eradication

52 Relationship Between Pharmacodynamics and

Clinical Efficacy

The effective treatment of patients suffering from an infectious disease is a multi-faceted process that depends upon many factors for a successful outcome Gener-ally speaking there is an inverse relationship between the MIC of an organismand clinical efficacy ie the lower the MIC the higher the cure rate This isillustrated in Table 1 What can also be seen in this table is that the oppositemdashidentifying the MIC that correlates to an effective clinical cure ratemdashis not soobvious and many MIC values (eg 1 10 20 30 40) can be consideredto result in an acceptable cure rate Table 2 illustrates that eradication of bacteriacan fail to occur even when the bacteria are susceptible to the antibiotic Reportshave been published eg in the treatment of S pneumoniae bacteremia wherethe death rate is substantial in spite of aggressive and appropriate antimicrobialtherapy [1516] Obviously the ability of the antibiotic to kill the organism isonly one factor affecting patient outcome Other factors such as the age of thepatient the vigor of the immune system and psychological factors such as thewill to live and the degree of confidence the patient has in the caregivers andthe health system will affect outcomes Although pharmacodynamic parametersare important and useful in guiding therapy they are not the only issues thataffect outcomes The clinical practice of medicine still remains somewhat of an

TABLE 1 General Relationship BetweenMICs and Clinical Cures (Claforan)

MIC Clinical cures(microgmL) ()

10 9480 90

160 77320 84

640 64

Source Ref 22


TA

BLE

2S

ucc

ess

and

Failu

reo

fR

esis

tan

tan

dS

ensi

tive

Str

ain

so

fH

aem

op

hilu

sIn

flu

enza

Clin

ical

corr

elat

ion

aN

um

ber

of

stra

ins

wit

hIn

dic

ated

MIC

An

tim

icro

bia

lN

um

ber

An

tib

ioti

co

utc

om

eb

of

Pat

ien

ts

025

05

12

48

16

32

Lore

carb

efC

118

64

752

7428

87

6R

F10

18

1A

mo

x

CI

137

753

4225

73

Cla

vR

F11

74

aH

aem

op

hilu

sin

flu

enza

AO

MS

A

MS

A

PE

CB

an

dp

neu

mo

nia

b

CI

cu

reo

rim

pro

ve

RF

re

lap

seo

rfa

ilure

S

ou

rce

Ref

23


art rather than a pure science Pharmacodynamic principles help move clinicalmedicine several steps toward the science side but they are not the only issueof concern in clinical medicine

Issues involving an antibioticrsquos properties such as protein binding PAEand volume of distribution are important properties of the drug however forlicensed or marketed drugs further consideration of these properties is rarelyneeded The rationale behind this statement is related to the process of drug li-censing Using the drug development process the first step is to do preclinicalstudies followed by clinical studies Phase I studies are designed to observe anddetect gross human toxicity and to explore the drug dose and dosing issues Oncethis is completed a value judgment is made concerning a useful and safe clinicaldose The next step phase II is to carry out small-scale clinical trials the mainpurpose of which is to confirm the efficacy of using the dose derived from preclin-ical and phase I studies When it is confirmed that the drug is safe and effectivewith the chosen dosage and dosing regimen the development proceeds to largerscale clinical trials phase III If the dose and dosing regimen are found to beacceptable phase III trials eventually lead to licensing The point of this is thatthe licensed drug has been found to be acceptable useful and nontoxic on thebasis of all its properties For an antibiotic these include its pharmacokineticsand pharmacodynamic properties which involve considerations of volumes ofdistribution half-life PAE protein binding and tissue penetration If the antibi-otic were not acceptable the dose would be changed until it met the criteria forlicensing In other words the observed (empirical) result is due to the dose usedbased upon the agentrsquos overall properties The issues of protein binding PAEand other issues although important have already been taken into account andcontribute to the net effect one observes Further consideration of these issueswith respect to clinical outcomes is rarely necessary Considering a drugrsquos proteinbinding as a separate and unrelated issue does not make an approved agent lessor more effective in clinical use It will change the pharmacodynamic parameterseg AUCMIC and on a comparative basis will affect the rank order of drugsdepending upon their protein binding

53 The Application of Pharmacodynamic Parameters to

Clinical Practice

All pharmacodynamic parameters are simply calculated numbers that have noinherent value by themselves In order for these numbers to have value one mustcorrelate them to an outcome Because most practitioners are interested in treatinginfected patients the most accepted correlation is between the pharmacodynamicparameter and the clinical outcome It is important to realize that if one correlatesthe pharmacodynamic parameter to an outcome that is different than clinical ef-fectiveness then the important or critical value of the pharmacodynamic parame-


ter will change For example if one correlates the AUCMIC to clinical curesone critical number appears such that if one exceeds that parameter one can ex-pect good clinical cures If one correlates the parameter to maximal eradicationof bacteria another AUCMIC number appears that will probably be higher thanthat calculated in the first example If one desires to correlate the pharmacody-namic parameter to the suppression of the emergence of resistance then a thirdcritical pharmacodynamic number will appear

Considering the pharmacodynamic parameter AUCMIC and correlating itto clinical cures when the target organism is S pneumoniae and the drug classis quinolones we can make two important observations Figure 5 represents aschematic hypothetical graph of the relationship between clinical cure rates andAUCMIC ratio This schematic is based upon a variant of a dosendashresponsecurve At some point (to the right of the vertical line) a maximum clinical re-sponse is observed Most clinical studies in humans lie within this plateau be-cause the usual clinical trials are designed for success and not failure Data tocomplete the curve (the data to the left of the vertical line) are generally obtainedfrom animal studies or in vitro experiments The breakpoint (the point where thecurve plateaus) is a value judgment based upon the composite of all of these data[17ndash19] Our value judgment is that an AUCMIC ratio of 30ndash40 is relatedto successful clinical outcomes If one examines Table 3 which is derived fromsuccessful clinical studies one can see that there are a variety of AUCMICratios for total drug that correlate with successful clinical outcomes [20] Thisis expected on the basis of Fig 5 and argues that a single AUCMIC ratio for aparticular drug class against a particular organism does not exist If one performsa similar analysis with the same or different drug classes against different organ-isms a different range of ratios will be found to correlate to acceptable clinicaloutcomes This argues that although pharmacodynamic parameters are useful indetermining proper dosing they should be used as a guide rather than as absolute

FIGURE 5 Dosendashresponse curve for quinoloones vs S pneumoniae


TABLE 3 Oral Quinolone Pharmacodynamics S pneumoniae(Pharmacodynamic Order)

Drug Dose MIC90 AUCMIC

Moxifloxacin 400 mg 025 142Trovafloxacin 200 mg 025 138Grepafloxacin 600 mg 025 908Gatifloxacin 400 mg 05 678Levofloxacin 500 mg 1 476Sparfloxacin 200 mg 05 374

numbers This concept is further strengthened by the realization that the AUCMIC is a ratio with a numerator and a denominator The numerator representsthe AUC but that is usually the AUC of the drug in normal healthy volunteersThe clinical use of antibiotics is in patients and it is the patientrsquos AUC thatshould be used in the ratio Unfortunately this value is not readily available Thenormal volunteerrsquos AUC represents the worst-case situation and this AUC isgenerally smaller than that of an actual patient The denominator has even moreerror and variability associated with it The MIC should be that of the drug andthe bacteria in the host body not in an artificial medium such as broth Brothaffords an ideal growth medium whereas bodily fluids do not The growth ratesof organisms will be different in the body than in broth and the interactionswith the antimicrobial agents will be affected ie the MIC will be different aspreviously discussed Unfortunately it is difficult or impossible to obtain theMIC in bodily fluids therefore we commonly use the incorrect MIC obtained inbroth A large degree of variability is associated with the usual technique ofdetermining the MIC Most MICs are measured using a twofold dilution tech-nique and one dilution to either side of the observed value is not considered tobe an important or real difference A reported MIC of 10 microgmL can be 0510 or 20 microgmL That represents 100 variability in the reported MIC valueIf the numerator is not accurate and the denominator is not accurate then theentire ratio cannot be accurate Reported AUCMIC ratios are too variable to beconsidered absolute numbers and should be used only as guides to therapy Asa guide the MIC is very useful but to consider it to be an absolute infalliblenumber is misleading and confusing

REFERENCES

1 HP Kuemmerle T Murakawa CH Nightingale eds Pharmacokinetics of Antimicro-bial Agents Principles Methods Applications Ecomed 1993


2 PR Murray Editor in Chief and EJ Baron MA Pfaller FC Tenover and RH Yolkeneds Manual of Clinical Microbiology 6th ed ASM Press Washington DC 1995

3 EJ Begg BA Peddie ST Chambers DR Boswell Comparison of gentamicin dosingregimens using an in-vitro model J Antimicrob Chemother 199229427ndash433

4 BD Davis Mechanism of the bactericidal action of the aminoglyocosides MicrobiolRev 198751341ndash350

5 DP Nicolau CD Freeman PP Beleveau CH Nightingale JW Ross R QuintilianiExperience with a once-daily aminoglycoside program administered to 2184 adultpatients Antimicrob Agents Chemother 199539650ndash655

6 MF Keating GP Bodey M Valdivieso V Rodriquez A randomized comparativetrial of three aminoglycosidesmdashcomparison of continuous infusions of gentamicinamikacin and sisomicin combined with carbenicillin in the treatment of neutropenicpatients with malignancies Medicine 197958159ndash170

7 RD Moore PS Lietman CR Smith Clinical response to aminoglycoside therapyImportance of the ratio of peak concentration to minimal inhibitory concentrationJ Infect Dis 198715593ndash99

8 JE Leggett S Ebert B Fantin WA Craig Comparative dose-effect relations at sev-eral dosing intervals for beta-lactam aminoglycoside and quinolone antibioticsagainst gram-negative bacilli in murine thigh-infection and pneumonitis modelsScand J Infect Dis 199174179ndash184

9 JE Leggett B Fantin S Ebert K Totsuka B Vogelman W Calame H Mattie WACraig Comparative antibiotic dose-effect relations at several dosing intervals in mu-rine pneumonitis and thigh-infection models J Infect Dis 1989159281ndash292

10 R Roosendaal IAJM Bakker-Woudenberg JC van den Berghe MF Michel Thera-peutic efficacy of continuous versus intermittent administration of ceftazidime in anexperimental Klebsiella pneumoniae pneumonia in rats J Infect Dis 1985156373ndash378

11 CO Onyeji DP Nicolau CH Nightingale R Quintiliani Optimal times above MICsof ceftibuten and cefaclor in experimental intra-abdominal infections AntimicrobAgents Chemother 1994381112ndash1117

12 GP Bodey SJ Ketchel V Rodriguez A randomized study of carbenicillin plus cefa-mandole or tobramycin in the treatment of febrile episodes in cancer patients AmJ Med 197967608ndash616

13 GP Bodey SJ Ketchel V Rodriguez A randomised trial of carbenicillin plus cefa-mandole or tobramycin in the treatment of febrile episodes in cancer patients Anti-microb Agents Chemother 199236540ndash544

14 I Lutsar IR Friedland L Wubbel CC McCoig HS Jafri WN Ng F Ghaffar GHMcCracken Jr Pharmacodynamics of gatifloxacin in cerebrospinal fluid in experi-mental cephalosporin-resistant pneumococcal meningitis Antimicrob Agents Che-mother 1998422650ndash2655

15 EW Hook III CA Horton DR Schaberg Failure of intensive care unit support toinfluence mortality from pneumococcal bacteremia JAMA 19832491055ndash1057

16 WR Gransden SJ Eykyn I Phillips Pneumococcal bacteremia 325 episodes diag-nosed at St Thomasrsquos Hospital BMJ 1985290505ndash508

17 PD Lister CC Sanders Pharmacodynamics of levofloxacin and ciprofloxacin againstStreptococcus pneumoniae J Antimicrob Chemother 19994379ndash86


18 Lacy M Quintiliani R et al AAC 199943672ndash67719 Lister P Sanders C AAC 1999431118ndash112220 P Ball A Fernald G Tillotson Therapeutic advances of new fluoroquinolones Exp

Opin Invest Drugs 19987761ndash78321 Craig WA et al Scand J Infect Dis 1991suppl 7422 Murry et al Abstract ICCAC meeting 198323 Doern G Pedi Infect Dis J 199514420ndash423

3

In Vitro Antibiotic PharmacodynamicModels

Michael J Rybak

Wayne State University Schools of Medicine and Pharmacy andDetroit Receiving Hospital and University Health Center Detroit Michigan

George P Allen

Wayne State University and Detroit Receiving Hospital andUniversity Health Center Detroit Michigan

Ellie Hershberger

William Beaumont Research Institute Royal Oak Michigan

1 INTRODUCTION

The traditional approach to in vitro assessment of antimicrobial action on organ-ism viability has been through the use of typical susceptibility testing such asagar diffusion assay or broth dilution techniques These tests give either a qualita-tive or quantitative assessment of antimicrobial activity at fixed concentrationsof drug at a single point in time usually 18ndash24 h The killing curve approachallows for periodic or continuous monitoring of viable organisms after antibioticexposure This method significantly improves the ability to assess the interactionbetween antimicrobials and organisms Factors such as the rate and extent of

41

42 Rybak et al

killing as a function of concentration can be determined In addition the impactof combination antibiotics can easily be evaluated However the major limitationof this method is the use of a fixed concentration of antibiotic throughout theexperimental period Therefore it does not represent the clinical setting whereantimicrobial concentrations continuously fluctuate as a function of delivery pen-etration metabolism and elimination

These limitations prompted the development of antibiotic pharmacody-namic models that are capable of simulating antibiotic pharmacokinetics in thepresence of viable bacteria Controlled dilution of media containing the drug andorganism inoculum is the basic function by which these dynamic models simulateantibiotic pharmacokinetics One of the earliest attempts to use an in vitro antibi-otic dynamic model system to simulate human infection was reported by Green-wood and OrsquoGrady [1] These investigators simulated bladder infection by usingglass flasks in which the effect of antibiotics on bacteria in artificial media couldbe studied photometrically Micturition was simulated by alterations in the vol-ume and flow rate of medium so that the concentrations of organism and antibioticin the model varied in a manner similar to in vivo infection Although it was avery novel approach there were a number of problems with this method includ-ing the use of photometric analysis The spectrophotometric method used in thisexperiment proved unreliable because it counted both viable and dead bacteriaand because of its dependence on light transmission through the artificial mediumit was not useful for dense bacterial populations of 106ndash109 CFUmL which areoften more relevant to human infection Another inherent problem was that bacte-ria grew more rapidly in artificial media than was observed in urine In subsequentmodels these limitations were corrected by plating and counting the number ofviable bacteria over the course of the experiment and by using filtered sterilizedurine as the growth medium There remain some mechanical problems with thismodel such as occasional adherence of organisms to the glass culture vesselsduring the washout phase and the inability to simulate cyclical variations in urineantibiotic concentrations due to renal clearance or micturition However thesemodelshave provided realistic simulationsof the responseoforganism andantibiot-ics among patients in clinical trials [2] In addition the bladder model had a muchhigher correlation with a mouse infection protection test than did a variety of stan-dard laboratory tests Furthermore conventional laboratory methods failed to pre-dict antibiotic synergy observed in both the bladder model and mouse models [34]

Over the years there have been a variety of in vitro antibiotic dynamic mod-els described in the literature that have attempted to characterize the pharmaco-kinetic profiles of various antibiotics while simulating an infection site Thesemodels have ranged from simple simulations of bloodstream infections with one-compartment dynamic models to sequestered infections with more sophisticatedmulticompartmental approaches or hybrid systems incorporating infected tissue

In Vitro Antibiotic Pharmacodynamic Models 43

or cell lines This chapter presents a description of these models and their inherentadvantages and disadvantages for modeling infection and antimicrobial pharma-cokineticspharmacodynamics

2 ONE-COMPARTMENT MODEL

In the one-compartment model the antimicrobial agent is added to a central com-partment that contains the appropriate culture medium and a known concentrationof the organism The medium inside the central compartment is then displacedvia a peristaltic pump set at a fixed rate to simulate the clearance and half-lifeof the antibiotic (Fig 1) This system represents a simple pharmacodynamicmodel that follows first order pharmacokinetics resulting in an exponential de-crease in the drug concentration Desired pharmacokinetic drug parameters canbe estimated using the equation

c c0eket

FIGURE 1 One-compartment model The bacterial inoculum and antibiotic areintroduced into the central compartment CA concentration of antibiotic ACC central compartment CIA clearance of antibiotic A FM fresh me-dium P peristaltic pump SP sampling and injection port VC volumeof distribution of antibiotic A SB magnetic stir bar WB water bath(375degC) WM waste medium

44 Rybak et al

where

c antibiotic concentrationc0 concentration following drug administrationke elimination rate constantt time

The elimination rate constant is defined by

ke ClVc (1)

and

ke (ln 2)t12 (2)

Therefore

(ln 2)t12 ClVc (3)

Solving for Cl

Cl (ln 2)(Vct12) (4)

where

Cl clearanceVc volume in the central compartmentt12 half-life of the drug

This system has the advantage of being able to simulate human kineticsby simply utilizing sterile flasks connected by tubing passed through a peristalticpump Because of their simplicity one-compartment models have been used ex-tensively throughout the years to evaluate pharmacodynamics of antimicrobialagents Zabinski et al [5] used a modified version of a one-compartment modelpreviously described by Garisson and his colleagues to determine the effect ofaerobic and anaerobic environments on the activities of different fluoroquinolonesagainst staphylococci The system used in this experiment consisted of a 1L glassvessel with inflow and outflow ports silicone tubing a peristaltic pump a freshmedia reservoir and a stir hot plate (to maintain a normal body temperature ofapproximately 37degC) The entire system was then placed in a chamber containingan anaerobic gas mixture

One of the disadvantages of this system is the clearance of bacteria fromthe central compartment along with the antibiotic This clearance may vary sig-nificantly depending on the half-life of the drug For example in an in vitroexperiment using vancomycin (half-life 6 h) against Staphylococcus aureus (start-ing inoculum of 106 CFUmL) an insignificant amount of organism will be lostbecause the doubling time of S aureus is greater than the clearance of vancomy-


cin However utilizing nafcillin (half-life 05 h) against the same isolate mightoverestimate the rate of kill because its clearance rate is greater than the doublingtime of S aureus Previously membranes were used in these models to preventthe elimination of bacteria from the central compartment however accumulationof antibiotic on the surface of the membrane may result in the antibiotic beingretained as well thus confounding interpretation of antibiotic killing effects [6]

In an effort to circumvent the problem of bacterial dilution while preventingconcomitant antibiotic retention Navashin et al [7] used a model that incorpo-rated a modified membrane system that is washed continuously throughout theexperiment Their model is similar in construction to the models described aboveconsisting of a supply of fresh medium that is pumped into a chamber into whichthe bacterial inoculum is injected (Fig 2) Medium is removed from this chamberby a second peristaltic pump however the medium is then eliminated througha filtration device that prevents the loss of bacteria The filtration device includes

FIGURE 2 Two-compartment model with membrane-wash system Mediumis pumped from the working unit (a photometric cell) through a filtration unitdesigned to prevent bacterial loss FM fresh medium FU filtration unitMF membrane filter P peristaltic pump WU working unit of model(Adapted from Ref 7)

46 Rybak et al

a membrane with a pore size of 045 microm To prevent blockage of this membranewith excreted antibiotic outflow medium is continuously run over the surface ofthe membrane before being eliminated to a final reservoir

In the absence of a membrane correction the loss of bacteria becomesimportant when the action of an antibiotic on the bacteria is being evaluatedThis is especially a problem when the activities of two drugs with considerablydifferent half-lives are being compared Haag et al [8] developed differentialequations to describe changes in the bacterial inoculum in the central compart-ment over time Keil et al [9] later examined these equations and developed anextension of them that can be used to compensate for the amount of bacteriabeing lost to the elimination rate

The following are the equations developed by Haag and Keil where N(t)represents the number of colony-forming units per milliliter in the free medium(liquid compartment) at a given time t NB(t) represents the number of CFUsper milliliter in the modelrsquos biofilm layer (solid compartment) at the same timedN(t)dt and dNB(t)dt are the first derivatives of N(t) and NB(t) respectivelykw is the apparent bacterial growth rate constant ka is the rate constant for absorp-tion of bacteria into the biofilm layer and kd is the rate constant for desorptionof cells from the biofilm into the liquid medium [910] The elimination rateconstant is represented by ke

Assuming that kw is the same in broth and biofilm ke ka and kd are constantover the course of the experiment and N(t) is far below the maximum bacterialdensity under nonflowing conditions we have

dN(t)dt

(kw ke ka)N(t) kdNB(t) (5a)

dNB(t)dt

(kw kd)NB(t) kaN(t) (5b)

When the bacterial culture is not diluted

dNprime(t)dt

(kw ka)N(t) kdNB(t) (6)

where dNprime(t)dt describes the change in CFUmL in an undiluted compartmentover time

The total time course of N(t) is described by a second-order differentialequation derived from Eqs (5a) and (5b) Solving this equation (not shown)results in

N(t) Ae m1t Be m2t (7)


and

A B N0 (8)

where N0 is the initial number of CFUmL in the model flask Ae m1t describesthe number of CFUmL coming from the biofilm Be m2t describes the number ofCFUmL coming from the liquid compartment m1 0 and m2 0

The factor Be m2t approaches 0 at high dilution rates and at t infin Assumingthat the development of a biofilm may be neglected (ke ka and kd or ka kd 0 A 0 and B N0) we arrive at

Nprime(t) N(t)e ket (9)

In cases where the desorption of bacteria from the biofilm to the liquid mediumfar exceeds the loss of bacteria due to dilution equation (9) becomes

Nprime(t) N(t) e(12)ket (10)

Comparison of Eqs (9) and (10) shows that the effect of the presence ofa biofilm layer on bacterial loss is compensated for by a factor f with a valuebetween 05 and 1 The value of f approaches 05 as the influence of the biofilmis magnified If ke is constant throughout the course of the investigation Eqs (9)and (10) may be combined to give

Nprime(t) N(t)e f ket (11)

If the antibiotic under study displays two different elimination rate con-stants for the α and β phases the above equations must be altered Assumingthat there is no biofilm in the model flasks manipulation of these equations resultsin

Nprime(t) N(t)esumkei ∆ti (12)

where ∆ti and kei represent any number of time periods ∆t and their correspond-ing elimination rate constants ke If there is a biofilm present we arrive at theequation

Nprime(t) N(t)e(12)sum(kei ∆ti) (13)

Combining Eqs (12) and (13) gives

Nprime(t) N(t)e f sum(kei ∆ti) (14)

Again the value of f is between 05 and 1 Equation (14) is valid only as longas bacteria are in the logarithmic phase of growth For cultures approaching thestationary phase of growth the following equation is valid

48 Rybak et al

Nprime(t) NmaxN(t)

N(t) [Nmax N(t)]eket(15)

Where there is an influence of biofilms and a variable ke the following equationshould be used

Nprime(t) NmaxN(t)

N(t) [Nmax N(t)]efsum(kei∆ti)(16)

where the value of f is again between 05 and 1Keil and Wiedemann [9] evaluated the validity of these equations in a study

of continuous infusions of meropenem with steady-state concentrations of 25ndash75 microgmL against Pseudomonas aeruginosa and Escherichia coli The resultingkill curves were compared to kill curves obtained from incubation of these bacte-ria in a static system with meropenem at constant concentrations of 25ndash75 microgmL Thus the sole difference between experiments was the presence of dilution

Equation (9) can be used to correct for bacterial loss when the duration ofstudy does not exceed 8 h However results of Keil and Wiedemannrsquos studyrevealed that Eq (15) should be used when the duration of the experiment exceeds12 h [9] In addition using a value of 1 for f results in kill curves representingthe worst-case scenario so that the antibacterial effect is not overestimated Un-fortunately this study additionally showed that individual corrections must beperformed for different bacterial species and model apparatuses

Routine application of the above mathematical equations may prove to becumbersome The inclusion of growth control experiments studying bacterialgrowth in the absence of antibiotic can also help to address the severity of theproblem of bacterial dilution A typical growth curve is set to simulate the half-life of the antibiotic under study To compensate for bacterial loss the killingactivity of the antibiotic can be compared to the growth control curve

3 TWO-COMPARTMENT MODEL

The two-compartment model simulates biexponential pharmacokinetics follow-ing intravenous administration Murakawa et al [11] designed a model represent-ing the central and peripheral compartments connected by tubing This modelconsists of a central compartment that contains the antibiotic and organism Me-dium constantly flows into this central compartment Medium is then pumpedfrom the central compartment to a peripheral compartment (free of antibiotic) andis subsequently pumped back into the central compartment via a second peristalticpump Finally it is pumped out of the central compartment to a reservoir forbacterial counts As with the one-compartment model this system allows theorganism to be pumped out of the central compartment therefore dilution of the


organism according to the half-life of the antibiotic is a potential problem Analternative design of the typical two-compartment model places the peripheralcompartment within the central compartment (Fig 3) The bacteria under studyare placed into this inner chamber and antibiotic is administered into the centralcompartment The antibiotic may diffuse into the peripheral compartment butbacteria are trapped within that compartment

In an attempt to mimic drug concentration profiles in tissue versus serumZinner et al [12] devised a model that uses a single capillary unit device as asystem for studying antimicrobial effects (29) Later Blaser et al [13] chose atwo-compartment capillary model that uses a series of artificial capillary unitsrepresenting extravascular infection sites (Fig 4) The Zinner and Blaser modelscan simulate human pharmacokinetics in interstitial fluid for one or two antibiot-ics Each of the models uses an artificial capillary (dialysis) unit connected bysilicone tubing to a peristaltic pump and a culture medium reservoir A samplingport with a bidirectional stopcock is inserted into the tubing and allows injection

FIGURE 3 Two-compartment model Bacteria are introduced into the periph-eral compartment and are unable to pass into the central compartment (illus-trated by heavy arrow) Antibiotic is injected into the central compartmentAB antibiotic B bacteria CC central compartment FM fresh me-dium PC peripheral compartment SB magnetic stir bar SP samplingand injection port WM water medium (Adapted from Ref 22)

50 Rybak et al

FIGURE 4 Two-compartment capillary model The capillary device (dialyzer)is pictured at the bottom of the illustration Inset shows a cross section ofthe capillary bundle Medium flow through the bundle is represented by theheavy arrow Bacteria are trapped in the outer compartment of the bundlewhile antibiotic (light arrows) flow through the dialysis membranes B bac-teria CB capillary bundle OC outer compartment P peristaltic pumpR reservoir SP sampling and injection port SS sampling site (septumfittings) (Adapted from Ref 12)

of antibiotics either toward the capillary unit (bolus injection) or toward the me-dium reservoir (for continuous infusion dosing simulations)

Each capillary unit consists of two chambers an outer chamber or culturetube and an inner chamber that comprises a bundle of 150 hollow polysulfonefibers Each fiber (lsquolsquocapillaryrsquorsquo) has an internal diameter of 200 microm and a wallthickness of 50ndash75 microm In the study reported by Zinner and his colleagues thecapillaries used retained proteins of a size greater than 10000 daltons Howeveradditional capillary units are available that retain proteins of sizes greater than50000 or 100000 daltons The entire capillary bundle is secured by siliconeO-rings which seal the outer chamber (extracapillary space) from the lumens ofthe capillary tubes

An inoculum of bacteria is introduced into the outer chamber of the capil-lary system Meanwhile the antibiotic under study is injected into the silicone


tubing and allowed to diffuse either directly to the capillary unit (to simulateintravenous bolus dosing) or indirectly through the medium reservoir (thus simu-lating a continuous infusion dosing strategy) Because bacteria do not diffuse outof the extracapillary space this model simulates an infected tissue site Dependingon the molecular size of the antibiotic used and the permeability characteristicsof the capillary tubing antibiotic diffuses into the extracapillary space from thecapillary tubing Again because the concentration of antibiotic obtained at thesite where bacteria are present will be lower than that in the capillary tubingthis system allows simulation of the treatment of infections at tissue sites

This model allows the study of a variety of pharmacokinetic and pharmaco-dynamic parameters including the effects on bacterial killing of bolus dosingversus continuous infusions of antibiotics In addition the effects of single orcombined agents can be assessed and the model allows simulation of the fluctua-tions in concentrations that typically occur after dosing in humans Furthermorethe model avoids the problem of bacterial dilution as the volume of bacteriain the extracapillary space is not significantly changed during the experimentHowever controlled variation of antibiotic concentrations in the two chambersof this model may be difficult given the variability inherent in relying on thediffusion of drugs through the walls of the capillary tubes

Blaser et al [13] compared the pharmacokinetics of netilmicin obtained ina two-compartment capillary model to those obtained in an experimental skinsuction blister model in healthy volunteers Netilmicin was administered ac-cording to two dosage schemes in the capillary model in order to achieve peakconcentrations of 16 or 24 microgmL In the human blister model netilmicin wasgiven as an intramuscular injection of 2 mgkg (lean body weight) The in vitromodel demonstrated linear two-compartment pharmacokinetics Peak concentra-tions obtained were higher than nominal for the central compartment and lowerthan nominal for the peripheral compartment One hour after the end of a 60 mininfusion netilmicin concentrations were equivalent in the central and peripheralcompartments Overall concentrations obtained in the in vitro model approxi-mated those obtained in the human blister model (Fig 5) Thus this capillarymodel successfully simulates human pharmacokinetics

4 COMBINATION THERAPY MODEL

In vitro models can also be used to simultaneously study the activity of two drugswith different half-lives in combination Blaser [14] described the procedures forsimulating combination therapy using both one- and two-compartment modelsIn these models both drugs are placed in the central compartment and the clear-ance pump rate is set to simulate the half-life of the drug with the shorter half-life (ClA drug A) (Fig 6) A supplemental compartment is used to replace the

52 Rybak et al

FIGURE 5 Comparison of in vitro and human models Netilmicin concen-trations in the central and peripheral compartments of an in vitro two-compartment capillary unit model are shown The 2 sets of curves representnetilmicin concentrations after administration of infusions attaining peakconcentrations of either 16 or 20 mgL The insert graph illustrates serum() and suction blister () netilmicin pharmacokinetics after administrationin healthy human volunteers Comparison of the main and insert graphs illus-trates the similarity in pharmacokinetics between the in vitro and humanmodels (Adapted from Ref 7)

antibiotic with the longer half-life (drug B) which has been overeliminated Thepump rate for the supplemental compartment (ClS) is set to make up the differencebetween the clearance of the two drugs

ClS ClA ClB

To maintain the same concentration of drug B in both compartments the centraland supplemental compartments should be dosed in a similar manner

Meanwhile drug-free medium is pumped into the central compartment sothat the total amount of medium pumped into the system (M in) equals the totalvolume of medium being pumped out of the system (Mout)


FIGURE 6 Combination therapy model In this example drug B is added froma supplemental chamber to the central reaction flask CA concentration ofdrug A CB concentration of drug B CC central compartment ClA clear-ance of drug A ClB clearance of drug B FM fresh medium Min rateof inflow of fresh medium P peristaltic pump SC supplemental compart-ment WM waste medium (Adapted from Ref 14)

M in ClA ClS

Therefore the pump rate for the drug free medium should be set as ClBThe advantage of this system is that it can be used to determine synergistic

additive or antagonistic effects of combinations of antibiotics simulating the ac-tual pharmacokinetics of each agent For example Houlihan et al [15] evaluatedthe activity of vancomycin alone and in combination with gentamicin againstmethicillin-resistant Staphylococcus aureusndashinfected fibrinndashplatelet clots in aninfection model Vancomycin was dosed at intervals of 6 12 and 24 h andgentamicin was given either as a single daily (every 24 h) dose or every 12 hIn plots of the bacterial inoculum versus time synergism was defined as a 2log increase in killing associated with a combination regimen in comparison tothe most active single agent

In preliminary fractional inhibitory concentration (FIC) testing the combi-

54 Rybak et al

nation of vancomycin and gentamicin displayed indifference However in thepharmacodynamic model several combinations (eg vancomycin every 24 h gentamicin every 24 h) demonstrated a synergistic effect Thus results from useof a static system (FIC testing) were not predictive of results obtained from amodel where simulation of human pharmacokinetics is achieved Blaser et al[16] obtained similar results in a study of ceftazidime-netilmicin combinationsagainst Pseudomonas aeruginosa Using a two-compartment capillary modelsynergism was noted for the combination against certain strains in the initial (46 and 8 h) and final (24 26 and 28 h) periods of evaluation Synergism testingusing the FIC method was predictive of the response seen in the in vitro modelduring the initial period only This serves to reinforce the fact that results obtainedfrom traditional susceptibility methodologies may not correlate with relationshipsseen with the use of pharmacodynamic models where in vivo pharmacokineticsare simulated

5 BIOFILM-CATHETER MODEL

Nickel et al [17] used a modified Robbins device to study the ability of tobra-mycin to penetrate a biofilm layer of Pseudomonas aeruginosa The model con-sisted of a 415 cm long acrylic block with a 2 10 mm inner lumen and 25evenly distributed sampling ports The block was connected via tubing to a reser-voir that was immersed in a 37degC water bath Medium containing the bacteriumunder study was pumped from this reservoir (simulated bladder) through the mod-ified Robbins device (simulated catheter) at a rate of 60 mLh The medium usedconsisted of artificial urine supplemented with nutrient broth Discs cut from acommercially available catheter were suspended within the model and could beremoved for analysis

In the study performed by Nickel et al a biofilm of Pseudomonas aerugi-nosa was formed by passing medium containing logarithmic-phase organismsthrough the model for 8 h Formation of the biofilm was verified by examinationof catheter surfaces using scanning electron microscopy (SEM) and epifluores-cence microscopy After the 8 h colonization period medium was removed andtransferred to flasks for subsequent exposure of these organisms to varying con-centrations of tobramycin for a period of 8 or 12 h Antibiotic-containing mediumwas then pumped through the model and discs of catheter material were removedat 8 and 12 h intervals for determination of colony counts and microscopic exami-nation Thus the killing of organisms in the sessile (biofilm) and planktonic statescould be compared as could the MICs and MBCs of these organisms This modeltherefore allows study of the development of resistance among organisms in abiofilm and of the ability of antibiotics to penetrate the biofilm layer and exertan antimicrobial effect

Walker et al [18] developed an alternative form of the above model that


consisted of a typical one-compartment vessel with plugged catheter segments(127 cm each) suspended within the chamber Catheter material was inoculatedwith 5 106 CFUmL of each organism and counts of sessile and planktonicbacteria were performed using standard plate dilution and SEM This model wasused to test the antiadherence characteristics of various catheter coatings andimpregnations

Prosser et al [19] developed a simplified adaptation of these models Inthis model 05 cm2 discs composed of silicon latex catheter material were spottedwith 80 microL of a bacterial suspension (confirmed to possess an optical density of08 at 540 nm) Discs were then incubated for 1 h washed with a buffer solutionand then transferred to broth-containing Petri dishes and incubated for an addi-tional 20ndash22 h The discs were then rewashed in buffer and placed in plain orantibiotic-containing broth incubated for 4 h washed and resuspended in broth(with or without antibotic) This procedure was repeated at 8 and 24 h At eachtime interval surface film on all discs was scraped off into 5 mL of saline andsamples were plated for colony count determinations Samples were also exam-ined by light microscopy and SEM One advantage of this model lies in the factthat planktonic bacteria are not contained within the model so inactivation ofantibiotic by these planktonic organisms does not occur In addition this modelmay be less cumbersome to operate than the modifed Robbins device

6 INFECTED FIBRIN CLOT MODEL

One modification of a one-compartment model is the infected fibrin clot modeldeveloped by Rybak et al [1520] which uses fibrin clots to simulate a deepsequestered infection such as those associated with cardiac vegetations (Fig 7)Fibrin clots made of human cryoprecipitate organisms and platelets (250000ndash500000 platelets per clot) are placed in 15 mL Eppendorf tubes Bovine throm-bin (5000 units) is then added to each tube after insertion of a sterile monofila-ment line into the mixture Each clot is then inserted into a sterile reaction vesseland removed at various time points for determination of bacterial counts Thismodel has been used to evaluate the activity of antimicrobial agents against or-ganisms embedded in a simulated human tissue With the incorporation of plate-lets and high inoculum of bacteria (109 CFUg) the fibrin clots represent condi-tions of simulated endocardial vegetations (SEVs) The system does not consist ofan airtight compartment therefore it requires two peristaltic pumps to pump themedium into and out of the central compartment The SEVs have been shown tobe viable in the models for up to 144 h

Several studies have been performed to compare this model to an in vivorabbit model of endocarditis McGrath et al [20] indirectly compared the resultsobtained from this model to those of a rabbit model of endocarditis done previ-ously They evaluated the effect of teicoplanin on colony-forming units (CFUs)

56 Rybak et al

FIGURE 7 Fibrin clot model Simulated fibrin clots or endocardial vegetationsare suspended within the central compartment The bacterial inoculum isintroduced into this central compartment FC simulated fibrin clots FM fresh medium ML monofilament lines P peristaltic pump SB mag-netic stir bar SP sampling and injection port WB water bath (375degC)WM waste medium (Adapted from Ref 20)

per gram of SEV in the model using human pharmacokinetics to the CFUg ofrabbit vegetations This study demonstrated that the CFUg of SEV were similarto those of the rabbit vegetations however the pharmacokinetics of teicoplaninvaried between the two models and samples were obtained at different timepoints for analysis

A more recent study has retrospectively compared the simulated endocar-dial vegetation model to four different rabbit studies [10] In this study authorscompared the activities of four different fluoroquinolonesmdashciprofloxacin cli-nafloxacin sparfloxacin and trovafloxacinmdashagainst various Staphylococcusaureus and enterococcal isolates simulating pharmacokinetics obtained in therabbit model Models were conducted in the same fashion as the rabbits weretreated and SEVs were evaluated at the same time points as those at which therabbit vegetations were assessed The bacterial inocula in the SEVs were similarto those achieved in the rabbitsrsquo vegetations prior to and following treatmentsuggesting that this model may have a role in initial studies of antibiotics in thetreatment of bacterial endocarditis (Fig 8) To further define the modelrsquos role inthe treatment of endocarditis prospective studies comparing the in vitro modelto rabbit models of endocarditis using various pathogens and antimicrobial agentsare necessary


FIGURE 8 Comparison of in vitro and animal models The activity of clina-floxacin against strains of methicillin-resistant (MRSA-494) and -susceptible(MSSA -1199) Staphylococcus aureus is illustrated in the graph Bacterial kill-ing observed with in vitro (lsquolsquomodelrsquorsquo) and animal (lsquolsquorabbitrsquorsquo) models of endo-carditis are shown for each organism Similarities between the animal andin vitro models in terms of the change in bacterial inoculum and final inocu-lum value (at 98 hours) may be seen (Adapted from Ref 16)

7 IN VITRO MODEL OF INTRACELLULAR PATHOGENS

The previously mentioned models evaluate the activity of antibiotics against ex-tracellular pathogens However many pathogens such as Salmonella spp Shi-gella spp Campylobacter spp Escherichia coli Proteus mirabilis Listeriamonocytogenes Haemophilus influenzae group B streptococci and Helicobacterpylori can survive intracellularly thereby escaping antimicrobial treatment Cer-tain antimicrobial agents such as azithromycin and clarithromycin possess intra-cellular activity and an in vitro model can be a valuable tool in determining suchactivity Several in vitro models using different cell types have been employedto determine intracellular activity of antimicrobial agents however continuousexposure to a fixed concentration of antibiotic does not reflect human pharmaco-kinetics

58 Rybak et al

Hulten et al [21] have designed an in vitro model to study the intracellularactivity of antimicrobial agents by simulating human pharmacokinetics (Fig 9)In this model a human epithelial cell line is grown in the appropriate mediumand seeded into cell culture inserts containing a 045 microm membrane Inserts areexposed to a fixed concentration of the bacteria and incubated for several hoursto allow the bacteria to penetrate into the cells Extracellular bacteria are thenremoved and inserts are placed in a metal rack that is fixed on a glass chamberconnected to a pump simulating pharmacokinetics of the antibiotic Inserts areremoved at various time points and cells are processed and lysed prior to deter-mination of bacterial inoculum This model was used previously to determinethe intracellular activity of amoxicillin clarithromycin and amoxicillin againstH pylori This model was also used to evaluate the activity of different agentsagainst Mycobacterium tuberculosis [21]

FIGURE 9 Intracellular pathogen model Medium is pumped through the cen-tral compartment (glass chamber) at a rate to simulate antibiotic pharmacoki-netics Inset shows a cross section of the central compartment with cell cul-ture inserts suspended within a metal rack B bottom section of centralcompartment CC cell culture insert FM fresh medium HP hot plate(375degC) P peristaltic pump SB magnetic stir bar SP sampling andinjection port SR stainless steel rack WM waste medium (Adapted fromRef 21)


8 LIMITATIONS OF IN VITRO MODELS

Although the in vitro models described in this chapter are widely used in the studyof antimicrobial pharmacokinetics and pharmacodynamics certain limitations totheir use must be noted The applicability of results obtained from in vitromodels to the treatment of infections in vivo is limited by the factors describedbelow

81 Effects of the Immune System

The majority of in vitro models commonly used are devoid of immune systemfactors that is antimicrobial effects are studied in an environment that does notcontain the host defense factors The antimicrobial effect may thus be underesti-mated because organisms are free from the inhibitory action of such immunesystem factors as leukocytosis phagocytosis and immunoglobulins

Shah [22] employed a model that incorporated fresh human blood fromhealthy volunteers in an effort to replicate in vivo immune system effects Themodel consisted of a glass chamber (similar to the one-compartment model previ-ously described) with an inner cell suspended within The inner cell composedof plexiglass was enclosed at each end with membrane filters designed to allowdiffusion of antibiotics Heparinized human blood was incorporated into the innerchamber and the outer chamber contained a nutrient medium The bacterial inoc-ulum was injected into the inner chamber and antibiotics were administered intothe outer vessel

Use of this model in a study of imipenem against Escherichia coli Kleb-siella pneumoniae and Pseudomonas aeruginosa revealed that in the presenceof blood alone the bacterial count was reduced by 90ndash99 but in all casesbacterial regrowth was noted As expected addition of antibiotic decreased thetime to achieve 99 reduction of the bacterial inoculum and the final bacterialcount was decreased in comparison to that obtained in antibiotic-free modelsAlthough the use of blood as a medium allowed incorporation of some aspectsof the human immune system the full range of host defense factors was notincluded in the model

Incorporation of blood as a medium into in vitro models has not beenwidely employed most likely due to the logistical concerns of working withhuman blood However it should be noted that the lack of an immune systemin typical in vitro models is advantageous in some respects in that the effectof antibiotic exposure alone on the development of bacterial resistance may bemeasured Also results from in vitro models lacking immune system effects maybe satisfactorily applied to infection occurring in neutropenic hosts althoughagain not without some concerns over the clinical applicability of these results

60 Rybak et al

82 Altered Bacterial Growth Characteristics

Multiple investigations have confirmed that the behavior of bacteria in an in vitroenvironment is not equivalent to that in vivo Microorganisms grown in vitrodiffer from those causing in vivo infection with respect to factors discussed inthe following paragraphs

821 Virulence

It has been noted in numerous experiments that bacterial virulence is progres-sively diminished with serial in vitro passage or cultivation [23] The cause ofthis is unknown but it may be due in part to adherence factors or derangementsin biochemical pathways The result of this loss of pathogenicity is potentiallyan overestimation of the effect of a given antimicrobial in an infection modelHowever this reduction in virulence may be strain-specific so the impact onclinical application of in vitro experimental results may be unclear

822 Morphology

Bacteria adapt to life in a blood environment and undergo such morphologicalchanges as encapsulation and mucus production [24] Thus bacteria in vivo pos-sess an enhanced resistance to killing by serum factors In vitro these alterationsin morphology are unstable and organisms rapidly revert to a normal morphol-ogy Thus the effect of an antimicrobial in an infection model may be overesti-mated because the above-described morphological defense factors are absent

823 Physiology

Microorganisms are known to rapidly develop adaptively to a variety of environ-ments possibly as a result of metabolic changes In an in vitro environmentbacteria differ from those cultivated in vivo in terms of amino acid compositionthe synthesis of toxic metabolites and metabolic rate [24] Overall bacteria aremore metabolically active in vivo than in vitro However replication generallyoccurs more rapidly in an in vitro environment possibly due to the lack of im-mune system effects For example it has been shown that the typical growth rateof microorganisms in an in vivo animal model of infection may be on the orderof 002ndash005 h1 wheras the growth rate in an in vitro batch culture may approach2ndash3 h1 [25] Continuous culture of microorganisms which allows precise controlof growth rate may allow maintenance of microorganisms in a state of growthmore closely approximating that of organisms in the natural state [25]

In addition to changes in growth characteristics differences in expressionandor structure of outer membrane proteins may occur such that microorganismsin vitro display reduced permeability These effects are dependent on the compo-sition of the nutrient medium used in model experiments For example the struc-ture and composition of the outer membrane proteins of Escherichia coli may


be altered by changes in growth medium and temperature as well as by the molec-ular size and osmolarity of sugars contained in the growth medium [24]

The overall effect of the above physiological changes on the applicabilityof results obtained from in vitro models is variable and may be difficult to deter-mine These alterations may be highly strain-specific so general conclusions re-garding methods to account for these changes are lacking

824 Sensitivity to Serum Factors

As noted above morphological changes of bacteria in vivo lead to a reducedsusceptibility of those organisms to serum immune factors For example bacteriacultured in vitro display an enhanced susceptibility to phagocytosis [26] There-fore use of models incorporating for example human blood as a medium mayfail to adequately account for immune effects because the bacterial strain maybe more susceptible to the effects of immune mediators than it would be in vivo

83 Microorganism Viability

Certain organisms are difficult to work with in in vitro systems because of viabil-ity problems that are not apparent in vivo For example it is difficult to keepStreptococcus pneumoniae viable for periods longer than 12 h because of thestationary-phase autolytic process that is common to this species For this reasonin vitro killing curves obtained in test tubes do not usually exceed 6ndash12 h soorganism death can be correctly attributed to antibiotic effects rather than to auto-lytic processes

In an in vitro infection model investigation Cappelletty et al [27] demon-strated that the viability of S pneumoniae during growth control experiments isdirectly affected by the clearance of growth medium through the model Simula-tions of antibiotics with longer half-lives were more likely to result in a slowerrate of medium turnover thus increasing the likelihood of autolysis inductionThis serves to reinforce the idea that growth control experiments without antibi-otic must always be performed to ensure that organism viability is optimal [27]

84 Effects of Protein Binding on Antimicrobial Action

Because plasma protein is usually not included in the nutrient broth medium theeffects of protein binding of antimicrobials may not be measured For examplethe antibacterial effect of drugs that are highly bound by plasma proteins maybe overestimated in models where such protein binding is not replicated becauseconcentrations of the drug in the model will exceed those obtained in vivo How-ever incorporation of protein such as human albumin may allow simulation ofthe free-drug concentrations that will be obtained after dosing in humans Forexample Garrison et al [28] assessed the effects of physiological concentrations(38ndash42) of human albumin on the activity of daptomycin and vancomycin

62 Rybak et al

against Staphylococcus aureus Albumin was directly incorporated into the two-compartment in vitro model It was found that the presence of albumin signifi-cantly diminished the bactericidal activity (as measured by kill rate and time to99 kill) of both daptomycin (low- and high-dose regimens) and vancomycinRather than include protein in a model if one knows the degree to which anantibiotic is bound to proteins one may simply dose in such a way as to simulatefree concentrations that will be obtained in the presence of protein This strategyis desirable because of the excessive cost associated with the use of albumin andother proteins in pharmacodynamic models

85 Bacterial Adherence

In a study conducted by Haag et al in vitro models with no dilution andorelimination of growth medium (growth control) were compared to models wherethe addition and elimination of medium took place (addition and elimination wereset to occur at the same rate) The investigators expected that given a dilutionrate higher than the bacterial growth rate an artificial decrease in bacterial countsdue to dilution of organisms would be observed However they instead notedthat an increase in colony-forming units occurred after 1ndash2 h of simulation [8]This phenomenon was ascribed to the presence of a subpopulation of bacteriathat adhered to the inner surfaces of the model in a biofilm layer It is thoughtthat bacteria residing within the biofilm resist dilution but that a certain proportionof bacteria diffuse into the medium at a quantifiable rate

As a result of this phenomenon the effect of an antibiotic on cells in theliquid medium may be measured only in the early stages of an experiment Afterthis time as the biofilm layer forms the effect of the agent on the nondilutableadherent population is measured instead Because organisms in this subpopula-tion typically display reduced antimicrobial susceptibility the effect of the agentunder study may be underestimated [8] On the other hand the effect of an agenton bacteria residing within sequestered sites of infection may be more reliablydetermined

Attempts have been made to prevent bacterial adherence to model surfacesFor example Gwynn et al [29] found that siliconization of their model preventedthe eventual increase in bacterial counts It was postulated that the siliconersquosprevention of the formation of an adherent population was responsible for thiseffect However others have been unable to replicate these findings Bacterialadherence may also depend on the geometry of vessels making up the model andon the material from which models are constructed

86 Dilutionary Artifacts

Although in vitro models incorporating dilution (ie addition and elimination ofmedium at a rate to simulate the half-lives of antibiotics) more closely replicate


the dosing of drugs in humans dilution of the bacterial inoculum is a problemassociated with these models Such dilution (elimination) of microorganisms willresult in an overestimation of the killing effect of an agent being studied Asdescribed previously investigators have employed a variety of strategies to over-come this effect including construction of models that prevent elimination ofbacteria the use of static models in which dilution does not occur and compensa-tion for the effects of dilution by the use of mathematical correction [9]

87 Other Effects

In addition to the above limitations associated with in vitro modeling problemsmay also arise during attempts to interpret results obtained from models incorpo-rating either very highly or minimally bactericidal agents With highly bacteri-cidal antibiotics possessing an extended elimination half-life complete eradica-tion of bacteria to the limit of assay detection will rapidly occur In this scenariothe extent of measurable effects of varying drug dosages will be minimized Incontrast in models in which an agent with minimal bactericidal activity and ashort elimination half-life is used organism regrowth will rapidly overwhelm themodel The effect of regrowth is unknown especially in light of the fact thatorganisms that regrow do not undergo changes in antimicrobial susceptibilityAgain the result of this phenomenon is that effects of the antimicrobial understudy are difficult to measure

9 SUMMARY

In vitro antibiotic pharmacodynamic models represent a significant advancementin the assessment of antimicrobial pharmacodynamics The ability to simulatehuman pharmacokinetics while evaluating the effect of an antibiotic on viablebacteria is clearly superior to more traditional assessments such as basic suscepti-bility testing or kill-curve experiments performed in test tubes These models areadaptable to a variety of conditions allowing for evaluations of different organ-isms environmental conditions dosing schemes combination therapies or resis-tance mechanisms Preliminary information suggests that results obtained fromcertain dynamic models correlate with those found in vivo Although there area number of limitations to the use and applicability of these models they continueto evolve and are likely to add to our overall understanding of antimicrobial andmicroorganism interactions

REFERENCES

1 D Greenwood F OrsquoGrady Potent combinations of beta-lactam antibiotics using thebeta-lactamase inhibition principle Chemotherapy 197521330ndash341

64 Rybak et al

2 JD Anderson KR Johnson MY Aird Comparison of amoxicillin and ampicillinactivities in a continuous culture model of the human urinary bladder AntimicrobAgents Chemother 198017554ndash557

3 JD Anderson Relevance of urinary bladder models to clinical problems and to anti-biotic evaluation J Antimicrob Chemother 198515(suppl A)111ndash115

4 JD Anderson F Eftekhar R Cleeland MJ Kramer G Beskid Comparative activityof mecillinam and ampicillin singly and in combination in the urinary bladder modeland experimental mouse model J Antimicrob Chemother 19818121ndash131

5 RA Zabinski KJ Walker AJ Larsson JA Moody GW Kaatz JC Rotschafer Effectof aerobic and anaerobic environments on antistaphylococcal activities of fivefluoroquinolones Antimicrob Agents Chemother 199539507ndash512

6 J Blaser BB Stone MC Groner SH Zinner Comparative study with enoxacin andnetilmicin in a pharmacodynamic model to determine importance of ratio of peakantibiotic concentration to MIC for bactericidal activity and emergence of resistanceAntimicrob Agents Chemother 1987311054ndash1060

7 SM Navashin IP Fomina AA Firsov VM Chernykh SM Kuznetsova A dynamicmodel for in-vitro evaluation of antimicrobial action by simulation of the pharmaco-kinetic profiles of antibiotics J Antimicrob Chemother 198923389ndash399

8 R Haag P Lexa I Werkhauser Artifacts in dilution pharmacokinetic models causedby adherent bacteria Antimicrob Agents Chemother 198629765ndash768

9 S Keil B Wiedemann Mathematical corrections for bacterial loss in pharmacody-namic in vitro dilution models Antimicrob Agents Chemother 1995391054ndash1058

10 E Hershberger EA Coyle GW Kaatz MJ Zervos MJ Rybak Comparison between arabbit model of bacterial endocarditis and an in vitro infection model with simulatedendocardial vegetations Antimicrob Agents Chemother 2000441921ndash1924

11 T Murakawa H Sakamoto T Hirse M Nishida New in vitro kinetic model forevaluating bactericidal efficacy of antibiotics Antimicrob Agents Chemother 198018377ndash381

12 SH Zinner M Husson J Klastersky An artificial capillary in vitro kinetic model ofantibiotic bactericidal activity J Infect Dis 1981144583ndash587

13 J Blaser BB Stone SH Zinner Two compartment kinetic model with multiple arti-ficial capillary units J Antimicrob Chemother 198515(suppl A)131ndash137

14 J Blaser In-vitro model for simultaneous simulation of the serum kinetics of twodrugs with different half-lives J Antimicrob Chemother 198515(suppl A)125ndash130

15 HH Houlihan RC Mercier MJ Rybak Pharmacodynamics of vancomycin aloneand in combination with gentamicin at various dosing intervals against methicillin-resistant Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infectionmodel Antimicrob Agents Chemother 1997412497ndash2501

16 J Blaser BB Stone MC Groner SH Zinner Impact of netilmicin regimens on theactivities of ceftazidime-netilmicin combinations against Pseudomonas aeruginosain an in vitro pharmacokinetic model Antimicrob Agents Chemother 19852864ndash68

17 JC Nickel JB Wright I Ruseska TJ Marrie C Whitfield JW Costerton Antibioticresistance of Pseudomonas aeruginosa colonizing a urinary catheter in vitro Eur JClin Microbiol 19854213ndash218

18 KJ Walker KJ Kelly AJ Larsson JC Rotschafer DRP Guay K Vance-Bryan Adhe-


sion of Staphylococcus epidermidis (SE) Staphylococcus aureus (SA) and Pseu-domonas aeruginosa (PA) to six vascular catheter materials (abstr) In Program andAbstracts of the 33rd Interscience Conference on Antimicrobial Agents and Chemo-therapy New Orleans LA 1993

19 BL Prosser D Taylor BA Dix R Cleeland Method of evaluating effects of antibiot-ics on bacterial biofilm Antimicrob Agents Chemother 1987311502ndash1506

20 BJ McGrath SL Kang GW Kaatz MJ Rybak Bactericidal activities of teicoplaninvancomycin and gentamicin alone and in combination against Staphylococcusaureus in an in vitro pharmacodynamic model of endocarditis Antimicrob AgentsChemother 1994382034ndash2040

21 K Hulten R Rigo I Gustafsson L Engstrand A new pharmacokinetic in vitro modelfor studies of antibiotic activity against intracellular microorganisms AntimicrobAgents Chemother 1996402727ndash2731

22 PM Shah Activity of imipenem in an in-vitro model simulating pharmacokineticparameters in human blood J Antimicrob Chemother 198515(suppl A)153ndash157

23 DL Watson Virulence of Staphylococcus aureus grown in vitro or in vivo MicrobiolImmunol 197923543ndash547

24 A Dalhoff Differences between bacteria grown in vitro and in vivo J AntimicrobChemother 198515(suppl A)175ndash195

25 P Gilbert The theory and relevance of continuous culture J Antimicrob Chemother198515(suppl A)1ndash6

26 A Dalhoff G Stubner Comparative analysis of the antimicrobial action of polymor-phonuclear leucocytes in vitro ex vivo and in vivo J Antimicrob Chemother 198515(suppl A)283ndash291

27 DM Cappelletty MJ Rybak Bactericidal activities of cefprozil penicillin cefaclorcefixime and loracarbef against penicillin-susceptible and -resistant Streptococcuspneumoniae in an in vitro pharmacodynamic infection model Antimicrob AgentsChemother 1996401148ndash1152

28 MW Garrison K Vance-Bryan TA Larson JP Toscano JC Rotschafer Assessmentof effects of protein binding on daptomycin and vancomycin killing of Staphylococ-cus aureus by using an in vitro pharmacodynamic model Antimicrob Agents Che-mother 1990341925ndash1931

29 MN Gwynn LT Webb GN Rolinson Regrowth of Pseudomonas aeruginosa andother bacteria after the bactericidal action of carbenicillin and other β-lactam antibi-otics J Infect Dis 1981144263ndash269

4

Animal Models of Infection for the Studyof Antibiotic Pharmacodynamics

Michael N Dudley

David Griffith

Microcide Pharmaceuticals Inc Mountain View California

1 INTRODUCTION

Animal models of infection have had a prominent place in the evaluation ofinfection and its treatment From early experiments demonstrating transmissionof infectious agents to satisfy Kochrsquos postulates to evaluation of chemotherapyin the antibiotic era animal models have proven useful for understanding humandiseases

More recently animal models for the study of infectious disease have beenfurther refined to consider the importance of pharmacokinetic factors in theoutcome of infection This allows for the study of the relationship betweendrug exposure (pharmacokinetics) and anti-infective activity (pharmacodynamics)Consideration of these aspects has enhanced the information provided by animalmodels of infection and made the results more predictive of the performance ofdrug regimens in humans This has largely been accomplished through a morethorough understanding of the importance of pharmacokinetics in the outcomeof an infection advances in quantitation of drug concentrations in biological ma-trices and the development of metrics to quantify antibacterial effects in vivo

67

68 Dudley and Griffith

This chapter will review approaches for design analysis and application of theseapproaches in animal models for the study of optimization of anti-infective ther-apy and the discovery and development of new agents

Use of animal models of infection to study the relationship between drugexposure in vivo and antibacterial effects dates back to the very early studies inpenicillin These early investigators noted that the duration of efficacy of penicil-lin in the treatment of streptococcal infections was dependent on the length oftime for which serum concentrations exceeded the MIC [1ndash3] How these earlyobservations ultimately impacted on penicillin therapy in humans is difficult toascertain however it is clear that pharmacodynamic studies in animal modelshave a crucial role in the preclinical evaluation of new anti-infectives optimiza-tion of marketed agents and the assessment of drug resistance

2 RELEVANCE OF ANIMAL MODELS TO INFECTIONS

IN HUMANS

It is largely assumed that efficacy of a drug in an animal model will correspondto that in humans However in many cases the induction and progression ofinfection in small animals does not correspond to that seen in the human settingFor example humans rarely suffer from large bolus challenges of bacteria bythe intravenous route A much more clinically relevant model for pathogenesisin humans involving translocation of bacteria from gastrointestinal membranesdamaged by cytostatic agents has been developed [4] Despite this major differ-ence the model of sepsis is a mainstay for early preclinical evaluation of anti-infectives in rodent models

A few animal models have been widely accepted for prediction of effectsin humans The rabbit model of endocarditis (see below) has proven faithful inmimicking damage to valvular surfaces similar to that reported in human patientsand colonization of these vegetations by bacteria and growth corresponds closelyto that observed in humans For several years prophylaxis regimens for endocar-ditis were based largely on experiments in this model

3 ENDPOINTS IN ANIMAL MODELS OF INFECTION

Examples of major endpoints used in pharmacodynamic studies in animal modelsof infection are shown in Table 1 For lethal infections the proportion of animalssurviving at each doseexposure level are determined to generate typical dosendashresponse curves With greater awareness of the potential for suffering in testanimals during the later stages of overwhelming infection from a failing drugregimen most investigators have substituted rapid progression to a moribundcondition as a more humane alternative endpoint Both endpoints require carefulmonitoring by the investigator and consistent application of these definitions to

Animal Models of Infection 69

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act

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and

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evan

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ensure reproducible results Further only the potency of the antimicrobial is esti-mated because the maximum response is generally bounded (100 survival)However given that death from infection often arises due to complex interplaybetween host factors organ damage etc or even other factors that may be unre-lated to antibacterial effects (eg a dropped cage) the investigator must be cau-tious in interpretating results

In contrast quantitation of bacteria in tissues at various time points allowsfor measurement of changes in bacterial numbers over time The antibacterialeffects often correlate with meaningful clinical outcomes in patients (eg relationbetween time to sterilization of CSF in meningitis and residual neurological ef-fects in children) Quantification of bacterial counts in tissues or fluids detectsmore subtle changes in bacterial killing with dosage regimens In additionchanges in susceptibility of the pathogens with treatment can be measured Thebest approach is validation of the number of pathogens in tissues or fluids thatcorrelates with survival in treated or untreated animals Identification of an earlierendpoint that predicts survival in both treated and untreated animals meets themost rigorous definition for a true surrogate marker (ie a marker that predictssurvival in treated and untreated groups)

31 Metrics for Quantifying Anti-Infective Effects In Vivo

Quantitation of antimicrobial effects in vivo usually measures the tissue or hostburden of organism at specified intervals after the initiation of treatment In viewof the differences among drugs in in vitro pharmacodynamic properties (egrate of bacterial killing and postantibiotic and subinhibitory effects) as well aspharmacokinetic properties several approaches have been developed to measurethe time course of antibacterial effects in vivo These metrics tend to focus eitheron the overall extent of bacterial killing over time or on the rate of bacterialkilling either by measuring the time to reach a particular level of bacteria (egtime to 3 log decrease) or by calculation of a killing rate (eg reduction in logCFUh) Table 2 summarizes several representative metrics used for describingthe pharmacodynamic effects on bacteria in vivo

4 PHARMACOKINETIC CONSIDERATIONS IN ANIMAL

MODELS OF INFECTION

As described in earlier chapters one objective of pharmacodynamic modeling isto relate the in vivo exposure of an anti-infective to the observed effects listedabove This requires careful study of antimicrobial pharmacokinetics in the testspecies

Our approach is to measure the pharmacokinetic properties of readily avail-able drugs in the infected animals Although literature data are often available


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the results in a preclinical pharmacokinetic study (where the goal is to carefullymeasure pharmacokinetic properties) may differ from the values obtained in sickanimals or with multiple doses For novel compounds the pharmacokinetics isnot known and studies in both infected and uninfected animals are needed tocharacterize the pharmacokinetic properties of the agent as well as to determinethe actual exposure to drug in the experimental model Nonlinear changes inexposure vs dose (due to changes in drug clearance or bioavailability for extra-vascular administration) should be probed When studying combinations of drugsone must ensure that pharmacokinetic drug interactions do not occur pharmaco-kinetic interactions could cause false interpretations of combination experiments(eg increased effects from antimicrobial synergism) where the effect was reallydue to elevated concentrations of an active component We have also found thatthe formulation used for a second agent may also influence the pharmacokineticproperties of antibiotics (eg PEG plus levofloxacin in mice unpublished obser-vations)

41 Design Issues in Pharmacokinetic Studies for

Animal Models

411 Sampling

Appropriate blood or tissue sampling strategies are key to getting robust estimatesof pharmacokinetic parameters in animals In mice it is usually necessary toeuthanize the animal using CO2 or another AVMA-approved method to obtainadequate volumes of blood for drug assay Cardiac puncture in a dead animalusually yields the greatest volume of blood Harvest of tissues or other samplesand immersion in a suitable matrix for sample processing (eg homogenization)can also be done to quantify tissue levels In larger animal species (eg ratsrabbits) an indwelling intravenous or intra-arterial catheter allows collection ofmultiple samples over time In these cases placement of two cannulas in separatevessels should be undertaken to avoid possible carryover of drug-containing infu-sate into samples

One can use d-optimality criteria to select sampling points [16] Howeverwe rarely use this because the pharmacokinetics of drugs are rapid in smallanimals and the slight advantage of narrowing sampling times is easily lost Fur-ther for novel compounds there is no information upon which to base selectionof sampling times The maximal volume of blood to be collected over a pharma-cokinetic study session should be estimated and approved upon consultation witha veterinarian and the Institutional Animal Care and Use Committee (IACUC)

After collection samples can be centrifuged to separate cells from plasmaor serum and aliquots of the sample transferred to screw-capped storage tubesPilot studies should consider possible loss of drug in polypropylene or other mate-rials that may result in falsely low levels of drug Administration of plasma ex-


panders or volume replacement should be considered for prolonged studies insmall animals Patency of catheters can be maintained by gentle flushing of linesusing heparin (10ndash100 UmL) in 09 saline but overzealous use can result ina systemic anticoagulant effect

412 Drug Assay

Assay of serum and tissue concentrations of drug in the test animal species can bedone using high performance liquid chromatographic (HPLC) or microbiologicalassays The more widespread availability precision sensitivity and rapidity ofHPLC methods [particularly those using detection by tandem mass spectrometry(MSMS)] has resulted in a shift toward the use of these methods particularlyin the early discovery or preclinical setting Metabolites and their antimicrobialactivity should be considered in the final pharmacokineticndashpharmacodynamicanalyses Preparation of standards in the appropriate biological matrix (egserum tissue homogenate) should be undertaken and the sensitivity speci-ficity reproducibility and ruggedness be validated to ensure reproducibility ofresults

413 Modeling Pharmacokinetic Data

Serum or tissue concentration vs time data can be analyzed using a variety ofmethods Although noncompartmental (SHAMmdashslope height area moment)methods are used by many investigators we have found that a more informativeanalysis results when compartmental models are used In addition full parameter-ization allows for better simulations Critical analysis of the selection of the ap-propriate pharmacokinetic model and generation of pharmacokinetic parametersand their confidence intervals is also helpful for performing simulations to calcu-late indices of pharmacokinetic parameters related to the MIC Compartmentalmodeling is also particularly important for estimating pharmacokinetic parame-ters from studies that generate a single composite lsquolsquopopulationrsquorsquo curve of singledata points gathered from destructive sampling (eg serum from mice) Manyinvestigators have adopted WinNONLIN as the fitting program but several othersuitable programs include ADAPT II and MKMODEL Even population model-ing programs (eg NOMEM NPEM) may be useful with some data sets

After estimation of pharmacokinetic parameters simulation of serum levelsfor various dosage regimens can be undertaken Unbound drug concentrationsshould be simulated using protein binding values generated in the same speciesIt is particularly important to correlate effects with unbound drug exposure forstudies where development of pharmacokinetic-pharmacodynamic relationshipswill be extended to other species particularly humans As will be discussed be-low several studies have shown that the free concentration of drug in serum islinked to the efficacy of several anti-infectives Differences in serum protein bind-ing between preclinical species and humans could lead to errors in conclusions


concerning target levels of total drug to obtain acceptable levels of efficacy Ex-amples include experience with cefonicid in the treatment of endocarditis due toS aureus [17]

42 Animal vs Human Pharmacokinetics

The pharmacokinetics of drugs are known to differ markedly between preclinicalanimal species (particularly small animals used in pharmacokinetic studies) andhumans These differences largely arise due to well-described allometric relation-ships that relate physiological variables measured within species to differencesin body weight [18] However the differences in pharmacokinetic properties be-tween animals and humans may arise due to differences in metabolism biliaryor renal transport or a combination of these factors For example although mero-penem is resistant to hydrolysis by human renal dehyropeptidases it is highlysusceptible to inactivation to a deydropeptidase produced in mouse tissues [19]Table 3 depicts examples of differences in pharmacokinetic properties betweenhumans and small animals for several classes of agents These differences areoften most marked for β-lactam agents

In conducting pharmacokinetic-pharmacodynamic investigations in animalmodels with drugs used in humans it is crucial that the differences betweenhumans and animals be considered in the design and interpretation of experi-ments This is particularly important for compounds whose activity in vivo isdependent upon the length of time concentrations exceed the MIC (eg β-lactamsand Pseudomonas aeruginosa) When pharmacokinetic differences between hu-mans and small animals are ignored the results from animal models of infectioncan underestimate the efficacy of compounds in humans

A vivid example of the importance of consideration of human vs mousepharmacokinetics was shown in studies with ceftazidime treatment of Pseudo-monas aeruginosa infection in the neutropenic mouse thigh infection model by

TABLE 3 Comparison of Pharmacokinetic Parameters of SelectedAntimicrobials in Small Animals and Humans

Clearance [L(h sdot kg)] Elimination half-life (h)

Drug Human Rat Mouse Human Rat Mouse Ref

Ceftizoxime 012 116 367 127 026 015 18 20Vancomycin 189 036 075 15 28 063 21 22Ofloxacin 015 045 NA 70 184 NA 23 24Azithromycin 129 385 333 659 68 072 25ndash27

NA not available


Gerber et al in a neutropenic mouse model of infection (see Fig 1) [28] Whenhuman pharmacokinetics were simulated in the mice the bacterial killing wasmuch more sustained than that observed under normal mouse pharmacokineticconditions

Generally two methods exist for simulation of human pharmacokineticparameters in animals In the simplest terms they involve changing drug inputor drug elimination

421 Changing Drug Input

Several investigators have used multiple frequent administration of drugs withrapid drug clearances and short serum elimination half-lives in mice or rats Thisapproach uses the principle of superposition of successive lsquolsquobolusrsquorsquo doses givenwhen concentrations are expected to have declined below target levels Thefrequency of administration may be as short as every 30 min around the clock(Fig 1)

The availability of computer-controlled pumps for delivery of intravenousfluids has enabled the use of these devices to use decreasing rates of drug deliveryto simulate concentrations in humans [29] Two approaches may be used contin-uous infusion of diluent into the infusate which is delivered as an infusion intothe animal (much like what is done in in vitro pharmacodynamic models) or acontinuously changing intravenous infusion rate of a fixed concentration of drugin infusate The former approach has particular usefulness if one wishes to simu-late human concentrations of two drugs in the animal Both of these approacheshave been used in larger animal species (rats rabbits) in the evaluation of treat-ment of endocarditis peritonitis pneumonia and meningitis

The theoretical advantage associated with continuous infusion of antibioticswith certain in vitro pharmacodynamic properties has been studied in animalmodels of infection Continuous infusion studies are considerably simpler thanintermittent administration One simply needs to divide the desired steady-statedrug concentration by the drug clearance in the animal species to determine theinfusion rate Studies have evaluated delivery by this route for up to 5 daysContinuous infusion of drugs may also be obtained in mice using Alzet peritonealor subcutaneous micro-osmotic pumps

422 Changing Drug Elimination

An alternative (and perhaps more direct) way to mimic human drug pharmacoki-netics in small animals is to slow drug elimination For drugs largely excretedunchanged in urine inducing renal impairment by administration of a nephro-toxin can result in slow excretion and allow for close simulation of human dosageregimens in mice Several investigators have administered a single dose of uranylnitrate (eg 10 mgkg given 2ndash3 days prior to antibiotic treatment) to inducetemporary renal dysfunction for studies of short-term duration (eg 24ndash30 h)


FIGURE 1 Comparison of the effects of a single dose of ceftazidime against astrain of Pseudomonas aeruginosa in the neutropenic mouse thigh infectionmodel When a single dose was given to a mouse bacterial killing occurredonly transiently followed by regrowth In contrast when multiple frequentdoses of drug were given to simulate the pharmacokinetic profile in humansmuch more sustained bacterial killing was observed (From Ref 28)


[8] Although convenient this substance is considered to have low-level radia-tion and some states require special permits for its handling and disposal Othersubstances have been reported to cause renal impairment in rodents (eg glyc-erol) [30] however we did not find that it significantly altered the pharmacokinet-ics of aztreonam in mice (Tembe Chen Griffith and Dudley unpublished obser-vations)

Figure 2 compares the serum pharmacokinetic profile for amoxicillin inmice pretreated with a single dose of uranyl nitrate with concentrations in hu-mans When proper doses are administered the serum concentration vs timeprofile is comparable to that observed in humans Simulation of human serumdrug levels allowed for testing of the relationship between amoxicillin MIC andeffects in a mouse model using drug exposures comparable to those observed inhumans [8]

Other approaches for reducing drug clearance include administration ofdrugs that block renal tubular secretion This requires that the agent of interestbe excreted to a high degree by net renal tubular secretion in the animal speciesto be tested Probenecid is one example however the magnitude of the effectmay be variable and highly dose-dependent In addition probenecid may haveeffects on other nonrenal pathways for elimination including phase II metabo-lism

FIGURE 2 Comparison of serum amoxicillin concentrations found in renallyimpared mice with those in human volunteers (From Ref 8)


5 PHARMACODYNAMIC MODELING USING

MATHEMATICAL INDICES FOR RELATING

PHARMACOKINETIC DATA WITH THE MIC

51 Pharmacokinetic-Pharmacodynamic Indices and

Relationship to Dose MIC and Drug Half-Life

As described in earlier chapters several indices for integrating pharmacokineticdata with the MIC have been applied for the study of anti-infective pharmacody-namics Figure 3 depicts several indices that have been used to express theserelationships As shown in the figure all of the parameters are highly correlatedeg an increase in dose results in an increase in all parameters However themagnitude of changes in each parameter with different doses dosing intervalsdrug clearance and MIC will not increase proportionately with all the pharmaco-kinetic-pharmacodynamic (PK-PD) indices This is especially important in thedesign and interpretation of studies of drugs with short half-lives in small animalswhere the percent of the dosing interval drug concentrations that exceed the MICis the most important parameter for describing in vivo antimicrobial effects

A brief consideration of the effects of dose on PK-PD parameters is helpfulin recognizing the relative importance of each of these variables For the ratiosCmaxMIC or AUCMIC a change in dose results in proportional and linear (as-suming linear pharmacokinetics) changes in AUCMIC Similarly the MIC af-fects these parameters (inversely) but the proportion of change is linear

FIGURE 3 Examples of (a) pharmacokinetic parameters and the MIC and (b)pharmacodynamic relationship between a PK-PD parameter and effects invivo


The picture is considerably more complicated in calculation of the numberof hours drug concentrations exceed the MIC For a one compartment pharmaco-kinetic model and bolus drug input

Hours concn exceeds MIC (T MIC) ln doseV ln MIC

ClV

In contrast to the ratio metrics CmaxMIC and AUCMIC the magnitude ofchange in T MIC with dose or MIC is not directly proportional but changesas the natural logarithm of these values Changes in drug clearance (half-life)produce the most marked change in this parameter

The practical implications of this relationship for the design of studies andthe evaluation of PK-PD data in animal models of infection for drugs whereactivity in vivo is dependent upon T MIC are profound Figure 4 depictschanges in T MIC according to MIC or drug dose When drug half-life is veryfast (ClV is large) even high doses of drug will produce only minor changesin T MIC Similarly differences in in vitro potency (MIC) will have littleeffect on T MIC Failure to incorporate these considerations into experimentsresults in erroneous conclusions concerning the effects of changes in MIC ordrug dose and in vivo response for drugs where in vivo activity is linked toT MIC

FIGURE 4 Relationship between MIC and T MIC and dose for vancomycinadministered subcutaneously in mice


52 Designing Experiments to Identify Relationships

Between Pharmacokinetics MIC and In Vivo Effects

In planning experiments in which many regimens can be evaluated (eg short-term studies in mouse models) simulations of serum drug concentrations vstime using pharmacokinetic parameters derived from infected animals should beperformed The parameters MICs and dosage regimens can easily be pro-grammed into a spreadsheet to generate PK-PD data One can assess a numberof lsquolsquowhat ifrsquorsquo scenarios for several doses dosage regimens and MICs and gener-ate two- or three-dimensional plots showing the correlation for each regimen Anexample of uncorrelated PK-PD parameters for several vancomycin regimens ina mouse is shown in Fig 5 One can finalize selection of dosage regimens thatwill minimize the covariance among different PK-PD parameters In selectingregimens it is also important to be mindful of the relationships of dose and MICwith the number of hours serum concentrations will exceed the MIC In somecases it may be virtually impossible to design dosage regimens that produce arange of PK-PD parameters that correspond to those possible in humans In these

FIGURE 5 Lack of correlation between T MIC and AUCMIC ratio for severalregimens of vancomycin administered every 2ndash12 h in a mouse model ofinfection


cases the use of techniques to alter drug input or retard drug clearance may berequired

6 ANIMAL MODELS USED IN PK-PD STUDIES

61 Selection of Appropriate Models for Study

of PK-PD Issues

For the study of pharmacokinetics and pharmacodynamics (PK-PD) in an animalmodel one must take into consideration that the time course of antimicrobialactivity will vary between different antimicrobial agents For example β-lactamantibacterials exhibit very little concentration-dependent killing and in the caseof staphylococci long in vivo postantibiotic effects (PAEs) For these antibacteri-als high drug levels will not kill bacteria more effectively or more rapidly thanlower drug levels because these agents should be bactericidal as long as the drugconcentrations exceed the MIC In contrast fluoroquinolones and aminoglyco-sides show concentration-dependent killing For these agents the peakMIC orAUCMIC ratios should be the PK-PD parameters that most effectively describetheir efficacy [1] With these caveats in mind the choice of an animal model tostudy PK-PD parameters will depend on the organism one wishes to study thepharmacokinetics of the antimicrobial agent and the type of infection one ulti-mately intends to treat in humans

Of the animal models used to study PK-PD relationships the most com-monly used is the neutropenic mouse thigh model fully described by Gerber andCraig in 1982 [20] Other animal models used to study PKndashPD relationshipsinclude animal models of pneumonia [21ndash23] kidney infectionspyelonephritis[24] peritonitissepticemia [25 26] meningitis [27 31] osteomyelitis [32] andendocarditis [33ndash35] In fact almost any model can be used as long as drugexposure can be correlated with organism recovery or response

62 Factors Influencing Endpoints

Factors that affect the results of all endpoints include the test strain and initialchallenge inoculum treatment-free interval (ie interval between bacterial inocu-lation and initiation of treatment) immunocompetence of test animal speciesand administration of adjuvants These all can contribute to outcome and theconclusion concerning the magnitude of the PK-PD parameter required for effi-cacy Gerber et al [36] showed that the efficacy of aminoglycosides and β-lac-tams was markedly affected by delaying treatment requiring a higher dose Influoroquinolone treatment of pneumococcal infection in mice the AUCMIC ra-tio associated with the static effect is similar in immunocompetent or neutropenicmice In contrast AUCMIC is considerably different between neutropenic andnonneutropenic mice in infections due to Klebsiella pneumoniae [37]


63 Animal Models

To study the pharmacodynamics of an antimicrobial agent the pharmacokineticsof the agent in question should be determined in the animal species and underthe same conditions that will be used in the infection model (eg neutropenicmice in the case of the neutropenic thigh model) The MICs of the organisms tobe tested should be determined by an NCCLS reference method

631 Thigh Infection Model

The thigh infection model has been used extensively to describe the pharmacody-namics of several classes of drugs particularly fluoroquinolones macrolidesβ-lactams and aminoglycosides Most investigators have used Swiss albino micefor this model although any mouse strain can be used Although occasional stud-ies are done in normal (nonneutropenic) animals most of the experience withthis model has been obtained in neutropenic mice [38] Most investigators induceneutropenia by the administration of 150 mgkg and 100 mgkg of cyclophospha-mide on days 0 and 3 respectively This results in severe neutropenia by day 4which is sustained over at least a 24ndash48 h period [38] Inocula should be preparedin a suitable medium and allowed to grow into log phase After dilution 01 mLof bacterial suspension (105ndash106 CFU) is injected into each thigh while the ani-mals are under light anesthesia (eg isoflurane sodium pentobarbital ether) Forsome experimental designs a different organism may be injected in the contralat-eral thigh (eg when studying resistance mechanisms in isogenic strains) ormixtures of organisms can be used The organisms are usually allowed to growfor 2 h prior to the start of treatment Treatment is given in multiple dosingregimens over 24 h based on the pharmacokinetics of the drug and PK-PD param-eters being tested After 24 h of treatment the animals are euthanized and theirthighs are removed homogenized in sterile saline and serially diluted and cul-tured on a suitable medium to perform colony counts In the event of agents withlong half-lives (eg azithromycin) thigh homogenates can be assayed for thedrug to exclude the possibility of drug carryover interfering with the results ora substance to inactivate drug (eg β-lactamase) may be added The CFUthighdetermined for each dosing regimen can be quantified and relationships to drugexposure fit to a suitable pharmacodynamic model

632 Pneumonia Models

The pneumonia model described below is a mouse model however there are anumber of different models in rats [21 39] hamsters [40] or rabbits [41] Organ-isms used in pneumonia models include but are not limited to S pneumoniaeK pneumoniae P aeruginosa S aureus P mirabilis E coli L pneumophilaand A fumigatus Adjuvants are often used to increase virulence

In the mouse model most Swiss albino mice of either sex can be used


Depending upon the organism the mice may or may not need to be renderedneutropenic Inocula should be prepared in a suitable medium and allowed togrow into log phase After dilution the animals are infected by intranasal instilla-tion of 005 mL of bacterial suspension (106ndash107 CFU) while the animals areunder anesthesia (isoflourane sodium pentobarbital ether etc) Treatment isgiven in multiple dosing regimens for up to 2 days starting 24 h after infectionAfter 24ndash48 h of treatment the animals killed and their lungs are removed ho-mogenized serially tenfold diluted and then cultured on a suitable medium toperform colony counts The CFUlungs determined for each dosing regimen canthen be fit to a suitable pharmacodynamic model The advantages of this modelare that the prolonged endpoint (24ndash48 h) allows for several cycles of bacterialgrowth

Using this model Woodnutt and Berry [21] found that the efficacy of amox-icillin-clavulanate (a combination of the two drugs) was best described by per-centage time above the MIC with the maximum bactericidal effect beingachieved at a T MIC of 35ndash40

634 PyelonephritisKidney Infection Models

Kidney infection models for study of antifungal activity have been described inboth mice [24] and rats [42] Organisms used in kidney infection models includebut are not limited to E faecalis E coli S aureus C albicans and A fumiga-tus

For the mouse model most Swiss albino Balbc and DBA2 mice can beused Swiss albino mice can be rendered neutropenic to support growth of theorganism Inocula should be prepared in a suitable medium and allowed to growinto log phase After dilution the animals are infected by intravenous injectionTreatment is started 2 h after infection in the case of C albicans but can bestarted as late as 24 h after infection in the case of E faecalis Treatment is givenin multiple dosing regimens for 24 h or longer After 24ndash120 h of treatment theanimals are killed and their kidneys are removed homogenized serially tenfolddiluted and then cultured on a suitable medium to perform colony counts TheCFUkidneys determined for each dosing regimen can then be fit to a suitablepharmacodynamic model The advantages of this model are endpoints that canrange anywhere from 24 to 120 h it generally uses an inoculum high enough tostudy resistance and it allows for several growth and regrowth cycles Using thismodel it has been shown that the AUCMIC best predicts the effects of flucona-zole against strains with a wide range of susceptibilities to this drug [244344]

635 PeritonitisSepticemia Models

The septicemia model described below is a mouse model [2645] however thereis a similar model in neutropenic rats [25] Organisms used in peritonitissepsis


models include but are not limited to E coli S aureus S epidermidis P aerugi-nosa K pneumoniae S pneumoniae E faecalis S marcescens P mirabilisgroup B Streptococcus C albicans and A fumigatus

For this model almost any mouse species can be used Mice may or maynot need to be rendered neutropenic Inocula should be prepared in a suitablemedium and allowed to grow into log phase After dilution the animals are in-fected by intraperitoneal injection of 01ndash05 mL of bacterial suspension (1ndash109 CFU mouse) Treatment is given in multiple dosing regimens for up to 72h After treatment animals still alive are considered long-term survivors Deathsrecorded throughout the experiment can be compared by KaplanndashMeier or probitanalysis Percent survival [(number alivenumber treated) 100] determined foreach dosing regimen can then be fit to a suitable pharmacodynamic model

Using this model Knudsen et al [45] found that the peakMIC ratio andtime above MIC were associated with survival for vancomycin and teicoplaninagainst S pneumoniae In a study using peritoneal washings and survival in micefor Emax modeling der Hollander et al [26] found that the peakMIC ratio wasassociated with maximum bactericidal effects for azithromycin against S pneu-moniae and was achieved at a ratio of 4 In a similar model in rats Drusano etal [25] found that for lomefloxacin a peakMIC ratio that was 201 or 101was associated with survival against P aeruginosa At peakMIC ratios of 101they found that the AUCMIC ratio was associated with survival

636 Meningitis Models

The meningitis model described below is a rabbit model [910] There are alsomodels in the rat guinea pig cat dog goat and monkey but studies in the higherspecies are discouraged Organisms used in meningitis include N meningitidisS pneumoniae H influenzae S aureus S pyogenes S agalactiae E aerogenesE coli K pneumoniae P mirabilis P aeruginosa and L monocytogenes

For this model male or female rabbits can be used A dental acrylic helmetis attached to the skull of each rabbit by screws while it is under anesthesia Thehelmet allows the animal to be secured to a stereotactic frame made for the punc-ture of the cisterna magna Meningitis is induced by injection of a bacterial sus-pension directly into the cisterna magna Treatment is started 18 h after infectionand is given in multiple dosing regimens for up to 72 h The CFUmL CSF fluidor cure rates (sterile CSF cultures) determined for each dosing regimen can thenbe fit to a suitable pharmacodynamic model

In this model Tauber et al [27] found that the peak CSF drug levelMBCratio emerged as an important factor contributing to the efficacy of ampicillinagainst S pneumoniae In earlier studies these investigators also found that therewas a linear correlation between β-lactam CSF peak concentrations and bacteri-cidal activity in rabbits with S pneumoniae meningitis [9] Antibiotic concentra-tions in the CSF in the range of the MBC produced static effects and concentra-tions of 10ndash30 times the MBC produced a maximal bactericidal effect


In the same model of pneumococcal meningitis Lutsar et al [31] foundthat the bactericidal activity of gatifloxacin in the CSF was closely related tothe AUCMBC ratio but that maximal activity was achieved only when drugconcentrations exceeded the MBC for the entire dosing interval In another studyof pneumococcal meningitis Lutsar et al [10] found that for ceftriaxone thetime above MBC predicted the bacterial killing rate and that there was a linearcorrelation between time above MBC and bacterial killing rate during the first24 h of therapy Sterilization of the CSF was achieved only with T MBC of95ndash100 Ahmed et al [46] found that concentrations of vancomycin neededto be at least four- to eightfold the MBC of S pneumoniae in order to obtainadequate bacterial clearance The suggestion was also that time above the MBCin the CSF was required for efficacy

637 Osteomyelitis Models

The osteomyelitis model described below is both a rat model [32] and a rabbitmodel [47] Organisms used in osteomyelitis models include S aureus and Paeruginosa For this model male or female rats or rabbits can be used Animalsare anesthetized then the tibia is exposed and a 1 mm hole is bored with a dentaldrill into the medullary cavity of the proximal tibia Bones are infected by firstinjecting 5 sodium morrhuate followed by an injection of bacterial suspensionThe hole is plugged with dental gypsum and the wound is closed Treatment isinitiated 10 days after surgery and is given in multiple dosing regimens for upto 21 days for each compound

In this model OrsquoReilly et al [32] chose single daily doses of 50 mgkgfor azithromycin single daily doses of 20 mgkg for rifampin and three 90 mgkg doses of clindamycin daily for treatment regimens against S aureus Despitehaving large bone peakMIC and troughMIC ratios azithromycin failed to steril-ize a single bone Clindamycin sterilized 20 of the animals treated and rifampinsterilized 53 of the animals treated These results underscore the difficulties inpredicting the efficacies of antibiotics in osteomyelitis based on in vitro studiesand antibiotic levels in bone

638 Endocarditis Models

The endocarditis model has been used extensively to evaluate antimicrobial regi-mens Its advantages are that it produces an infection comparable to that observedin humans The endocarditis model described below is a rabbit model [48] how-ever there is also a rat model [49] Organisms used in osteomyelitis modelsinclude but are not limited to S aureus P aeruginosa S epidermidis E aero-genes E faecalis E faecium S sanguis S mitis C albicans and A fumigatus

For this model New Zealand rabbits are usually used Rabbits are anesthe-tized then a catheter is placed across the heart valve and left in place for theduration of the study Animals are infected after 24 h by an intravenous injection


of bacterial inoculum Treatment begins 24 h after infection and is given as multi-ple dosing regimens for 3ndash10 days Animals are killed then heart valve vegeta-tions are collected homogenized serially tenfold diluted and then cultured ona suitable medium to perform colony counts Terminal blood samples are takento check for sepsis The CFUg vegetation determined for each dosing regimencan be fit to a suitable pharmacodynamic model

In the rabbit model Powell et al [50] found that single daily doses orcontinuous infusion of tobramycin provided equally efficacious results sug-gesting that the AUCMIC ratio may be the variable that best describes the effi-cacy of this compound In another rabbit model of endocarditis Fantin et al [51]found that RP 59500 was efficacious against S aureus despite the fact that itwas above the MIC for only 33 of the time However it was found that RP59500 penetrated vegetations well with a vegetationblood ratio of 41 and aprolonged in vitro postantibiotic effect

In a rat model of endocarditis Entenza et al [49] found that despite havingconcentrations of the quinolone Y-688 that were the same as human concentra-tions the compound failed to be efficacious against quinolone-resistant S aureusThis failure may be due to insufficient vegetation penetration [52] In separatein vitro time-kill studies with S aureus low concentrations of Y-688 selectedfor drug resistance Poor drug penetration into vegetations could have providedideal conditions for selection of resistance in vivo and hence the failure of Y-688 in this model

In an analysis of 19 publications on the treatment of experimentally inducedendocarditis caused by S aureus S epidermidis viridians streptococci E aero-genes and P aeruginosa in rabbit or rat models Andes and Craig [35] foundthat for fluoroquinolones a 24 h AUCMIC of 100 a peakMIC of 8 anda time above the MIC of 100 were all associated with significant bactericidalactivity after 3ndash6 days of therapy However they determined that the 24 h AUCMIC ratio exhibited the best linear correlation A conclusion was that the resultsfound in endocarditis with quinolones were similar to those found in the neutro-penic thigh model

7 EXAMPLES OF USE OF ANIMAL MODELS TO STUDY

PHARMACOKINETIC-PHARMACODYNAMIC ISSUES

71 In Vivo Postantibiotic Effects

Postantibiotic effects have been recognized since the very early studies of penicil-lin Although in vitro studies can describe the persistent effects of a drug follow-ing its complete removal from a growing culture they often have little relevanceto the in vivo setting when drug concentrations fall more slowly over time Incontrast demonstration of persistent antibiotic effects following a single dose can


be helpful in determining the optimal dosage interval Single dose experiments inanimal models can be used to define the in vivo postantibiotic effect (in vivoPAE) [5354] In these experiments a single dose of drug is given and the serumconcentration and tissue burden of the test organisms are measured over timeThe in vivo PAE is calculated by the equation

PAE T C M

where M is the time for which plasma levels exceeded the MIC T is the timerequired for the bacterial counts of treated mice to increase by 1 log10 CFUthighabove the count at time M and C is the time necessary for the counts in controlanimals to increase by 1 log10 CFUthigh An example is shown in Fig 6

There are some important differences between the in vitro and in vivoPAEs Most notably the in vivo PAE tends to be longer than the in vitro PAEA possible explanation for the differences between the in vitro and in vivo PAEsis the effect of subinhibitory drug concentrations on bacterial growth and re-

FIGURE 6 Example of an in vivo PAE for cefazolin Growth curves of control() and antibiotic-exposed () S aureus ATCC 25923 in mouse thighs aftera single dose of cefazolin 125 mgkg Serum levels of cefazolin after a singledose at 125 mgkg(∆) (From Ref 38)


growth Sub-MIC concentrations can increase the length of the PAE [55] asreflected in the in vitro measurement PAE-SME Measurements of the in vivoPAE from single-dose experiments can then be used to better define the durationof antimicrobial effects in vivo with declining concentrations

72 Serum Protein Binding

Unfortunately the effect of serum protein binding on antimicrobial activity stillattracts considerable controversy and confusion among many clinicians and re-searchers The proportional reduction of antimicrobial activity in the presence ofserum or binding proteins has been thoroughly demonstrated for several anti-infectives in susceptibility testing

Although of high interest there are too few examples of well-controlled ex-periments that demonstrate the importance of serum protein binding on efficacyin vivo The difficulty in showing the importance of protein binding in animalmodels largely lies in the fact that the class of anti-infectives with the greatestvariability in serum protein binding are the β-lactam antibiotics Since the in vivoefficacy of these agents is dependent upon T MIC and they have relatively shorthalf-lives in small animals large differences in serum protein binding are requiredto producesignificant differences in free-drug T MIC Merrikenet al [56] demon-strated the importance of serum protein binding on the efficacy of several structur-ally related analogs of penicillin in a mouse model of sepsis due to Staphylococcusaureus All of the agents had similar in vitro potency against the test organism (MICbetween 025 and 05 mgL) and pharmacokinetic properties but the percent boundto serum proteins ranged between 36 and 98 Although the differences in phar-macokinetic properties of total drug were small (25-fold range) there was a 70-fold difference among agents in dose required for survival in 50 of animals (ED50

ranged from 07 to 497) In a neutropenic mouse thigh model we also comparedthe efficacy of three cephalosporin analogs with varying MICs to 2 strains of methi-cillin-resistant Staphylococcus aureus pharmacokinetics and protein binding Asshown in Fig 7 bacterial killing was best described by the number of hours thatfree-drug concentrations exceeded the MIC

73 Drug Resistance

The increasing problem of resistance to anti-infective drugs has required criticalanalysis of the significance of novel resistance mechanisms The study of drugpharmacodynamics in animal models of infection either using isogenic strainswith or without a resistance factor or using relevant clinical isolates can assessthe importance of resistance under in vivo conditions This information can beimportant in establishing resistance breakpoints for in vitro MIC testing (see be-low) as well as for formulating optimal dosage regimens to prevent or overcomeestablished resistance Generally if resistance in vivo is less than that observed


in vitro this will be reflected by the disruption of expected relationships betweendrug exposure (eg AUC) and MIC

The clinical relevance of resistance to extended spectrum cephalosporinsby novel plasmid-mediated β-lactamases was studied by Craig and colleagues inthe neutropenic mouse thigh model against several isogenic strains producingextended-spectrum β-lactamases Mice were pretreated with uranyl nitrate to sim-ulate human exposures to the drug The results showed that although MICs wereelevated at a high inoculum in vivo results were best correlated with MICs ob-tained at the lower inoculum (W Craig personal communication)

Reduced susceptibility to vancomycin in enterococci and more recentlyStaphylococcus aureus is of increasing clinical concern because of the absenceof alternative therapies In experiments in the neutropenic mouse thigh modelwith S aureus strains that were vancomycin-susceptible or intermediate (VISA)the efficacy was best described by the CmaxMIC or AUCMIC ratio When resultsfor the vancomycin susceptible strains were compared with those for VISA onlyslightly higher vancomycin exposures (Cmax or AUC) were required for the samelevel of efficacy despite the higher MICs Although further studies are requiredthis suggests that these strains have a reduced level of susceptibility in vivo tovancomycin that is less than that predicted by the in vitro MIC [57] Optimizationof vancomycin dosage regimens could be a successful strategy for the clinicalmanagement of strains with reduced susceptibility to vancomycin

Drug efflux is increasingly recognized as an important mechanism of resis-tance in bacteria and fungi However little is known concerning the efficiencyof these pumps to produce resistance under in vivo conditions Using isogenicstrains of Pseudomonas aeruginosa with varying levels of expression of themulticomponent mexAB-oprM efflux pump a reduced response to levofloxacinand ciprofloxacin in the neutropenic mouse thigh model and mouse sepsis modelwas observed the reduction in efficacy due to efflux was proportional to thechange in AUCMIC [58] In contrast Andes and Craig [59] reported that AUCMIC ratios associated with response in the neutropenic mouse thigh model forNOR-A efflux-related resistance to fluoroquinolones in Streptococcus pneumon-iae were lower than that in susceptible strains or strains with reduced susceptibil-ity due to non-efflux (eg gyrA) mechanisms These data suggest that effluxmechanisms of resistance may be significant in some bacteria but not in others

Resistance to fluconazole due to target modifications andor efflux has alsobeen shown to be significant in vivo using pharmacodynamic modeling Sorensenet al studied several strains of C albicans with over a 2000-fold range in MICin a mouse model of disseminated candidiasis The reduction in counts in kidneysat 24 h following fluconazole was found to be described by the AUCMIC ratiofor all doses and strains tested [44] suggesting that elevated fluconzole MICsdue to target or efflux-based mechanisms correspond to similar levels of reducedactivity in vivo


74 Establishing Susceptibility Breakpoints

Given the usefulness of pharmacokinetic-pharmacodynamic relationships for pre-dicting efficacy in vivo animal models using these analyses have been used forestablishing breakpoints for in vitro susceptibility testing The Antimicrobial Sus-ceptibility Testing Subcommittee of the National Committee for Clinical Labora-tory Standards (NCCLS) has used such analyses for consideration of breakpointsfor certain classes of drugs where clinical data are scant as well as for new agentsA combined approach where data from human clinical trials involving treatmentwith organisms with varying MICs along with animal model PK-PD data pro-vides a rational basis for establishing susceptibility breakpoints

Pharmacokinetic-pharmacodynamic data derived in animal models of in-fection are used to establish susceptibilityresistance breakpoints for MIC testingin several ways Studies in a relevant model of infection can be used to character-ize the parameter that best describes efficacy in the model The lsquolsquotargetrsquorsquo PK-PD parameter (eg 24 h AUCMIC ratio) for producing a bacteristatic drug50 or even 90 of the maximum response can be derived from the experimentsBased on the pharmacokinetic properties in humans at safe doses PK-PD parame-ters for various dosage regimens can be calculated for various MIC values The

FIGURE 7 Relationship between exposure in vivo to free drug concentrationsand bacterial killing in a neutropenic mouse thigh infected model due to 2strains of methicillin-resistant Staphylococcus aureus and three cephalospo-rin analogs ( for each compound) with varying MICs and serum pro-tein binding


highest MIC value that still achieves the target PK-PD parameter for the drugwould correspond to a suitable MIC breakpoint for susceptibility testing Forexample a drug whose efficacy in an animal model of infection is maximal whenthe 24 h free-drug AUCMIC ratio exceeds 30 and safe regimens in humansproduce a 24 h AUC of 60 mg sdot hL then a susceptibility breakpoint of 2 mgLcould be recommended Of note is that although the exact serum concentration vstime curve dosing frequency and protein binding may differ between humans andsmall animals these differences are considered in reducing the exposure relativeto the MIC by using pharmacokinetic measures (eg free-drug AUC Cmax)

The rigor of the selected breakpoint can be further evaluated using simula-tion The availability of population pharmacokinetic parameters and their vari-ability in target patient groups can be used to determine the probability of individ-ual patients attaining a PK-PD target parameter using a selected breakpoint Thesimulation can be even further developed by incorporating the distribution ofMICs in organisms of interest and serum concentration data for individual pa-

FIGURE 8 Effect of simulated human dosage regimens of amoxicillin on re-covery of bacteria from the thighs of neutropenic mice according to amoxicil-lin MIC Mice were pretreated with uranyl nitrate to enable simulation ofamoxicillin levels corresponding to that observed in humans with usualdoses as shown in Fig 2 Organisms with an MIC 2 mgL all showed a reduc-tion in CFUthigh thus supporting a susceptibility breakpoint of this value(From Ref 8)


tients for the population means and variances [60] This corresponds to the equiv-alent of a simulated clinical trial

An alternative approach employs direct simulation of human pharmacoki-netics in the animal and testing of strains with varying MICs to the test agentOne would expect to see a graded response according to MIC and ultimately alsquolsquono effectrsquorsquo at a threshold MIC value This approach is shown in Fig 8 Humandosage regimens of amoxicillin were simulated in neutropenic mice whose thighswere then infected with several strains of S pneumoniae with varying levels ofsusceptibility to the drug For strains with MICs exceeding 2 mgL little or noeffect was seen on bacterial counts recovered from mice at 24 h thus supportinga susceptibility breakpoint of 2 mgL or less

8 CONCLUSIONS

Animal models of infection are a pivotal tool in the study of PK-PD propertiesof anti-infectives Consideration of PK-PD issues in the design and interpretationof experiments in animals have strengthened the usefulness of these models forthe study of human infection Animal experimentation has been greatly improvedbecause of recognition of the importance of these issues In addition many ofthe recognized limitations of animal models for application to treatment of humaninfections have been overcome by recognition of the importance of pharmacoki-netics in the outcome of infection These principles are routinely applied in thestudy of new drugs in all phases of drug discovery and development as well asin the optimization of dosage in the pre- and postmarketing evaluation of agents

ACKNOWLEDGMENTS

We acknowledge the excellent assistance of scientists and animal care personnelin Discovery and Preclinical Pharmacology at Microcide Pharmaceuticals whocontributed to generating data for some of the examples provided

REFERENCES

1 H Eagle R Fleishman M Levy lsquolsquoContinuousrsquorsquo vs lsquolsquodiscontinuousrsquorsquo therapy withpenicillin N Engl J Med 1953 248481ndash488

2 H Eagle R Fleishman M Levy On the duration of penicillin action in relation toits concentration in the serum J Lab Clin Med 1953 40122ndash132

3 H Eagle R Fleishman A Musselman Effect of schedule of administration on thetherapeutic efficacy of penicillin Am J Med 1950 9280ndash299

4 H Collins A Cross A Dobek S Opal J McClain J Sadoff Oral ciprofloxacin anda monoclonal antibody to lipopolysaccharide protect leukopenic rats from lethal in-fection with Pseudomonas aeruginosa J Infect Dis 1989 1591073ndash1082


5 J Tisdale M Pasko J Mylotte Antipseudomonal activity of simulated infusion ofgentamycin alone or with piperacillin assessed by serum bactericidal rate and areaunder the killing curve Antimicrob Agents Chemother 1989 19899

6 A Firsov S Vostrov A Shevchenko G Cornaglia Parameters of bacterial killingand regrowth kinetics and antimicrobial effect examined in terms of area under theconcentration-time curve relationships Action of ciprofloxacin against Escherichiacoli in an in vitro model Antimicrob Agents Chemother 1997 19976

7 M Dudley D Gilbert T Longest S Zinner Dose ranging of the pharmacodynamicsof cefoperazone plus sulbactam in an in vitro model of infection Comparison usinga new parametermdasharea under the survival fraction versus time curve Pharmacother-apy 1989 9189

8 D Andes W Craig In vivo activities of amoxicillin and amoxicillin-clavulanateagainst Streptococcus pneumoniae Application to breakpoint determinations Anti-microb Agents Chemother 1998 422375ndash2379

9 M Tauber C Doroshow C Hackbarth M Rusnak T Drake M Sande Antibacterialactivity of β-lactam antibiotics in experimental meningitis due to Streptococcuspneumoniae J Infect Dis 1984 149568ndash574

10 I Lutsar A Ahmed I Friedland M Trujillo L Wubbel K Olsen GH McCracken JrPharmacodynamics and bactericidal activity of ceftriaxone therapy in experimentalcephalosporin-resistant pneumococcal-meningitis Antimicrob Agents Chemother1997 412414ndash2417

11 K Madaras-Kelly A Larsson J Rotschafer A pharmacodynamic evaluation of ci-profloxacin and ofloxacin against two strains of Pseudomonas aeruginosa J Antimi-crob Agents Chemother 1996 37703ndash710

12 H Hanberger Pharmacodynamic effects of antibiotics Studies on bacterial morphol-ogy initial killing postantibiotic effect and effective regrowth time Scand J InfectDis Suppl 1992 811ndash52

13 H Mattie A predictive parameter of antibacterial efficacy in vivo based on efficacyin vitro and pharmacokinetics Scand J Infect Dis Suppl 1990 74133ndash136

14 J Zhi C Nightingale R Quintiliani A pharmacodynamic model for the activity ofantibiotics against microorganisms under measurable conditions J Pharm Sci 1986751063ndash1067

15 M Dudley S Zinner Simultaneous mathematical modeling of the pharmacokineticand pharmacodynamic properties of cefoperazone 89th Meeting of the AmericanSociety of Clinical Pharmacology and Therapeutics San Diego CA 1988

16 D DrsquoArgenio A Schumitsky ADAPT II A program for simulation identificationand optimal experimental design User manual Biomedical Simulations ResourceUniv Southern California Los Angeles 1992

17 M Dudley W Shyu C Nightingale R Quintiliani Effects of saturable protein bind-ing on the pharmacokinetics of unbound cefonicid Antimicrob Agents Chemother1986 30565ndash569

18 J Mordenti Pharmacokinetic scale-up Accurate prediction of human pharmacoki-netic profiles from animal data J Pharm Sci 1985 741097ndash1099

19 M Tsuji Y Ishii A Ohno S Miyazaki K Yamaguchi In vitro and in vivo antibacte-rial activities of S-4661 a new carbapenem Antimicrob Agents Chemother 19984294ndash99


20 A Gerber W Craig Aminoglycoside-selected subpopulations of Pseudomonas aeru-ginosa Characterization and virulence in normal and leukopenic mice J Lab ClinMed 1982 100671ndash681

21 G Woodnutt V Berry Two pharmacodynamic models for assessing the efficacy ofamoxicillin-clavulanate against experimental respiratory tract infections causedby strains of Streptococcus pneumoniae Antimicrob Agents Chemother 1999 4329ndash34

22 R Roosendaal I Bakker-Woudenber J v d Bergh M Michel Therapeutic efficacyof continuous versus intermittent administration of ceftazidime in an experimentalKlebsiella pneumoniae pneumonia in rats J Infect Dis 1985 152373ndash378

23 E Azoulay-Dupuis E Vallee J Bedos M Muffat-Joly J Pocidalo Prophylactic andtherapeutic efficacies of azithromycin in a mouse model of pneumococcal pneumo-nia Antimicrob Agents Chemother 1991 351024ndash1028

24 D Andes M v Ogtrop Characterization and quantitation of the pharmacodynamicsof fluconazole in a neutropenic murine disseminated candidiasis infection modelAntimicrob Agents Chemother 1999 432116ndash2120

25 G Drusano D Johnson M Rosen M Standiford Pharmacodynamics of a fluoroqui-nolone antimicrobial agent in a neutropenic rat model of Pseudomonas sepsis Anti-microb Agents Chemother 1993 37483ndash490

26 J der Hollander J Knudsen J Mouton K Fuurstad N Frimodt-Moller H VerbrughF Espersen Comparison of pharmacodynamics of azithromycin and erythromycinin vitro and in vivo Antimicrob Agents Chemother 1998 42377ndash382

27 M Tauber S Kunz O Zak M Sande Influence of antibiotic dose dosing intervaland duration of therapy on outcome in experimental pneumococcal meningitis inrabbits Antimicrob Agents Chemother 1989 33418ndash423

28 A Gerber H Brugger C Feller T Stritzko B Stalder Antibiotic therapy of infectionsdue to Pseudomonas aeruginosa in normal and granulocytopenic mice Comparisonof murine and human pharmacokinetics J Infect Dis 1986 15390ndash97

29 L Mizen Methods for obtaining human-like pharmacokinetic patterns in experimen-tal animals In O Zak M Sande eds Handbook of Animal Models of InfectionAcademic Press New York 1999 pp 93ndash103

30 J Lin T Lin Renal handling of drugs in renal failure I Differential effects of uranylnitrate- and glycerol-induced acute renal failure on renal excretion of TEAB andPAH in rats J Pharmacol Exp Ther 1988 246896ndash901

31 I Lutsar I Friedland L Wubbel C McCoig J Jafri W Ng F Ghaffar GH McCrackenJr Pharmacodynamics of gatifloxacin in cerebrospinal fluid in experimental cephalo-sporin-resistant Pneumococcal meningitis Antimicrob Agents Chemother 1998 422650ndash2655

32 T OrsquoReilly S Kunz E Sande O Zak M Sande M Tauber Relationship betweenantibiotic concentration in bone and efficacy of treatment of staphylococcal osteomy-elitis in rats Azithromycin compared with clindamycin and rifampin AntimicrobAgents Chemother 1992 362693ndash2697

33 F Genko T Mannion C Nightingale J Schentag Integration of pharmacokineticsand pharmacodynamics of methicillin in curative treatment of experimental endocar-ditis J Antimicrob Chemother 1984 14619ndash631

34 T Carpenter C Hackbarth H Chembers M Sande Efficacy of ciprofloxacin for


experimental endocarditis caused by methicillin-susceptible or -resistant strains ofStaphylococcus aureus Antimicrob Agents Chemother 1986 30382ndash384

35 D Andes W Craig Pharmacodynamics of fluoroquinolones in experimental modelsof endocarditis Clin Infect Dis 1998 2747ndash50

36 A Gerber W Craig H-P Brugger C Feller A Vastola J Brandel Impact of dosingintervals on activity of gentamicin and ticarcillin against Pseudomonas aeruginosain granulocytopenic mice J Infect Dis 1983 145296ndash329

37 D Andes M V Ogtrop W Craig Impact of neutrophils on the in-vivo activity offluoroquinolones 37th Annual Meeting of the Infectious Disease Society ofAmerica Infect Dis Soc Am Philadelphia PA 1999

38 W Craig S Gudmundsson The postantibiotic effect In V Lorian ed Anti-biotics in Laboratory Medicine Williams and Wilkins Baltimore MD 1996pp 296ndash329

39 G Smith K Abbott Development of experimental respiratory infections in neutro-penic rats with either penicillin-resistant Streptococcus pneumoniae or β-lactamase-producing Haemophilus influenzae Antimicrob Agents Chemother 1994 38608ndash610

40 S Arai Y Gohara A Akashi K Kuwano N Nishimoto T Yano K Oizumi KTakeda T Yamaguchi Effects of new quinolones on Mycoplasma pneumoniae-in-fected hamsters Antimicrob Agents Chemother 1993 37287ndash292

41 L Piroth L Martin A Coulon C Lequeu M Duong M Buisson H Portier P Chava-net Development of an experimental model of penicillin-resistant Streptococcuspneumoniae pneumonia and amoxicillin treatment by reproducing human pharmaco-kinetics Antimicrob Agents Chemother 1999 432484ndash2492

42 T Rogers J Galgiani Activity of fluconazole (UK 49858) and ketoconazole againstCandida albicans in vitro and in vivo Antimicrob Agents Chemother 1986 30418ndash422

43 A Louie G Drusano P Banerjee Q-F Liu W Liu P Kaw M Shayegani H TaberH Miller Pharmacodynamics of fluconazole in a murine model of systemic candidia-sis Antimicrob Agents Chemother 1998 421105ndash1109

44 K Sorensen E Corcoran S Chen D Clark V Tembe O Lomovskaya M DudleyPharmacodynamic assessment of efflux- and target-based resistance to fluconazole(FLU) on efficacy against C albicans in a mouse kidney infection model 39th Inter-science Conference on Antimicrobial Agents and Chemotherapy San Francisco CA1999

45 J Knudsen K Fuursted F Esperson N Frimodt-Moller Activities of vancomycinand teicoplanin against penicillin-resistant pneumococci in vitro and in vivo andcorrelations to pharmacokinetic parameters in the mouse peritonitis model Antimi-crob Agents Chemother 1997 411910ndash1915

46 A Ahmed H Jafri I Lutsar C McCoig M Trujillo L Wubbel S Shelton GHMcCracken Jr Pharmacodynamics of vancomycin for the treatment of experimentalpenicillin- and cephalosporin-resistant pneumococcal meningitis AntimicrobAgents Chemother 1999 43876ndash881

47 M Smeltzer J Thomas S Hickmon R Skinner C Nelson D Griffith TR Parr JrR Evans Characterization of a rabbit model of staphylococcal osteomyelitis J Or-thop Res 1997 15414ndash421


48 M Sande M Johnson Antimicrobial therapy of experimental endocarditis causedby Staphylococcus aureus J Infect Dis 1975 131367ndash375

49 J Entenza O Marchetti M Glauser P Moreillon Y-688 a new quinolone activeagainst quinolone-resistent Staphylococcus aureus Lack of in vivo efficacy in exper-imental endocarditis Antimicrob Agents Chemother 1998 421889ndash1894

50 S Powell W Thompson M Luthe R Stern D Grossniklaus D Bloxham D GrodenM Jacobs A Discenna H Cash J Klinger Once-daily vs continuous aminoglycosidedosing Efficacy and toxicity in animal and clinical studies of gentamicin netilmicinand tobramicin J Infect Dis 1983 147918ndash932

51 B Fantin R Leclercq M Ottaviani J Valois B Maziere J Duval J Pocidalo CCarbon In vivo activities and penetration of the two components of the strepto-gramin RP 59500 in cardiac vegetations of experimental endocarditis AntimicrobAgents Chemother 1994 38432ndash437

52 A Cremieux B Maziere J Vallois M Ottaviani A Azancot H Raffoul A BouvetJ Pocidalo C Carbon Evaluation of antibiotic diffusion into cardiac vegetations byquantitative autoradiography J Infect Dis 1989 159938ndash944

53 W Craig Post-antibiotic effects in experimental infection models Relationship toin-vitro phenomena and to treatment of infections in man J Antimicrob Chemother1993 31149ndash158

54 B Vogelman S Gudmundsson J Turnidge J Leggett W Craig In vivo postantibioticeffect in a thigh infection in neutropenic mice J Infect Dis 1988 157287ndash298

55 O Cars I Odenholt-Tornqvist The post-antibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 1993 31159ndash166

56 D Merrikin J Briant G Rolinson Effect of protein binding on antibiotic activity invivo J Antimicrob Chemother 1983 11233ndash238

57 M Dudley D Griffith E Corcoran C Liu K Sorensen V Tembe S ChamberlandD Cotter S Chen Pharmacokinetic-pharmacodynamic (PK-PD) indices for vanco-mycin (V) treatment of susceptible (VSSA) and intermediate (VISA) S aureus inthe neutropenic mouse thigh model 39th Interscience Conference on AntimicrobialAgents and Chemotherapy San Francisco CA 1999

58 D Griffith O Lomovskaya L Harford A Lee MN Dudley V Lee Effect of effluxpumps (EPs) on the activity of fluoroquinolones against Pseudomonas aeruginosain murine models of infection 37th Interscience Conference on AntimicrobialAgents and Chemotherapy Toronto Canada 1997

59 D Andes W Craig Pharmacodynamics of gemifloxacin (GEM) against quinolone-resistant strains of S pneumoniae (SP) with known resistance mechanisms 39thInterscience Conference on Antimicrobial Agents and Chemotherapy San Fran-cisco CA 1999

60 M Dudley PG Ambrose Pharmacodynamics in the study of drug resistance andestablishing in vitro susceptibility breakpoints ready for prime time Curr Opin Mi-crobiol 2000 3515ndash521

5

β-Lactam Pharmacodynamics

JoCarol J McNabb

University of Nebraska Medical Center Omaha Nebraska

Khanh Q Bui

Bristol-Myers Squibb Company Chicago Illinois

1 INTRODUCTION

11 Historical Overview

Penicillin G the original β-lactam antibiotic was discovered by Fleming in 1928and used for the first time in 1941 to treat a staphylococcal infection in a Britishpoliceman By the end of World War II penicillin was commercially availablein the United States [1] Although initially given as a continuous infusion due tothe abundance of penicillin and the difficulties with the intravenous drip deliverysystem long-acting repository forms of penicillin G became popular with clini-cians The pharmacodynamic concepts that apply to β-lactams were actually pion-eered in the late 1940s by Harry Eagle an immunologist at the National Institutesof Health Calling upon both in vitro and in vivo animal studies Eagle was thefirst investigator to propose the concept of time-dependent bactericidal killingfor β-lactams Eagle demonstrated in vivo that a penicillin-free interval prolongsthe duration of treatment necessary for cure and that less total daily drug givenin frequent multiple doses is more effective than larger infrequent doses Eagle

99

100 McNabb and Bui

also demonstrated in vivo with nonneutropenic mice and rabbits that the durationof the penicillin concentration above the MIC of a specific pathogen correlatesto drug efficacy In Eaglersquos experiments a serum concentration 2ndash5 times theMIC of the bacteria correlated with penicillinrsquos bactericidal activity [2] The con-cept of dividing antibacterials into groups based on the pattern of bactericidalactivity was not formally proposed by investigators until the late 1970s As earlyas 1953 however Eagle clearly discussed not only the time-dependent bacteri-cidal activity of penicillin but also the concentration dependence of streptomycinthe first aminoglycoside [3]

In like manner Eagle also elucidated the effect of antibiotic concentrationon the rate at which a pathogen is killed both in vitro and in vivo Marked differ-ences in the effective concentration of penicillin and the maximal rate of killexist between organisms depending on the generation time and the MIC of theorganism For gram-positive bacteria however 3ndash10 times the MIC of the patho-gen kills at a maximal rate that does not continue to increase as the concentrationof penicillin increases [4ndash7] Excessively high concentrations of penicillin donot increase its bactericidal effect In fact in relation to S pneumoniae Eagleobserved a paradoxical decrease in the rate of killing in vitro at very high concen-trations of penicillin [8] Although two case reports of streptococcal endocarditiscured by reducing the dose of penicillin appear in the literature the clinical sig-nificance of this paradoxical phenomenon now termed the lsquolsquoEagle effectrsquorsquo hasnever been studied in a clinical trial [9ndash10]

As experience with penicillin increased clinicians soon realized that inpractice the concentration did not have to be continuously maintained above theMIC of a given pathogen to effect a cure in humans [11] Several investigatorsmost notably Bigger and Parker demonstrated in vitro that once gram-positivebacteria were exposed to a lethal dose of penicillin a period of delayed organismregrowth ensued during which surviving organisms did not immediately beginto multiply [12ndash14] Schmidt and Eagle were the first investigators to report thisphenomenon in animal studies of S pneumoniae infection [15ndash17] Eagle pro-posed an elegant hypothesis explaining what is now known as the post antibioticeffect Calling this phenomenon the lsquolsquoslow recovery of bacteria from the toxiceffects of penicillinrsquorsquo he proposed that bacteria that are damaged but not killedby exposure to penicillin remain susceptible to host defenses for several hoursafter the penicillin is removed [1819] Eaglersquos in vitro and in vivo experimentsare the foundation for β-lactam pharmacodynamics His experiments howeverwere not designed to answer one of the most crucial pharmacodynamic questionsWhat is the optimal time above the MIC for the β-lactams Although not specifi-cally proven by his own experiments Eagle consistently asserted throughout hiswork that the best dosing strategy for penicillin is to maintain antibiotic concen-trations above the pathogenrsquos MIC for the entire dosing interval This conceptguided the dosing of β-lactams for the next 50 years

-Lactam Pharmacodynamics 101

12 Pharmacokinetics Versus Pharmacodynamics

The differentiation between pharmacokinetics and pharmacodynamics has beendiscussed earlier in this book To review these concepts the reader is encouragedto refer back to Chapter 2 Because of the large number of β-lactam antibioticsavailable a complete review of each product will not be undertaken Where possi-ble generalizations will be made to simplify the discussion as it pertains to thisclass β-Lactams are best represented by a two-compartment model in which theyundergo rapid distribution followed by an elimination or terminal phase Thepharmacokinetic parameters of the β-lactams show many similarities in relationto the steady-state volume of distribution In a review of eight β-lactams (includ-ing members of the semisynthetic penicillin cephalosporin and carbapenemgroup) Drusano [20] showed that the volume of distribution following the post-distributive phase for these agents is much less than 1 Lkg (range 015ndash024 Lkg) indicating that they remain in the extracellular water as opposed to movinginto mammalian cells β-Lactams can be found in most body sites and secretionsmaking them an option for many infections

Although the volumes of distribution are similar for the compounds in thisclass other pharmacokinetic parameters such as clearance route of eliminationand half-life show more variability The clearance can vary by threefold from7 to 24 Lh [20] Because the volume of distribution falls within a narrow rangethis change in clearance is the primary determinant of half-life variation for thisclass The terminal half-life for penicillin and its analogs tends to be shorter (08ndash12 h) than those of the cephalosporins (14ndash8 h) [20ndash23] This is the rationalefor different dosing regimens as some require administration every 4 h whereasothers produce reliable outcomes with more extended dosing intervals of every12 or 24 h

The renal route of elimination is the primary pathway for the clearance ofβ-lactams The notable exceptions for this would be ceftriaxone cefoperazoneand nafcillin which undergo extensive nonrenal elimination as well [22]

2 FACTORS AFFECTING PHARMACODYNAMICS

21 Postantibiotic Effect

The postantibiotic effect (PAE) is the suppression of bacterial growth that persistswhen drug is removed after a short exposure of a microorganism to an antimicro-bial All antimicrobials appear to have a PAE to gram positive bacteria in vitroThe mechanism of the PAE is not well understood It may relate to nonlethaldamage of the bacteria or limited persistence of the drug at a cellular site ofaction Factors that have been shown to influence the PAE include the type oforganism the dose and concentration of antibiotic the duration of exposure andthe size of the bacterial inoculum

102 McNabb and Bui

In vitro one determines the PAE by observing bacterial growth kineticsafter antibiotic is removed The PAE retards the growth of antibiotic-exposedculture in relation to a control and is usually defined as the difference betweenthe time for each culture (exposed and control) to increase in concentration by1 log10 (tenfold) after removal of the drug [24] There is no standard protocol formeasurement of the PAE in vitro however Some investigators measure the PAEindirectly by viable colony counts Others use direct methods including biolum-inescense or optical density [25] The methodology will affect the results A PAEobtained from a bioluminescense assay is typically longer than the PAE deter-mined by viable counts [26] This knowledge is important in evaluating resultsfrom different studies The magnitude of the PAE depends upon drug exposuretime and antibiotic concentration up to some maximal response [27 28] Byviable counts the PAE of penicillins and cephalosporins to gram-positive bacteriais consistently 1ndash3 h [29] Imipenem and meropenem are the only β-lactams thatdemonstrate a PAE to gram-negative bacteria primarily P aeruginosa [30ndash32]The PAE of these carbapenems to P aeruginosa appears to be strain-dependentand varies up to 2 h in length depending on the particular strain [33]

In general the PAE observed in vivo is of greater duration than the PAEobserved in vitro for the same bacteria Several theories have been proposedto explain this phenomenon The sub-MIC effect is one possible explanationInvestigators evaluating the effects of residual drug on bacterial growth haveshown that in vitro a post antibiotic sub-MIC effect will prolong the durationof the PAE For example the PAE of benzylpenicillin is 24 h to S pyogenesWhen exposed to sub-MIC concentrations of penicillin (03 MIC) the PAEincreases to 22 h Continued exposure to antibiotics at sub-MIC concentrationsis actually closer to the reality of declining in vivo drug levels than the in vitromodels where antibiotic is completely removed from the system when PAE istested [34] Another possible explanation for the longer in vivo PAE is the postan-tibiotic leukocyte enhancement (PALE) effect As long as there is otherwise aPAE leukocytes have been shown in vitro to enhance the killing of antibiotic-damaged bacteria and prolong the PAE [35]

The neutropenic mouse thigh model and the neutropenic mouse pneumoniamodel are the two most widely accepted animal models used by investigators tostudy the PAE in vivo By eliminating the effects of white blood cells one candetermine the PAE attributable solely to the antibiotic In a neutropenic mousethigh model of infection all β-lactams induce a PAE of 12ndash45 h to S aureusno PAE to gram-negative bacteria and no PAE to S pneumoniae [36] Theseresults are consistent with in vitro results with the exception of penicillin in rela-tion to S pneumoniae [37] Even though penicillin does exert an in vitro PAEto S pneumoniae investigators have not been able to reproduce the effect in vivoUsing the mouse thigh model to evaluate the effect of combination antibiotics


investigators have also shown that when both drugs (an aminoglycoside plus aβ-lactam to treat S aureus) induce a PAE alone then the combination prolongsthe PAE beyond the longer of the individual PAEs [38]

The most important clinical application of the PAE as a pharmacodynamicparameter is in defining optimal dosing schedules for β-lactams The PAE iscrucial in determining the optimal time above the MIC for any given drugndashpatho-gen combination The existence of a PAE implies that the time above the MICcan be less than 100 of the dosing interval and in fact the PAE is the theoreticalrationale for the intermittent dosing of β-lactams

22 Protein Binding

The effect of protein binding continues to be a topic of debate even as more islearned about the pharmacodynamics of β-lactams It is recognized that proteinbinding is a rapid process that produces a reversible interaction between antibioticand protein principally albumin [39] A constant equilibrium exist between thetotal (T) drug concentration and freeunbound (F) and protein bound (DB) frac-tions T harr F DB It is accepted that only the free drug is able to diffuse fromthe bloodstream to the site of infection and subsequently into the bacteria Thisconcept is important for the time-dependent antibiotics that rely on maintainingunbound serum concentrations in excess of the MIC for a prolonged period oftime For highly protein bound drugs failures may be predicted as free-drugconcentrations drop below the MIC

In vivo models with mice and rabbits have demonstrated that the degreeof β-lactam protein binding correlates with the penetration into implanted tissuecages When table tennis balls were implanted into rabbits it was found that thepenetration of cephalothin (65 protein bound) was 27 versus 12 that ofcefazolin (86 protein bound) [40] This was further substantiated with work ina mouse peritonitis infection model with S aureus showing that the degree ofprotein binding was inversely proportional to the dose of antibiotic required toproduce a protective dose [41] Merrikin et al [41] used a set of β-lactams thathad the same intrinsic activity to the test strain while maintaining the pharmacoki-netics relatively constant to derive their data They also noted that the more highlyprotein bound drugs had a longer half-life but that this did not correlate with theprotective dose because the MICs of all agents were identical

Studies evaluating the effect of protein binding and clinical outcome inhumans are difficult to perform Instead the skin blister fluid model has been usedin healthy subjects to estimate the relationship between serum protein binding andtissue penetration This is interpreted as a movement of drug from the intravascu-lar to the extravascular space that is necessary during the treatment of an activeinfection When flucloxacillin (95 protein-bound) was compared to amoxicillin

104 McNabb and Bui

(17 protein-bound) penetration into blister fluid was markedly less for thehighly protein bound antibiotic [42] This suggests that amoxicillin is a betteragent for treatment but many other factors (intrinsic antimicrobial activity solu-bility) must be considered when making a determination for the appropriatenessof use The authors also pointed out that the effect of protein binding on penetra-tion becomes more pronounced only when the proportion of bound drug is highBecause most β-lactams exhibit less than 70 protein binding their penetrationwill be minimally affected [42]

The in vitro and in vivo reduction in cephalosporin activity is less predict-able when cephalosporins are tested against gram-negative organisms Leggettand Craig [43] found that the ceftriaxone cefoperazone moxalactam and cefti-zoxime MICs for E coli and K pneumoniae in human serum ultrafiltrate wereless than predicted by simply examining the protein binding alone The sameeffect was not observed with S aureus or P aeruginosa A disproportionate risein the MIC was observed when the antibiotics were placed into 25 50 and95 serum The hypothesis for this observation was that the serum containedproducts that enhanced the killing of gram-negative bacilli In essence a proteinfound in the serum effectively lowered the MIC when these cephalosporins wereexposed to Enterobacteriaceae This finding may explain why ceftriaxone (95protein bound) provides activity at or slightly below its MIC even though failurewould be predicted

Clinical data in humans to show that protein binding can alter the outcomeof an infection were presented in a case report by Chambers et al [44] Theseworkers reported three therapeutic failures with cefonicid used once daily for thetreatment of S aureus endocarditis This is a highly bound (95) cephalosporinand it was suggested that free drug concentrations were not sustained for a longenough period of time The measured total drug concentrations (free and bound)exceeded 150 microgmL but the serum bactericidal titer (SBT) was 18 Success-ful outcomes are predicted when the SBTs are 18 Another consequence ofthe high protein binding was the MIC difference of cefonicid in broth versusserum When the organisms were originally tested in broth the geometric meanfor all patients was 46 microgmL whereas use of 50 serum as the diluent resultedin a six fold increase (279 microgmL) As noted previously protein binding particu-larly in situations with highly bound cephalosporins against gram-positive bacte-ria will result in an increase of the MIC This latter observation is often over-looked when susceptibility tests are performed From these data it appears thatthis regimen allowed free-drug concentrations to remain above the MIC for onlya very short period of time The role of protein binding may affect the pharmaco-dynamics of specific antibioticndashbacterium combinations especially in situationswhere the free drug concentrations do not exceed the time above the MIC for β-lactams


3 RESEARCH STUDIES

31 In Vitro Studies

The in vitro evidence supporting the role of β-lactams as concentration-indepen-dent (time-dependent) agents was reported by Craig and Andes [45] who useda standard time-kill method with ticarcillin ciprofloxacin and tobramycin Fol-lowing the addition of antibiotic to the experimental strain of Pseudomonas aeru-ginosa they found that only ticarcillin failed to produce a faster rate of bacterialkilling after concentrations of 4 MIC were achieved The rate and extent ofbacterial killing between 4 MIC as compared to 16 MIC and 64 MIC weresimilar leading them to suggest that exceeding the MIC by more than fourfold isnot necessary

32 In Vivo Animal Studies

The in vivo data with animals correlate with observations from in vitro experi-ments An undertaking to describe and highlight all of the previous work in thisarea will not be attempted here Instead studies describing important results thatcontribute to the understanding of β-lactam pharmacodynamics will be used Tominimize the confusion related to host factors most of the experimental datawere derived using a neutropenic infection model Eliminating the activity ofwhite blood cells from these models has a distinct advantage over using normalanimals only antibioticndashorganism interactions are studied without any additionalinterference from neutrophils

Using a mouse model Gerber et al [46] correlated bacterial regrowth tothe time above the MIC (T MIC) When ticarcillin was administered as eithera single bolus or a fractionated dose (more closely simulating human pharmacoki-netics resulting in similar AUCs) neutropenic animals were found to have re-duced bacterial growth with more frequent dosing extending the T MIC Theextremely large peakMIC ratio produced from a bolus dose (201) did not reducethe bacteria count more than the fractionated doses (51) This same study evalu-ated the effect of these two dosing schemes in normal mice Unlike previousresults the bacterial regrowth with the bolus dose was not demonstrated sug-gesting that the effect of the combination with antibiotic and white blood cellswas significant in suppressing P aeruginosa No attempt was made to determinethe optimal T MIC required to produce a good outcome but an examinationof these data show that concentrations during bolus dosing were above the MICfor only 25 of the dosing interval

The required T MIC for obtaining the best outcome may vary for individ-ual antibioticndashpathogen combinations A univariate analysis of ticarcillin cefa-zolin and penicillin all demonstrated that the T MIC was the most important

106 McNabb and Bui

parameter in determining outcome as opposed to the AUC or peak concentrations[47] To produce a bactericidal effect E coli required a longer exposure to cefa-zolin (60 versus 20) compared to S aureus This large difference is theresult of a significant PAE of cefazolin for S aureus This evidence supportsearlier work with cephalosporins and S pneumoniae that found a relationshipbetween T MIC and the effective dose required for protection of 50 of theanimals [48]

Studies comparing normal and neutropenic animals can show the pharma-codynamic influence of host defenses As described previously the work ofGerber et al [46] demonstrated that normal mice could suppress bacterial growthregardless of the optimal dosing scheme Roosendaal et al [49] also discovereda striking difference between normal and leukopenic rats For normal rats theintermittent (every 6 h) and continuous administration of ceftazidime requiredequivalent doses (035 and 036 mgkg respectively) to reach the protective dosefor 50 (PD50) of the animals For leukopenic rats the continuous infusion re-quired 375 mgkg to produce the PD50 whereas 30 mgkg of ceftazidime wasneeded with the intermittent dosing This study highlights two important differ-ences between normal and neutropenic animals (1) Host defenses can overcomethe deficiencies when the pharmacodynamics are not maximized and (2) neutro-penic hosts require larger doses than normal hosts to eradicate organisms

33 Clinical Studies

Human studies to determine the optimal pharmacodynamics of β-lactams havefocused on infections in acute otitis media (AOM) or continuous infusion (CI)settings AOM is an attractive model because of the relative ease of fluid collec-tion that can be used for determining antibiotic concentation or MIC over timeA more in-depth discussion of AOM is presented in Section 7 Clinical data withcontinuous infusion provide important information because the pharmacodynam-ics are maximized with a prolonged time above the MIC The following sectionpresents a summary of relevant continuous infusion studies

4 CONTINUOUS INFUSION

The goal of antibiotic therapy is to achieve the best possible clinical outcomeswhile consuming the least amount of hospital resources Health care systems areunder intense pressure to increase quality of care and at the same time reducecosts Pressure to reduce the cost of antimicrobial therapy is especially intensebecause these drugs may account for up to 50 of a hospital pharmacy budgetAlthough β-lactam antibiotics have traditionally been given by intermittent infu-sion administration by continuous infusion is gaining popularity because it takesfull advantage of the known pharmacodynamics of the β-lactams and potentially


consumes the least amount of hospital resources Other options that will alsomaintain the time above MIC for the entire dosing interval are the use of antibiot-ics with longer half-lives or more frequent larger doses An advantage of continu-ous infusion however is that it can be given at a lower total daily dose thanstandard dosing schedules Although the IV drip method used in the past wascumbersome and inaccurate today because of lightweight portable and accurateinfusion pumps a continuous infusion is relatively easy and cost-effective toadminister Giving a loading dose prior to starting the continuous infusion willbring the antibiotic concentration into the therapeutic range immediately and min-imize any lag time in drug tissue equilibration Constant infusion of β-lactamantibiotics has the potential for appreciable cost reductions because it may repre-sent the best method to maintain levels above the MIC during the entire dosinginterval using the least amounts of drug labor and supplies

A number of in vitro and animal models substantiate the equivalent orsuperior efficacy of continuous infusion of β-lactams compared to standard dos-ing These models have been used extensively to elucidate the pharmacodynamicsof β-lactams because they hold a significant advantage over studies in humansThe doses and dosing intervals of antibiotic are easily varied reducing the inter-dependence of the pharmacodynamic parameters Using an in vitro model inves-tigators have demonstrated that a continuous infusion (CI) at 5 MIC of Paeruginosa is as efficacious as intermittent dosing (II) using less total daily drug[5051] Experiments in animal models have confirmed these in vitro results anddemonstrated further that a continuous infusion may be the better dosing strategyif the same total daily dose is used [5253]

The mouse thigh model has been used to evaluate the difference betweenneutropenic and nonneutropenic host response [54] The real difference in effi-cacy between CI and II shows up in the neutropenic host Even with a lowertotal daily dose CI provides much better efficacy than bolus dosing [5556] Inneutropenic rats with gram-negative infection a ceftazidime bolus dose that pro-tects 50 of the animals from death (PD50) had to be 65-fold higher than thePD50 dose for a continuous infusion of the same drug Once again this experimentconfirms that time above MIC correlates to efficacy for the β-lactams and whenhost defenses are not present the β-lactam concentration should exceed the MICof the pathogen for the entire dosing interval [57]

Continuous infusion is a practical way to maintain 100 time above MICwith less total daily drug ( eg 3 g24 h CI of ceftazidime versus 1ndash2 g every8 h) Although clinical efficacy data are sparse the pharmacodynamics of contin-uous infusion are well-characterized in both normal volunteers and critically illpatients and several small clinical trials have shown the equivalence of continu-ous infusion and standard dosing [58ndash62] In one of the first clinical trials ofcontinuous infusion Bodey et al [63] compared the efficacy of cefamandoledosed as either a continuous infusion or an intermittent dose given in combination

108 McNabb and Bui

with carbenicillin There was no significant difference in clinical cure betweenthe two regimens By subgroup analysis patients with persisting severe neutro-penia had a better clinical outcome (65 versus 21 p 003) with continuousinfusion [63] In a study of CI benzylpenicillin versus daily IM procaine penicillinG in 123 patients with pneumococcal pneumonia there was also no differencein clinical cure rates [64] A nonrandomized trial of continuous versus intermit-tent dosing of cefuroxime showed that the CI results in a lower total antibioticdose The CI results in equivalent efficacy shorter length of hospital stay andoverall cost savings to the institution [65]

At Hartford Hospital there have been two continuous infusion β-lactamclinical studies The investigators compared cefuroxime given either as a continu-ous infusion of 1500 mg or intermittently as 750 mg every 8 h (n 25 in eachgroup) to treat hospitalized community-acquired pneumonia (CAP) [66] Steadystate cefuroxime serum concentrations were 1325 629 microgmL more than 2ndash4 times the MIC90 of typical CAP pathogens There was no difference in clinicalcure rates but the CI regimen was associated with a shorter length of treatmentdecreased length of stay lower total cefuroxime dose and overall cost savingsThe average amount of intravenous cefuroxime per patient decreased significantly(p 004) from 80 34 g for intermittent dosing to 59 32 g for thecontinuous infusion The average daily costs (including antibiotics labor andsupplies) decreased significantly (p 004) $6364 3095 for the continuousinfusion compared with $8385 3482 for intermittent dosing

The second Hartford Hospital study was a prospective randomized trial ofthe efficacy and economic impact of ceftazidime given as either a continuousinfusion (3 gday) or intermittent infusion (2 g q8h) plus once-daily tobramycinfor the treatment of nosocomial pneumonia in the ICU [67] The investigatorsevaluated 35 patients 17 in the CI group and 18 in the II group Clinical efficacydid not vary significantly between groups (94 success in CI vs 83 in II)Number of adverse events duration of treatment and total length of hospital stayalso did not vary significantly The continuous infusion regimen used half of theintermittent dose and maintained concentrations above the MIC of the pathogenfor 100 of the dosing interval The intermittent dosing regimen maintainedconcentrations above the MIC for 76 of the dosing interval The costs (includ-ing drug acquisition antibiotic preparation and administration adverse eventsand treatment failures) associated with the CI of ceftazidime $62569 38784were significantly lower (p 0001) than with the II $100464 42995

There are some potential disadvantages to giving antibiotics by continuousinfusion For patients with limited IV access the continuous infusion may requiretheir only IV line Drug compatibility will be an issue if two drugs must beinfused simultaneously through one line Although the cost of infusion pumpsshould be considered in any cost-effectiveness analysis of drug delivery by con-tinuous infusion since most manufacturers will provide the use of the infusion


pumps with a supply contract the cost to the hospital is usually limited to theprice of the administration sets Overall the advantages of the continuous infu-sion both pharmacodynamically and economically far outweigh the disadvan-tages The continuous infusion of β-lactams in place of frequent intermittentdosing is a good example of how the knowledge and application of pharmacody-namic concepts can lead to cost-effective antibiotic therapy

5 TISSUE PENETRATION

Because of the difficulties associated with measuring tissue concentrations ofdrugs we generally use serum concentration as a surrogate marker in pharmaco-dynamic models Although most common bacterial pathogens are extracellularinfections occur in the tissues and it is the pharmacodynamic profile of an antibi-otic at the site of infection that ultimately determines its clinical efficacy Antibi-otic concentrations can vary significantly depending on the type of tissue Drugpenetration into tissues depends on the properties of the specific tissue type theproperties of the antibiotic and the interactions that occur at the tissuedrug inter-face Protein binding tissue permeability tissue metabolic processes and drugphysiochemical properties such as lipid solubility pKa and molecular weight allinfluence an antibioticrsquos movement into human tissues Only the free fraction(non-protein-bound) of an antibiotic is available to exert a pharmacological ef-fect For β-lactams the percent tissue penetration is inversely proportional to theprotein binding of the drug Animal studies show a direct correlation betweenserum protein binding and penetration into peripheral lymph The penetrationinto rabbit lymph of ceftriaxone a drug that is highly protein bound is 673 Incomparison the penetration of amoxicillin a drug with much less serum proteinbinding is 976 [68]

Methodological problems in obtaining tissue samples are the limiting factorin characterizing antibiotic tissue concentrations A complete pharmacodynamicpicture would include a concentrationndashtime curve for the various tissue compart-ments Most studies done in humans to date however simply characterize drugpenetration into various tissue compartments In spite of the limitations thesestudies have provided important information about the extracellular and intracel-lular disposition of these drugs Tissue penetration studies consistently show thatpenicillins and cephalosporins have rapid and good penetration into interstitialfluid [69ndash72] The opposite occurs in relation to intracellular space There isessentially no uptake of β-lactams into peripheral blood mononuclear cells poly-morphonuclear cells or alveolar macrophages thus explaining the ineffec-tiveness of β-lactams against intracellular pathogens such as Mycoplasma orChlamydia [7374]

Standard methods of characterizing tissue concentrations by using wholetissue homogenates tend to underestimate the interstitial concentration of β-

110 McNabb and Bui

lactams A promising new technique currently being developed for studying thepharmacodynamics of drug tissue penetration in both animals and humans hasbeen applied to the study of β-lactams with some success Microdialysis is anin vivo sampling technique for the continuous monitoring of drugs or other ana-lytes in the extravascular space The advantage of microdialysis is that it can beused in a variety of tissue compartments with minimal invasiveness and can there-fore be used in subjects that are awake and freely moving Because it overcomesthe limitations of other methods microdialysis offers a unique opportunity tocharacterize the pharmacokinetic profile of drugs in tissue compartments such asthe interstitial fluid adipose tissue muscle or dermis

Two microdialysis studies in animals have evaluated the tissue pharmacoki-netic profile of various β-lactams In the rat thigh model investigators found anexcellent correlation between piperacillin and ceftriaxone concentrations inplasma and predicted free levels of drug in the tissue [7576] In a separate studyusing awake and freely moving rats the investigators measured ceftriaxone andceftazidime concentrations in two regions of the animalrsquos brain Interestinglynot only are the half-lives of the antibiotics in the brain different from their re-spective half-lives in serum but drug distribution within the brain differs byregion and is not homogenous Based on this study antibiotic distribution appearsto differ by specific tissue compartment and region [77] Similar differences bytissue compartment in equilibration rates and pharmacokinetic profiles have beenobserved in human subjects [78] Microdialysis promises to be a useful tool inpharmacodynamic studies of the future

The central nervous system presents special problems in relation to study-ing the pharmacodynamics of drugs The brain is a unique tissue compartmentTight junctions of endothelial cells and a low rate of transcellular drug transportboth act to exclude antibiotics from the CNS Impaired host defenses and theslow rate of bacterial growth in the CNS decrease the effectiveness of antibioticsThe primary determinants of antibiotic blood-brain barrier penetration are adrugrsquos lipid solubility degree of protein binding ionization and active transportinto or out of the CSF [79] Although highly lipophilic compounds pass readilyinto the CSF β-lactams are hydrophilic weak organic acids and their penetrationis normally less than 10 This penetration significantly increases during theinflammation associated with meningitis [80ndash83] An additional issue in relationto β-lactams is the active transport mechanism that pumps these drugs out of theCSF Benzylpenicillin has the highest affinity for the transport pump but otherβ-lactams are also subject to elimination by this route

Antibiotic concentrations are especially difficult to measure in the braindue to the invasiveness in obtaining either spinal fluid (CSF) or tissue samplesand relatively few pharmacodynamic studies have examined the activity of β-lactams in the CNS Although there is limited evidence that antibiotic pharmaco-dynamics in the brain differ from the dynamics in other tissue compartmentsbecause we cannot easily characterize this in human subjects antibiotic dosing


in CNS infections is still largely empirical The data we do have have beenobtained from in vitro models or animal studies

In vitro for cefotaxime investigators have shown a limited PAE to E coliof about 05 h in pooled human CSF The same studies do not demonstrate aPAE for cefotaxime to E coli in Mueller-Hinton broth (MHB) indicating thatthere may be a mechanism for PAE that is unique to the CSF [8485] There areconflicting results from animal studies measuring the PAE of β-lactams in theCSF [86] In one study adding β-lactamase to the CSF reversed the PAE indicat-ing that small amounts of residual drug in the CSF may actually be responsiblefor the in vivo PAE [87]

Although studies in animals have suggested that the β-lactam pharmacody-namic parameter in CSF that correlates to efficacy is actually the peak concentra-tion rather than the time above MIC the results from these studies are inconclu-sive [88ndash90] Using a rabbit pneumococcal meningitis model to investigate therelationship between CSF penicillin concentration and bactericidal rate of killingthe investigators demonstrated that maximal killing occurred at 10ndash30 times theminimal bactericidal concentration (MBC) of the pathogen [91] Unfortunatelythey did not also determine the duration of time above MBC As the concentrationof drug increases the time above MBC in the CSF will also increase and willmost likely approach 100 of the dosing interval When the dose of the antibioticis high enough to provide 100 time above MIC the pharmacodynamic parame-ters of time and concentration cannot be separated to determine which parameterreally correlates to efficacy

In contrast the one study to examine the relationship between the pharma-codynamic parameters of time above MBC peakMBC and AUCMBC demon-strated that T MIC continues to be the important pharmacodynamic parameterfor β-lactams even in the CSF By varying the doses and dosing intervals ofceftriaxone in a rabbit meningitis model of cephalosporin-resistant S pneumoniae(MIC and MBC 40 microgmL) the investigators determined that T MBC isthe only pharmacodynamic parameter that independently correlated with ceftriax-onersquos bactericidal activity During the first 24 h the highest rate of bactericidalkilling occurred when T MBC exceeded 95 of the dosing interval Also inthe first 24 h twice-daily administration of the same total ceftriaxone dose re-sulted in longer time MBC and higher killing rates [92] On the basis of thisstudy it appears that the same pharmacodynamic model time-dependent killingthat defines β-lactam activity in serum and other tissue compartments also definesβ-lactam efficacy in the CSF [93]

6 -LACTAM AND -LACTAMASE INHIBITOR

COMBINATIONS

The incidence of gram-negative bacterial resistance has been on the rise in recentyears Escherichia coli the leading cause of gram-negative bacteremia in both

112 McNabb and Bui

the community and hospital setting develops resistance to β-lactams by the pro-duction of TEM-1 and TEM-2 plasmid-mediated β-lactamases β-Lactamases area large family of enzymes ubiquitous to bacteria that hydrolyze the β-lactamring of penicillins and cephalosporins The incidence of gram-negative bacterialresistance as well as the increasing incidence of nosocomial gram-positive infec-tions (due to bacteria such as S aureus that are intrinsically resistant to β-lactams)have led to the search for new antibiotics One approach is to combine a β-lactamwith a potent β-lactamase inhibitor Currently in the United States there are threeβ-lactamase inhibitors on the market (clavulanate sulbactam and tazobactam)that are used in combination with a β-lactam antibiotic The goal of β-lactamndashβ-lactamase inhibitor combinations is to expand the coverage of the β-lactam togram-positive bacteria and overcome drug resistance to gram-negative bacteria[9495]

Strategies for the optimal dosing of β-lactamndashβ-lactamase inhibitors basedon pharmacodynamic principles have not been established or even extensivelystudied In addition to the pharmacodynamic issues that relate to the individualβ-lactam several additional pharmacodynamic questions arise in relation to com-bination drugs These questions include Would the sequential dosing of inhibitorand β-lactam affect the bactericidal activity of the combination Second is it theratio of the combination of drugs that matters or is it the time above the MICof the component drugs that determines efficacy Finally if it is the time abovethe MIC of each drug that correlates to overall efficacy what is the optimal timeabove the MIC for the inhibitor and how does that affect the time above theMIC required for the β-lactam

Two pharmacodynamic studies have addressed the issue of sequential dos-ing In vitro the sequential dosing of tazobactam followed by piperacillin doesnot enhance the bactericidal activity of piperacillin [96] Similarly in vivo asstudied in E coli bacteremia in mice the pharmacodynamics of ampicillin-sul-bactam does not depend on whether sulbactam is dosed sequentially or simulta-neously with ampicillin [97]

The question of optimal T MIC is complicated for β-lactamndashβ-lactamaseinhibitor combinations because the turnover rate of both the enzyme and theinhibitor will affect the inactivation of the β-lactamase [98] The amount andtype of enzyme produced by the bacteria have a marked effect on the dynamicsof the inhibitor [99] Subsequently the amount of inhibitor present determinesrestoration of susceptibility to the partner drug [100] Current dosing regimensprovide concentrations of inhibitor that exceed the in vitro susceptibilitybreakpoint for only 2ndash3 h not the entire dosing interval Inasmuch as these drugshave been shown to work clinically the unique pharmacodynamics of the β-lactamndashβ-lactamase inhibitor combination is clearly the determining factor [101]

One explanation for the clinical efficacy of these drugs may relate to apost-β-lactamase inhibition effect In an adaptation of the model used to deter-mine post-antibiotic effect the effect of tazobactam was evaluated in β-lacta-


mase-producing strains of E coli Preincubation of bacteria with tazobactam andpiperacillin resulted in piperacillin-induced killing during a second exposure topiperacillin alone Bacteria not initially exposed to tazobactam were not killedby piperacillin during the second exposure [102] Similarly other investigatorshave reported a post-β-lactamase inhibition effect in which regrowth of amoxicil-lin-resistant bacteria is prevented by amoxicillin alone following exposure to andremoval of amoxicillin and clavulanate [103]

In an in vitro study of piperacillin versus piperacillintazobactam as ex-pected the addition of tazobactam to piperacillin did not alter the killing of anE coli piperacillin-susceptible strain By adding tazobactam to piperacillin thekilling of an isogenic TEM-3 E coli resistant to piperacillin became equivalentto the killing of the susceptible strain even though the concentration of tazobac-tam fell below 4 mgL (the concentration used in susceptibility testing) at 3 hAs an explanation the investigators suggest that several hours of an essentiallyβ-lactamase-negative state during which bacteria must reaccumulate sufficientquantities of β-lactamase to inactivate the drug follow exposure to high levelsof inhibitor Therefore the dosing interval of β-lactamndashβ-lactamase inhibitorcombinations can be extended for some finite period of time limited by the reaccumulation of β-lactamase by persisting bacteria [104] On the basis of thissmall number of in vitro studies it appears that dosing piperacillin-tazobactamevery 8 h instead of every 6 h is a reasonable dosing regimen

7 CORRELATION TO IN VITRO RESISTANCE

Clinical studies evaluating resistance rates and patient outcomes have been pri-marily conducted in respiratory tract infections In comparison to other infectionsites respiratory tract infections are more prevalent and recovery of bacteria isprimarily noninvasive allowing more data to be compiled A limitation to inter-preting these data is that only a handful of the most commonly isolated bacteriaare studied often excluding serious pathogens such as vancomycin-resistant En-terococcus faecium (VRE) or methicillin resistant Staphylococcus aureus(MRSA) which are not associated with these body sites Although limited thesestudies provide clues for the treatment of other infections that require aggressiveantibiotic therapy The reporting of resistance continues to rise but the impacton clinical outcomes may not always correlate with microbiological reports

Using a retrospective review of pneumonia caused by Streptococcus pneu-moniae Pallares and colleagues compared cases involving penicillin-nonsensi-tive strains (MIC 012ndash8 microgmL) to a matched control group with penicillin-sensitive strains (MIC 01 microgmL) [105] Of the 24 cases 14 presented withintermediate-resistant pneumococci (012 MIC 1 microgmL) and the remaining10 were resistant (MIC 2 microgmL) A higher mortality rate (54 vs 25 P 00298) was observed when these cases were compared to the control groupThis difference may have been multifactorial because cases also had statistically

114 McNabb and Bui

significant more episodes of pneumonia in the past year (P 001) exposureto β-lactam therapy in the past 3 months (P 00008) recent hospitalizations(P 00038) and nosocomial pneumonia (P 00032) and were initially criti-cally ill (P 00030) These factors provide evidence that cases with penicillin-nonsensitive strains had more comorbidities When the authors examined theirdata they reported that of the 19 cases that received a β-lactam as treatment 11recovered Furthermore all eight deaths occurred in patients who were initiallycritically ill suggesting that this parameter not the choice of drug therapy ac-counted for mortality differences Two strains with a higher MIC (4 and 8 microgmL) resulted in therapeutic failures providing support that current breakpointsneed to be revised upward to correlate with clinical outcomes The authors con-cluded that penicillin-resistant strains with MIC 2 microgmL responded well toβ-lactam therapy if patients were not critically ill at the initial presentation

As a followup the same group performed a 10-year prospective study[106] It was shown that mortality was higher (38 vs 24 P 0001) whena penicillin-resistant S pneumoniae was isolated As described previously whenall other factors relating to severity were included no statistical significance wasfound (P 032) The mortality resulting from the administration of penicillinG ampicillin ceftriaxone or cefotaxime was in the range of 19ndash25 regardlessof the susceptibility patterns or agent used These data suggest that the use ofcephalosporin or high dose intravenous penicillin may be effective for treatmenteven when the MIC of penicillin predicts resistance (2 microgmL)

These studies have since been substantiated by the work of other investiga-tors [107108] Gress et al [107] compared the outcomes of intermediate-resistantand susceptible S pneumoniae and found no difference in length of stay (P 096) fever (P 074) or mortality (P 015) The treatment in this study waspenicillin or ampicillin and the conclusion was that both agents provided ade-quate serum and tissue concentrations to effectively treat organisms that had anMIC of 1 microgmL Cabellos et al [108] confirmed the usefulness of procainepenicillin in the treatment of pneumococcal pneumonia Fifteen of 16 patientswere cured and the only failure occurred in a patient who had a S pneumoniaewith penicillin MIC of 4 microgmL The findings were supported with pharmacoki-netic and serum bactericidal activity data

Friedland [109] prospectively studied children with pneumococcal pneu-monia and found that after 3 days of therapy clinical improvement was notedin 70 (1217) of patients with penicillin-resistant strains compared to 62 (2845) of patients with penicillin-susceptible strains [109] At the day 7 evaluation88 (1517) of patients with a penicillin-resistant strain had improved along with93 (4245) in whom a penicillin-susceptible S pneumoniae was isolated Twodeaths occurred in each group and all were treated with intravenous therapyEvery patient treated with oral β-lactam therapy improved regardless of the peni-cillin susceptibility profile These data show poor correlation between penicillin


resistance (2 microgmL) and clinical outcome for non-CNS infections caused byS pneumoniae

Although resistance data for pneumococcal pneumonia do not correlatewell with clinical outcomes studies in acute otitis media (AOM) have producedmore predictable outcomes In a comparative study utilizing either cefuroximeaxetil or cefaclor for the treatment of AOM Dagan et al [110] found that cefuro-xime axetil consistently produced better outcomes These data showed that in-creasing penicillin resistance correlated with statistically significant (P 0001)bacteriological failures despite cephalosporin therapy The penicillin MIC rangesof 01 0125ndash025 and 038ndash1 microgmL resulted in overall failure rates of 621 and 64 respectively When further stratified to the drug regimens thefailure rate of cefuroxime axetil was 9 8 and 50 respectively In thesesame ranges the cefaclor failure rate was 4 43 and 80 The escalatingfailure rate in these patients probably resulted from poor antibiotic penetrationinto the middle ear fluid because the pharmacodynamics was not optimized withthese dosage regimens

Gehanno et al [111] also reported the diminished activity of cefuroximeaxetil as the penicillin MIC increased When 84 children with S pneumoniae weretreated with 30 mgkg of cefuroxime suspension for 8 days a clinical success rateof 86 (7284) was observed for the penicillin MIC range of 0015 to 4 microgmL The clinical success rate was 92 (3942) for penicillin-susceptible strains90 (910) for penicillin-intermediate strains and 75 (2432) for penicillin-resistant strains The conclusion was that cefuroxime axetil was clinically effec-tive for AOM but may not be the drug of choice when resistant strains emergedue to the higher failure rate

Craig and Andes [112] had predicted this scenario when they evaluatedthe pharmacokinetics and antimicrobial effects of these agents in AOM Againstpenicillin-intermediate and -resistant S pneumoniae failure was expected withcefaclor because the usual dosage regimen did not produce any appreciable timeabove the minimal inhibitory concentration (T MIC) that is required of β-lactams for clinical success Cefuroxime was more likely to produce a successfuloutcome because the T MIC against penicillin-intermediate S pneumoniaeranged from 33 to 53 whereas the T MIC for penicillin-resistant strainswas 0ndash23 The T MIC can be thought of as a surrogate marker for resistancebecause as the MIC increases the T MIC is reduced predicting failure In theanalysis of AOM an inverse correlation was observed between resistant strainsand clinical outcomes

The previous examples do not provide a strong correlation between micro-biological resistance and clinical outcomes The primary reason for these discrep-ancies is that microbiological tests are performed with only bacteria and antibiot-ics whereas clinical outcomes include an additional component of host-specificfactors When the host has a functional immune system or is not critically ill

116 McNabb and Bui

outcomes may be much better than predicted by pharmacodynamic relationshipsalone Conversely a host with comorbidities or situations that compromise thepharmacodynamics can unmask the true effectiveness of an antibiotic Lindenet al [113] reported that the poor outcomes between vancomycin-resistant and-susceptible Enterococcus faecium (VREF vs VSEF) were multifactorial withthe antibiotic accounting for a partial effect The effect of reduced antibioticsusceptibility also increased the number of adverse events for patients infectedwith Pseudomonas aeruginosandashresistant isolates [114] These patients simulta-neously presented with additional problems that may have added to their pooroutcomes The in vitro reporting of bacterial resistance should be used as a guidebut cannot serve as the sole determinant of therapeutic decision making becauseit is the triad of antibiotic host and bacteria that determines outcomes

8 CONCLUSION

β-Lactam antibiotics are time-dependent agents that display bactericidal activitywithin the therapeutically achievable dosages Because most agents in this drugclass have short half-lives it is often necessary to administer frequent doses or usenovel dosing strategies to optimize their pharmacodynamic and pharmacokineticproperties The data gathered over the past half century using in vitro animaland human studies clearly show their usefulness in the treatment of numerousinfections when factors such as tissue penetration postantibiotic effect and pro-tein binding are considered

REFERENCES

1 A Fleming On the antibacterial action of cultures of a penicillium with specialreference to their use in the isolation of B influenzae Br J Exp Pathol 192910226

2 H Eagle R Fleischman AD Musselman Effect of schedule of administration ontherapuetic efficacy of penicillin Importance of aggregate time penicillin remainsat effectively bactericidal levels Am J Med 19509280ndash299

3 PM Shah W Junghanns W Stille Dosis-Wirkuns-Beziehung der Bakterizidie beiE coli K pneumoniae und Staphylococcus aureus Dent Med Wochenschr 1976101325ndash328

4 H Eagle R Fleischman AD Musselman Effective concentrations of penicillin invitro and in vivo for streptococci pneumococci and Treponema pallidum J Bacte-riol 195059625ndash43

5 H Eagle R Fleischman M Levy On duration of penicillin action in relation to itsconcentration in serum J Lab Clin Med 195341122ndash132

6 H Eagle HJ Magnuson R Fleischman Effect of method of administration on thera-


peutic efficacy of sodium penicillin in experimental syphilis Bull Johns HopkinsHosp 194679168ndash89

7 H Eagle AD Musselman The rate of bactericidal action of penicillin in vitro asfunction of its concentration and its paradoxically reduced activity at high concen-trations against certain organisms J Exp Med 19488899ndash131

8 WMM Kirby Bacteriostatic and lytic actions of penicillin on sensitive and resistantstaphylococci J Clin Invest 194524165ndash169

9 LR Griffiths HT Green Paradoxical effect of penicillin in-vivo (letter) J Antimi-crob Chemother 198515507ndash508

10 RH George A Dyas Paradoxical effect of penicillin in vivo (letter) J AntimicrobChemother 198717684ndash685

11 H Eagle R Fleischman M Levy lsquolsquoContinuousrsquorsquo vs lsquolsquodiscontinuousrsquorsquo therapy withpenicillin N Engl J Med 1953248481ndash488

12 RF Parker S Luse Action of penicillin on Staphylococcus Further observationsof effect of short exposure J Bacteriol 19485675ndash81

13 RF Parker HC Marsh Action of penicillin on staphylococcus J Bacteriol 194651181ndash186

14 JW Bigger Bactericidal action of penicillin on Staphylococcus pyogenes Irish JMed Sci 1944228585ndash595

15 E Jawetz Dynamics of the action of penicillin in experimental animals Arch InternMed 1946771

16 LH Schmidt A Walley RD Larson The influence of the dosage regimen on thetherapeutic activity of penicillin G J Pharmacol Exp Ther 194996258ndash268

17 LH Schmidt A Walley The influence of the dosage regimen on the therapeuticeffectiveness of penicillin G in experimental lobar pneumonia J Pharmacol ExpTher 1951103479ndash488

18 H Eagle R Fleischman AD Musselman Bactericidal action of penicillin in vivoParticipation of host and slow recovery of surviving organisms Ann Intern Med195033544ndash571

19 H Eagle AD Musselman The slow recovery of bacteria from the toxic effects ofpenicillin J Bacteriol 194958475ndash490

20 GL Drusano Role of pharmacokinetics in the outcome of infections AntimicrobAgents Chemother 198832289ndash297

21 GR Donowitz GL Mandell Beta-lactam antibiotics (part 1) N Engl J Med 1988318419ndash426


23 M Fassbender H Lode T Schaberg K Borner P Koeppe Pharmacokinetics ofnew oral cephalosporins including a new carbacephem Clin Infect Dis 199316646ndash653

24 A Kumar MB Hay GA Maier JW Dyke Post-antibiotic effect of ceftazidimeciprofloxacin imipenem piperacillin and tobramycin for Pseudomonas cepacia JAntimicrob Chemother 199230597ndash602

25 H Hanberger LE NilssonM Nilsson R Maller Post-antibiotic effect of β-lactamantibiotics on gram-negative bacteria in relation to morphology initial killing andMIC Eur J Clin Microbiol Infect Dis 199110927ndash934

118 McNabb and Bui

26 H Hanberger E Svensson LE Nilsson M Nilsson Pharmacodynamic effects ofmeropenem on gram-negative bacteria Eur J Clin Microbiol Infect Dis 199514383ndash390

27 PJ McDonald WA Craig CM Kunin Persistent effect of antibiotics on Staphylo-coccus aureus after exposure for limited periods of time J Infect Dis 1977135217ndash223

28 RW Bundtzen AU Gerber DL Cohn WA Craig Postantibiotic suppression ofbacterial growth Rev Infect Dis 1981328ndash37

29 F Baquero E Culebras C Patron JC Perez-Diaz JC Medrano MF Vicente Postan-tibiotic effect of imipenem of gram-positive and gram-negative micro-organismsJ Antimicrob Chemother 198618(suppl E)47ndash59

30 H Erlendsdottir S Gudmundsson The post-antibiotic effect of imipenem and peni-cillin-binding protein 2 J Antimicrob Chemother 199230231ndash232

31 HL Nadler DH Pitkin W Sheikh The postantibiotic effect of meropenem andimipenem on selected bacteria J Antimicrob Chemother 198924(suppl A)225ndash231

32 CI Bustamante GL Drusano BA Tatem HC Standiford Postantibiotic effect ofimipenem on Pseudomonas aeruginosa Antimicrob Agents Chemother 198426678ndash682

33 S Gudmundsson B Vogelman WA Craig The in-vivo postantibiotic effect of imi-penem and other new antimicrobials J Antimicrob Chemother 198618(suppl E)67ndash73

34 O Cars I Odenholt-Tornqvist The post-antibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 199331(suppl D)159ndash166

35 PJ McDonald BL Wetherall H Pruul Postantibiotic leukocyte enhancement in-creased susceptibility of bacteria pretreated with antibiotics to activity of leuko-cytes Rev Infect Dis 1981338ndash44

36 B Vogelman S Gudmundsson J Turnidge J Leggett WA Craig In vivo postantibi-otic effect in a thigh infection in neutropenic mice J Infect Dis 1988157287ndash298

37 WA Craig Post-antibiotic effects in experimental infection models Relationshipto in vitro phenomena and to treatment of infections in man J Antimicrob Chemo-ther 199331(suppl D)149ndash158

38 S Gudmundsson S Einarsson H Erlendsdottir J Moffat W Bayer WA Craig Thepost-antibiotic effect of antimicrobial combinations in a neutropenic murine thighinfection model J Antimicrob Chemother 199331(suppl D)177ndash191

39 R Wise Protein binding of β-lactams The effects on activity and pharmacologyparticularly tissue penetration I J Antimicrob Chemother 1983121ndash18

40 DN Gerding WH Hall The penetration of antibiotics into peritoneal fluid BullNY Acad Med 1975511016ndash1019

41 DJ Merrikin J Briant GN Rolinson Effect of protein binding on antibiotic activityin vivo J Antimicrob Chemother 198311233ndash238

42 R Wise AP Gillett B Cadge SR Durham S Baker The influence of protein bindingupon tissue fluid levels of six β-lactam antibiotics J Infect Dis 198014277ndash82

43 JE Leggett WA Craig Enhancing effect of serum ultrafiltrate on the activity of


cephalosporins against gram-negative bacilli Antimicrob Agents Chemother 19893335ndash40

44 HF Chambers J Mills TA Drake MA Sande Failure of a once-daily regimen ofcefonicid for treatment of endocarditis due to Staphylococcus aureus Rev InfectDis 19846S870ndash874

45 WA Craig SC Ebert Killing and regrowth of bacteria in vivo A review ScandJ Infect Dis Suppl 19916463ndash70

46 AU Gerber H-P Brugger C Fell T Stritzko B Stadler Antibiotic therapy of infec-tions due to Pseudomonas aeruginosa in normal and granulocytopenic mice Com-parison of murine and human pharmacokinetics J Infect Dis 198615390ndash97

47 B Vogelman S Gudmundsson J Leggett J Turnidge S Ebert WA Craig Correla-tion of antimicrobial pharmacokinetic parameters with therapeutic efficacy in ananimal model J Infect Dis 1988158831ndash847

48 N Frimodt-Moslashller MW Bentzon VF Thomsen Experimental infection with Strep-tococcus pneumoniae in mice Correlation of in vitro activity and pharmacokineticparameters with in vivo effect for 14 cephalosporins J Infect Dis 1986154511ndash517

49 R Roosendaal IAJM Bakker-Woudenbert MVDBV Raffe MF Michel Continu-ous versus intermittent administration of ceftazidime in experimental Klebsiellapneumoniae pneumonia in normal and leukopenic rats Antimicrob Agents Chemo-ther 198630403ndash408

50 JW Mouton JG den Hollander Killing of Pseudomonas aeruginosa during continu-ous and intermittent infusion of ceftazidime in an in vitro pharmaocokinetic modelAntimicrob Agents Chemother 199438931ndash936

51 DM Cappelletty SL Kang SM Palmer MJ Rybak Pharmacodynamics of ceftazi-dime administered as continuous infusion or intermittent bolus alone and in combi-nation with single daily-dose amikacin against Pseudomonas aeruginosa in an invitro infection model Antimicrob Agents Chemother 1995331797ndash1801

52 JJ Mordenti R Quintiliani CH Nightingale Combination antibiotic therapy Com-parison of constant infusion and intermittent bolus dosing in an experimental animalmodel J Antimicrob Chemother 198515313ndash321

53 DH Livingston MT Wang Continuous infusion of cefazolin is superior to intermit-tent dosing in decreasing infection after hemorrhagic shock Am J Surg 1993165203ndash207

54 AU Gerber Impact of the antibiotic dosage schedule on efficacy in experimentalsoft tissue infections Scand J Infect Dis 1991suppl 74147ndash154

55 IAJM Bakker-Woudenberg JC van den Berg P Fontijne MF Michel Efficacyof continuous versus intermittent administration of penicillin G in Streptococcuspneumoniae pneumonia in normal and immunodeficient rats Eur J Clin Microbiol19843131ndash135

56 K Totsuka K Shimizu J Leggett B Vogelman WA Craig Correlation betweenthe pharmacokinetic parameters and the efficacy of combination therapy with anaminoglycoside and a β-lactam In Y Ueda ed Proceedings of the InternationalSymposium on Netilmicin Professional Postgraduate Services Japan 198541ndash48

57 R Roosendaal IAJM Bakker-Woudenberg M van den Berghe-van Raffe MF Mi-

120 McNabb and Bui

chel Continuous versus intermittent administration of ceftazidime in experimentalKlebsiella pneumoniae pneumonia in normal and leukopenic rats AntimicrobAgents Chemother 198630403ndash408

58 DP Nicolau JC McNabb MK Lacy J Li R Quintiliani CH Nightingale Pharma-cokinetics and pharmacodynamics of continuous and intermittent ceftazidime dur-ing the treatment of nosocomial pneumonia (in press)

59 DP Nicolau CH Nightingale MA Banevicius Q Fu R Quintiliani Serum bacteri-cidal activity of ceftazidime Continuous infusion versus intermittent injectionsAntimicrob Agents Chemother 19964061ndash64

60 S Daenen Z Erjavec DRA Uges HG de Vries-Hospers P de Jonge MR HalieContinuous infusion of ceftazidime in febrile neutropenic patients with acute my-eloid leukemia Eur J Clin Microbiol Infect Dis 199514188ndash192

61 AS Benko DM Cappelletty JA Kruse MJ Rybak Continuous infusion versus in-termittent administration of ceftazidime in critically ill patients with suspectedgram-negative infections Antimicrob Agents Chemother 199640691ndash695

62 H Lagast F Meunier-Carpenter J Klastersky Treatment of gram-negative bacillarysepticemia with cefoperazone Eur J Clin Microbiol 19832554ndash558

63 GP Bodey SJ Ketchel V Rodriguez A randomized study of carbenicillin pluscefamandole or tobramycin in the treatment of febrile episodes in cancer patientsAm J Med 197967608ndash611

64 A Brewin L Arango WK Hadley JF Murray High-dose penicillin therapy andpneumococcal pneumonia JAMA 1974230409ndash413

65 JA Zeisler JD McCarthy WA Richelieu MB Nichol Cefuroxime by continuousinfusion A new standard of care Infect Med 19921154ndash60

66 PG Ambrose R Quintiliani CH Nightingale DP Nicolau Continuous vs intermit-tent infusion of cefuroxime for the treatment of community-acquired pneumoniaInfect Dis Clin Prac 19987463ndash470

67 DP Nicolau J McNabb MK Lacy J Li R Quintiliani CH Nightingale Pharmaco-kinetics and pharmacodynamics of continuous and intermittent ceftazidime duringthe treatment of nosocomial pneumonia Clin Drug Invest 1999in press

68 G Woodnutt V Berry L Mizen Effect of protein binding on penetration of β-lactams into rabbit peripheral lymph Antimicrob Agents Chemother 1995392678ndash2683

69 DM Ryan B Hodges GR Spencer SM Harding Simultaneous comparison of threemethods for assessing ceftazidime penetration into extravascular fluid AntimicrobAgents Chemother 198222995ndash998

70 T Kalager A Digranes T Bergan CO Solberg The pharmacokinetics of ceftriax-one in serum skin blister and thread fluid J Antimicrob Chemother 198413479ndash485

71 R Wise IA Donovan MR Lockley J Drumm JM Andrews The pharmacokineticsand tissue penetration of imipenem J Antimicrob Chemother 198618(suppl E)93ndash101

72 JW Mouton MF Michel Pharmacokinetics of meropenem in serum and suctionblister fluid during continuous and intermittent infusion J Antimicrob Chemother199128911ndash918

73 H Koga High-performance liquid chromatography measurement of antimicrobial


concentrations in polymorphonuclear leukocytes Antimicrob Agents Chemother1987311904ndash1908

74 WL Hand N King-Thompson JW Holman Entry of roxithromycin (RU 965) imi-penem cefotaxime trimethoprim and metronidazole into human polymorphonu-clear leukocytes Antimicrob Agents Chemother 1987311553 -1557

75 A Nolting TD Costa R Vistelle KH Rand H Derendorf Determination of freeextracellular concentrations of piperacillin by microdialysis J Pharm Sci 199685369-372

76 A Kovar TD Costa H Derendorf Comparison of plasma and free tissue levels ofceftriaxone in rats by microdialysis J Pharm Sci 19978652ndash56

77 L Granero M Santiago J Cano A Machado JE Peris Analysis of ceftriaxone andceftazidime distribution in cerebrospinal fluid of and cerebral extracellular spacein awake rats by in vivo microdialysis Antimicrob Agents Chemother 1995392728ndash2731

78 M Muller O Haag T Burgdorff A Georgopoulos W Weninger B Jansen G Sta-nek H Pehamberger E Agneter HG Eichler Characterization of peripheral-compartment kinetics of antibiotics by in vivo microdialysis in humans AntimicrobAgents Chemother 1996402703ndash2709

79 RA Fishman Blood-brain and CSF barriers to penicillin and related organic acidsArch Neurol 196615113ndash124

80 LJ Strausbaugh CR Bodem PR Laun Penetration of aztreonam into cerebrospinalfluid and brain of noninfected rabbits and rabbits with experimental meningitiscaused by Pseudomonas aeruginosa Antimicrob Agents Chemother 198630701ndash704

81 E Marmo L Coppola R Pempinello G di Nicuolo E Lampa Levels of amoxicillinin the liquor during continuous intravenous administration Chemotherapy 198228171ndash175

82 R Yogev WE Schultz SB Rosenman Pentrance of nafcillin into human ventricularfluid Correlation with ventricular pleocytosis and glucose levels AntimicrobAgents Chemother 198119545ndash548

83 DT Mullaney JF John Cefotaxime therapy Evaluation of its effect on bacterialmeningitis CSF drug levels and bactericidal activity Arch Intern Med 19831431705ndash1708

84 GG Zhanel JA Karlowsky RJ Davidson DJ Hoban Effect of pooled human cere-brospinal fluid on the postantibiotic effects of cefotaxime ciprofloxacin and genta-micin against Escherichia coli Antimicrob Agents Chemother 1992361136ndash1139

85 JA Karlowsky GG Zhanel RJ Davidson SR Zieroth DJ Hoban In vitro postantibi-otic effects following multiple exposures of cefotaxime ciprofloxacin and genta-micin against Escherichia coli in pooled human cerebrospinal fluid and Mueller-Hinton broth Antimicrob Agents Chemother 1993371154ndash1157

86 MA Sande OM Korzeniowski GM Allegro RO Brennan O Zak WM ScheldIntermittent or continuous therapy of experimental meningitis due to Streptococcuspneumoniae in rabbits Preliminary observations on the postantibiotic effect in vivoRev Infect Dis 1981398ndash109

87 MG Tauber O Zak WM Scheld B Hengstler MA Sande The postantibiotic effect

122 McNabb and Bui

in the treatment of experimental meningitis caused by Streptococcus pneumoniaein rabbits J Infect Dis 1984149575ndash583

88 MG Tauber S Kunz O Zak MA Sande Influence of antibiotic dose dosing inter-val and duration of therapy on outcome in experimental pneumococcal meningitisin rabbits Antimicrob Agents Chemother 198933418ndash423

89 JM Decazes JD Ernst MA Sande Correlation of in vitro time-kill curves andkinetics of bacterial killing in cerebrospinal fluid during ceftriaxone therapy of ex-perimental Escherichia coli meningitis Antimicrob Agents Chemother 198324463ndash467

90 I Lustar GH McCracken Jr IR Friedland Antibiotic pharmacodynamics in cere-brospinal fluid Clin Infect Dis 1998271117ndash1129

91 MG Tauber CA Doroshow CJ Hackbarth MG Rusnak TA Drake MA SandeAntibacterial activity of β-lactam antibiotics in experimental meningitis due toStreptococcus pneumoniae J Infect Dis 1984149568ndash574

92 I Lutsar A Ahmed IR Friedland M Trujillo L Wubbel K Olsen GH McCrackenJr Pharmacodynamics and bactericidal activity of ceftriaxone therapy in experi-mental cephalosporin-resistant pneumococcal meningitis Antimicrob Agents Che-mother 1997412414ndash2417

93 I Lustar A Ahmed IR Friedland M Trujillo L Wubbel K Olsen GH McCrackenJr Pharmacodynamics and bactericidal activity of ceftriaxone therapy in experi-mental cephalosporin-resistant pneumococcal meningitis Antimicrob Agents Che-mother 1997412414ndash2417

94 MR Jacobs SC Aronoff S Johenning DM Shlaes S Yamabe Comparative activi-ties of the β-lactamase inhibitors YTR 830 clavulanate and sulbactam combinedwith ampicillin and broad-spectrum penicillins against defined β-lactamase-produc-ing aerobic gram-negative bacilli Antimicrob Agents Chemother 198629980ndash985

95 EE Stobberingh In vitro effect of YTR 830 (tazobactam) in plasmid and chromo-somally mediated β-lactamases Chemotherapy 199036209ndash214

96 PD Lister AM Prevan CC Sanders Importance of β-lactamase inhibitor pharmaco-kinetics in the pharmacodynamics of inhibitor-drug combinations Studies with pip-eracillin-tazobactam and piperacillin-sulbactam Antimicrob Agents Chemother199741721ndash727

97 M Alexov PD Lister CC Sanders Efficacy of ampicillin-sulbactam is not depen-dent upon maintenance of a critical ratio between components Sulbactam pharma-cokinetics in pharmacodynamic interactions Antimicrob Agents Chemother 1996402468ndash2477

98 K Bush C Macalintal BA Rasmussen VJ Lee Y Yang Kinetic interactions oftazobactam with β-lactamases from all major structural classes Antimicrob AgentsChemother 199337851ndash858

99 KS Thomson DA Weber CC Sanders WE Sanders Jr β-lactamase productionin members of the family Enterobacteriaceae and resistance to β-lactam-enzymeinhibitor combinations Antimicrob Agents Chemother 199034622ndash627

100 DM Livermore Determinants of the activity of β-lactamase inhibitor concentra-tions J Antimicrob Chemother 199331(suppl A)9ndash21

101 RN Jones MN Dudley Microbiologic and pharmacodynamic principles applied


to the antimicrobial susceptibility testing of ampicillinsulbactam Analysis of thecorrelations between in vitro test results and clinical response Diagn MicrobiolInfect Dis 19971595ndash18

102 AB Maderazo DH Gilbert MN Dudley Post-exposure inhibition of TEM-3 β-lactamase by high concentrations of tazobactam In Program and Abstracts of the36th Interscience Conference on Antimicrobial Agents and Chemotherapy SanFrancisco 1996 Am Soc Microbiol Washington DC Abstract A95 p 19

103 CE Thorburn J Molesworth The post β-lactamase inhibitor effect A novel aspectof the activity of clavulanic acid in antibacterial tests In Program Abstracts of32nd Interscience Conference on Antimicrobial Agents and ChemotherapeuticsAbstract 540

104 AH Strayer DH Gilbert P Pivarnik AA Medeiros SH Zinner MN Dudley Phar-macodynamics of piperacillin alone and in combination with tazobactam againstpiperacillin-resistant and -susceptible organisms in an in vitro model of infectionAntimicrob Agents Chemother 1994382351ndash2356

105 R Pallares F Gudiol J Linares J Ariza G Rufi L Murgui J Dorga PF ViladrichRisk factors and response to antibiotic therapy in adults with bacteremic pneumoniacaused by penicillin-resistant pneumococci N Engl J Med 198731718ndash22

106 R Pallares J Linares M Vadillo C Cabellos F Manresa PF Viladrich R MartinF Gudiol Resistance to penicillin and cephalosporin and mortality from severepneumococcal pneumonia in Barcelona Spain N Engl J Med 1995333474ndash480

107 TW Gress KW Yingling RJ Stanek MA Mufson Infection with Streptococcuspneumoniae moderately resistant to penicillin does not alter clinical outcome InfectDis Clin Pract 19965435ndash439

108 C Cabellos J Ariza B Barreiro F Tubau J Linares R Pallares F Manresa FGudiol Current usefulness of procaine penicillin in the treatment of pneumococcalpneumonia Eur J Clin Microbiol Infect Dis 199817265ndash268

109 IR Friedland Comparison of the response to antimicrobial therapy to penicillin-resistant and penicillin-susceptible pneumococcal disease Ped Infect Dis J 199514885ndash890

110 R Dagan O Abramson E Leibovitz R Lang S Goshen D Greenberg P YagupskyA Leiberman DM Fliss Impaired bacteriologic response to oral cephalosporins inacute otitis media caused by pneumococci with intermediate resistance to penicillinPed Infect Dis J 199615980ndash986

111 P Gehanno G Lenoir P Berche In vivo correlates for Streptococcus pneumoniaepenicillin resistance in acute otitis media Antimicrob Agents Chemother 199539271ndash272

112 WA Craig D Andes Pharmacokinetics and pharmacodynamics of antibiotics inotitis media Pediat Infect Dis J 199615255ndash259

113 PK Linden AW Pasculle R Manez DJ Dramer JJ Fung AD Pinna S KusneDifferences in outcomes for patients with bacteremia due to vancomycin-resistantEnterococcus faecium or vancomycin-susceptible E faecium Clin Infect Dis 199622663ndash670

114 Y Carmeli N Troillet AW Karchmer MH Samore Health and economic outcomesof antibiotic resistance in Pseudomonas aeruginosa Arch Intern Med 19991591127ndash1132

6

Aminoglycoside Pharmacodynamics

Myo-Kyoung Kim and David P Nicolau


1 INTRODUCTION

Aminoglycosides are highly potent broad-spectrum antibiotics that have re-mained an important therapeutic option for the treatment of life-threatening in-fections Since the introduction of this class of agents into clinical practice somefive decades ago the major obstacle to their use is the potential for drug-relatedtoxicity However over the last decade new information concerning the phar-macodynamic profile of these agents has been revealed that leads not only tothe potential for improved antibacterial effectiveness but also to the minimiza-tion of their toxicodynamic profile As a result of our contemporary understand-ing of these principles parenteral dosing techniques for the aminoglycosideshave been modified from the administration of frequent small intermittent dos-ages to once-daily regimens that not only optimize the pharmacodynamic andtoxicodynamic profiles but also substantially reduce the expenditure associatedwith this therapeutic option Application of these new principles together withthe aminoglycosidesrsquo in vitro activity proven clinical effectiveness and syner-gistic potential form the rationale for their continued use in the management ofserious infections

125

126 Kim and Nicolau

2 HISTORY AND MECHANISM OF ACTION

OF AMINOGLYCOSIDES

The aminoglycosides include an important group of natural and semisyntheticcompounds The first parenterally administered aminoglycoside streptomycinwas introduced in 1944 and was followed by a number of other naturally oc-curring compounds including neomycin kanamycin tobramycin gentamicinsisomicin and paromomycin Amikacin and netilmicin are semisynthetic deriva-tives of kanamycin and sisomicin respectively whereas isepamicin is a semisyn-thetic derivative of gentamicin

Like that of many antibiotics (ie macrolides tetracyclines streptogram-ins) the bactericidal activity of the aminoglycosides is thought to be ribosomallymediated Existing data suggest that their antibacterial activity results from inhi-bition of protein biosynthesis by irreversible binding of the aminoglycoside tothe bacterial ribosome The intact bacterial ribosome is a 70S particle that consistsof two subunits (50S and 30S) that are assembled from three species of rRNA(5S 16S and 23S) and 52 ribosomal proteins The smaller 30S ribosomal submitwhich contains the 16S rRNA has been identified as a primary target for amino-glycoside that ultimately induces mistranslation on prokaryotic ribosomes [12]

To reach their cytoplasmic ribosomal target aminoglycosides must initiallycross the outer membrane (in gram-negative organisms) and the cytoplasmicmembrane (in gram-negative and gram-positive bacteria) In gram-negative bac-teria the initial step involves ionic binding of the highly positively charged amino-glycosides to negatively charged phosphates mainly in lipopolysaccharides onthe outer membrane surface uptake across this membrane is likely due a lsquolsquoself-promoted uptakersquorsquo mechanism [34] The cationic aminoglycosides may act bycompetitively displacing the divalent cations in the membrane resulting in theentry of the antibiotic [3] The rapid initial binding of the aminoglycosides tothe cell accounts for the rapid bactericidal activity which appears to increase withincreasing aminoglycoside concentration This characteristic of concentration- ordose-dependent killing explains in part the recent attention given to administeringthe entire dose of the aminoglycoside on a once-daily basis in order to maximizebacterial killing

Aminoglycoside uptake across the cytoplasmic membrane is the result ofits electrostatic binding to the polar heads of phospholipids whereas the drivingforce for aminoglycoside entry is provided by a cellular transmembrane electricalpotential The combination of these effects is characterized by rapid binding to theribosome and an acceleration of aminoglycoside uptake across the cytoplasmicmembrane

Aminoglycosides of the gentamicin kanamycin and neomycin familiesinduce misreading of mRNA codons during translation and also inhibit transloca-tion [5] Streptomycin induces misreading of the genetic code in addition to inhib-

Aminoglycosides 127

iting translational initiation By contrast spectinomycin an agent with only bacte-riostatic activity does not cause translation errors but inhibits translocationThese findings support the notion that translational misreading is at least partlyresponsible for the bactericidal activity characteristic of aminoglycosides [5ndash8]

Although the ribosome has been identified as a primary target for theseagents the precise mechanism by which aminoglycosides exert their bactericidalactivity has remained elusive because these drugs manifest pleiotropic effectson bacterial cells These effects include but are not limited to disruption of theouter membrane irreversible uptake of the antibiotic and blockade of initiationof DNA replication [9]

3 MICROBIOLOGIC SPECTRUM

Although the aminoglycosides are highly potent broad-spectrum antibiotics theirin vitro activity is considered to be most notable against a variety of gram-negative pathogens These pathogens include common clinical isolates of Acinet-obacter spp Citrobacter spp Enterobacter spp Escherichia coli Klebsiellaspp Serratia spp Proteus spp Morganella spp and Pseudomonas aeruginosaHowever although these agents are generally considered active against thesemicrobes substantial differences in antimicrobial potency exist among the vari-ous aminoglycosides For example even though the antimicrobial spectra of gen-tamicin and tobramycin are quite similar tobramycin is generally more active invitro against P aeruginosa whereas gentamicin is more active against Serratia

Although streptomycin has been used extensively for many years theemergence of resistance against Mycobacterium tuberculosis and aerobic gram-negative bacilli and the relatively frequent occurrence of vestibular toxicity com-bined with the availability of less toxic antibiotics have greatly diminished itsclinical utility The introduction of kanamycin provided a broader spectrum ofactivity against gram-negative bacilli including streptomycin-resistant strainsbut it was not active against P aeruginosa As with streptomycin extensive useof kanamycin quickly led to the emergence and widespread dissemination ofkanamycin resistance among Enterobacteriaceae The development of otheragents within the class (ie gentamicin tobramycin netilmicin and amikacin)further expanded the spectrum of antimicrobial activity of this class to covermany kanamycin-resistant strains including P aeruginosa

Although the aminoglycosides are also active against Salmonella spp Shi-gella spp N gonorrhea and Haemophilus influenzae they are not recommendedfor infections caused by these species because of the wide availability of effectiveand less toxic drugs Also although they are generally active against Staphylo-cocci aminoglycosides are not advocated as single agents for infections due tothis genera However an aminoglycoside usually gentamicin is frequently ad-ministered in combination with a cell-wall-active agent to provide synergy in the

128 Kim and Nicolau

treatment of serious infections due to Staphylococci Enterococci and Viridansstreptococci

4 PHARMACOKINETIC CHARACTERIZATION

Although the focus of this chapter is related to the pharmacodynamics of amino-glycosides and their application in the management of systemic infection itshould be noted that although these agents are poorly absorbed after oral adminis-tration the prolonged use of large oral doses in patients with altered renal functionmay result in detectable aminoglycoside serum concentrations and the develop-ment of toxicity In addition although they penetrate poorly through intact skintheir use as a topical antibacterial for large areas of denuded skin (ie thermalinjury) may lead to substantial systemic absorption Similarly the use of amino-glycosides for local irrigation of closed body cavities may result in considerablesystemic accumulation and potential toxicity

As a consequence of poor oral bioavailability the aminoglycosides mustbe given parenterally in order to achieve a consistent serum concentration profileThe intramuscular route is well tolerated and results in essentially complete ab-sorption but intravenous administration is generally preferred because of therapid and predictable serum profile The importance of the rapid and reliableattainment of sufficient peak concentrations will be further discussed in subse-quent sections of this chapter

The aminoglycosides are weakly bound to serum proteins and thereforefreely distribute into the interstitial or extracellular fluid The apparent volumeof distribution of this class of agents is approximately 25 of the total bodyweight which corresponds to the estimated extracellular fluid volume Althoughthe volume of distribution is generally approximated at 025ndash03 Lkg patientswho are malnourished obese or pregnant are in the intensive care unit or haveascites may have substantial alterations in this parameter that require dosage andor schedule modifications to maintain the desired serum profile In general theconcentrations of the aminoglycosides attained in tissue and body fluids are lessthan that obtained in serum with the notable exceptions of the kidney perilymphof the inner ear and urine Approximately 20ndash50 of the serum concentrationcan be achieved in bronchial sputum pleural or synovial fluid and unobstructedbile Although low aminoglycoside bronchial fluid concentrations have been re-ported the administration of large single daily doses versus conventional dosingsubstantially improves drug penetration into this fluid while reducing drug accu-mulation in the renal tissue [10ndash12] Compared with other body sites the penetra-tion into prostate tissue and bone is poor Penetration of aminoglycosides intocerebral spinal fluid in the presence of inflammation or into the fluid of the eye isinadequate and variable therefore direct instillation is often required to providesufficient concentrations at these sites Additionally these agents cross the pla-

Aminoglycosides 129

centa therefore the potential risk to the fetus and mother must be consideredprior to use

The kidneys via glomerular filtration are responsible for essentially allaminoglycoside elimination from the body As a result there is a proportionalrelationship between drug clearance and glomerular filtration rate which is rou-tinely used to assist with aminoglycoside dosage modification [13] In adults andchildren older than 6 months with normal renal function the elimination half-life is approximately 2ndash3 h In premature or low birth weight infants less than1 week old the half-life is 8ndash12 h whereas the half-life decreases to 5 h forneonates whose birth weight exceeds 2 kg As a result of the primary renal elimi-nation it should be expected that substantial increases in the half-life would beobserved in patients with renal dysfunction

5 PHARMACODYNAMIC OVERVIEW

Over the last several decades new data emerged that extended our understandingof the complex interactions that take place among the pathogen drug and hostduring the infection process Much of the focus has been on the influence of drugconcentration on bacterial cell death The pharmacodynamic properties or thecorrelation of drug concentration and the clinical effect (eg bacterial killing)of a specific antibiotic class are therefore an integration of two related areas onebeing microbiological activity and the other pharmacokinetics (refer to Chapter2 for a more complete review of this topic) Distinct pharmacodynamic profilesexist for all antimicrobials because the influence of drug concentration on therate and extent of bactericidal activity differs among the various classes of drugs

The pharmacodynamic profile of the aminoglycosides has been character-ized both in vitro and in vivo Utilization of both static (ie time-kill studies)and dynamic (ie pharmacokinetic modeling) in vitro techniques has providedfundamental information concerning the pharmacodynamic profile of the amino-glycosides These data indicate that a general pharmacodynamic division amongantimicrobials occurs between agents whose rate and extent of bactericidal activ-ity is dependent upon drug concentration (aminoglycosides and fluoroquinolones)and agents such as the β-lactams whose bactericidal activity is independent ofdrug concentration when their concentration exceeds four times the minimuminhibitory concentration (MIC) [14ndash17]

Figure 1 depicts these principles as illustrated with ciprofloxacin tobra-mycin and ticarcillin In this experiment bacteria are exposed to various multiplesof the MIC in vitro and as shown with ticarcillin little difference in the rate ofbactericidal activity is noted when its concentration exceeds 4 times the MICTherefore this type of killing which is characteristic of β-lactams is termedconcentration- or dose-independent bactericidal activity (refer to Chapter 5 fora more complete discussion of β-lactam pharmacodynamics)

130 Kim and Nicolau

FIGURE 1 Influence of drug concentration on the bactericidal activity of tobra-mycin ciprofloxacin and ticarcillin against Pseudomonas aeruginosa (FromRef 15)

By contrast when the same multiples of the MIC are studied with tobra-mycin and ciprofloxacin the number of organisms is seen to decrease more rap-idly with each rising MIC interval Because these agents eliminate bacteria morerapidly when their concentrations are appreciably above the MIC of the organismtheir killing activity is referred to as concentration- or dose-dependent bacteri-cidal activity [15] These in vitro data indicate that optimum bactericidal activityfor the aminoglycosides is achieved when the exposure concentration is approxi-mately 8ndash10 times the MIC [14151819] In addition to maximal bactericidalactivity Blaser et al [20] demonstrated that the peakMIC ratio of 81 is corre-lated with a decrease in the selection and regrowth of resistant subpopulationsoccurring during treatment with netilmicin

Antimicrobial activity in vivo is a complex and multifactorial process Asdescribed elsewhere in this text to be effective an antimicrobial must reach andmaintain adequate concentrations at the target site and interact with the targetsite for a period of time so as to interrupt the normal functions of the cell Invivo this description of the interaction between the pathogen and the drug (iemicrobiological activity) is influenced by the drug disposition or pharmacokinetic

Aminoglycosides 131

profile of the host species As a result of the complex interactions occurringamong the pathogenndashdrugndashhost triad in vivo pharmacodynamic characterizationof compounds is required

Because we are not yet able to measure drug concentrations at the site ofaction (ie ribosome for the aminoglycosides) we commonly employ a microbio-logical parameter (ie the MIC) as the critical value in the interpretation of thesein vivo pharmacodynamic relationships When integrating the microbiologicalactivity and pharmacokinetics several parameters appear to be significant constit-uents of drug efficacy The pharmacokinetic parameters AUC (area under theconcentrationndashtime curve) maximum observed concentration (Cmax or peak) andhalf-life are often integrated with the MIC of the pathogen to produce pharmaco-dynamic parameters such as the AUCMIC peakMIC and the time for whichthe drug concentration remains above the MIC (T MIC) For the aminoglyco-sides the AUCMIC peakMIC and T MIC have all been shown to be pharma-codynamic correlates of efficacy [142021] However it is not surprising thatseveral pharmacodynamic parameters have been related to efficacy with theseagents because all of these parameters are related (Fig 2) Therefore since theamount of drug delivered against the pathogen is proportional to the amount ofdrug delivered to the host (AUC) the AUC is the primary pharmacokinetic

FIGURE 2 Antimicrobial pharmacodynamics integration of microbiologicalpotency and selected pharmacokinetic parameters

132 Kim and Nicolau

parameter associated with efficacy However because the AUC is a product ofconcentration and time under certain conditions the influence of concentrationwill appear to be a predominant factor whereas under a different set of conditionsthe exposure to the drug or the time above the MIC may assume a larger rolein bacterial eradication In the case of the aminoglycosides which display concen-tration-dependent killing and a relatively long PAE the influence of the timeabove the MIC is small compared to the influence of peak concentration As aresult the pharmacodynamic parameter that is believed to best characterize theprofile of the aminoglycosides in vivo is the peakMIC ratio

In support of the concentration-dependent bactericidal activity of theaminoglycosides displayed in in vitro studies and animal models of infectionseveral studies in humans have also demonstrated the importance of achievingsufficient peakMIC ratios as related to defined treatment success Aminoglyco-side concentrations have been associated with treatment success in a three studiesreported by Moore and colleagues [22ndash24] In the first report by this group higherCmax concentrations were associated with improved outcomes in gram-negativepneumonia whereas in the second report higher concentrations were associatedwith improved survival in gram-negative bacteremia [2223] Results of thesestudies suggest the importance of achieving adequate and early aminoglycosideconcentrations in severely ill patients with gram-negative infection Their laststudy published in 1987 evaluated the relationship between the ratio of the peakconcentration to MIC and clinical outcome through data collection from fourrandomized double-blind controlled clinical trials that used gentamicin tobra-mycin or amikacin for the treatment of gram-negative bacterial infections [22]For the purposes of the study the maximal peak concentration (Cmax) was definedas the highest concentration determined during therapy and the mean peak con-centration was calculated as the average of all Cmax values during the course oftreatment The investigators demonstrated that ratios of high maximal and meanpeak aminoglycoside concentrations (85 50 microgmL and 66 39 microgmLrespectively) to MIC were significantly (p 000001 and p 00001 respec-tively) correlated with clinical response Of the 188 patients who had a clinicalresponse to therapy the CmaxMIC average value was 85 50 microgmL whereasthe 48 nonresponders had a ratio of 55 46 microgmL (p 000001) Althoughthese studies used fixed dosing intervals and were not designed to assess the invivo pharmacodynamic parameters and their relationship to outcome the dataprovide the backbone for our commitment to the value of the peakMIC ratio inclinical practice Deziel-Evans et al [25] demonstrated that a 91 cure rate wasobserved in patients with peakMIC ratios greater than 8 whereas only a 125cure rate was observed for patients with ratios of 4 in a retrospective studywith 45 patients In a study by Keating et al [26] response rates of 57 67and 85 were observed in neutropenic patients with mean serum aminoglycosideconcentrationMIC ratios of 1ndash4 4ndash10 and 10 respectively Williams et al

Aminoglycosides 133

[27] have also reported that CmaxMIC ratios correlated significantly with cure in42 patients undergoing amikacin treatment who could be evaluated for clinicaloutcome [27] Additionally Fiala and Chatterjee [28] noted that infection wascured more frequently in patients with severe gram-negative infections whoachieved higher peakMIC ratios The association of high peakMIC ratios withimproved outcomes has also been noted in orthopedic patients receiving genta-micin and recently a similar relationship was noted for 61 febrile neutropenicpatients with hematological malignancies [2930] Other investigators have alsoobserved beneficial correlations between serum concentrations or pharmacody-namic parameters and therapeutic outcomes in patients treated with aminoglyco-sides [31ndash33]

More recently Kashuba et al [3435] reported that achieving an aminogly-coside peakMIC of 10 within 48 h of initiation of therapy for gram-negativepneumonia resulted in a 90 probability of therapeutic response by day 7 oftherapy They also note that aggressive aminoglycoside dosing (initial dose of 7mgkg) followed by individualized pharmacokinetic monitoring should maximizethe rate and extent of response in this patient population

Additionally many once-daily aminoglycoside trials and their meta-analy-sis studies (which will be described in a later section in this chapter) also provedthe importance of the correlation between the peakMIC ratio and clinical out-come

6 POSTANTIBIOTIC EFFECT

The postantibiotic effect (PAE) is defined as the persisting suppressive activityagainst bacterial growth after limited exposure of bacteria to an antibiotic Inorder to measure the in vitro PAE of aminoglycosides after a 1ndash2 h exposureof an antibiotic or a combination antibiotics to bacteria the drug is removed rap-idly by dilution drug inactivation [eg cellulose phosphate powder or tobra-mycin-acetylating enzyme AAC (3)-II and acetyl coenzyme A] or filtration(045 microm pore size filters) After this drug elimination process viable counts(CFUmL) at each time point are required to develop viability curves The invitro PAE is calculated with the equation PAE T C where T is the timerequired for the count of CFUs in the test culture to increase by 1 log10 abovethe count observed immediately after drug removal and C is the time neededfor the count of CFUs in an untreated control culture to increase by 1 log10

above the count observed immediately after the same procedure used on the testculture for drug removal [36]

Like quinolones aminoglycosides represent a antibiotic class that has aclinically meaningful PAE however several factors may affect the presence andduration of the PAE A range of 05ndash75 h has been reported for the PAE ofaminoglycosides [3637] Major factors influencing PAE include organism con-

134 Kim and Nicolau

centration of antibiotic duration of antimicrobial exposure and antimicrobialcombinations Minor factors include size of the inoculum growth phase of theorganism at the time of exposure mechanical shaking of the culture type ofmedium pH and temperature of the medium and the effect of re-exposure [36]

The examples below illustrate how the above factors affect the presenceand duration of the PAE Unlike β-lactam antibiotics with PAEs against onlygram-positive organisms aminoglycosides exhibit a PAE on both gram-positiveand gram-negative organisms [3738] However the PAE duration depends onthe type of bacterium For example the duration of the PAE following exposureof Pseudomonas aeruginosa to gentamicin and tobramycin is 22 h and 21 hrespectively whereas that of Escherichia coli is 18 h and 12 h respectively[36] Additionally it has been shown that there is a positive correlation betweensubsequent dose or concentration of aminoglycosides and duration of the PAE[39ndash42] The maximum concentration to exert the maximal PAE effect of amino-glycosides is difficult to determine because most bacteria are completely andrapidly killed at high drug concentrations In contrast the PAE of penicillin Ggradually increases up to a point of maximal effect at a concentration 8ndash16 timesthe MIC [42ndash44] The same theory also applies to duration of exposure

The duration of PAE also varies depending on concurrently applied antibi-otics when they are tested as a combination therapy The combined effect ofaminoglycosides and cell wall inhibitors on the duration of the PAE was studiedby several researchers [45ndash48] In general these combinations produced additiveeffects (ie similar to the sum of PAEs for individual drugs) or synergistic effects(ie at least 1 h longer than the sum of PAEs for individual drugs) in Staphylococ-cus aureus and various streptococci The effects of antibiotic combinationsagainst gram-negative bacilli were mainly additive or indifferent (ie no differentfrom the longest of the individual PAEs) As an exception the addition of tobra-mycin to rifampin which can achieve prolonged PAEs in gram-negative bacillishowed synergism of the PAE in P aeruginosa E coli and K pneumonia

However there are unavoidable limitations in this in vitro determinationof the PAE duration One major drawback is that bacteria undergo a single expo-sure for a short period of time to a fixed concentration of a testing antimicrobialagent However in a clinical setting the antimicrobial agent should be used multi-ple times In addition it should maintain the concentration above the MIC for arelatively longer time period than that which occurs during PAE testing andthe concentration should decline continuously throughout the dosing intervalKarlowsky et al [4950] demonstrated that multiple exposures of E coli and Paeruginosa to aminoglycosides significantly decreased the duration of PAE alongwith attenuating bacterial killing activity McGrath et al [51] suggested that thereasons for this phenomenon may be adaptive resistance or the selection of drug-resistant variants Li et al [52] demonstrated that P aeruginosa organism ex-posed to constant tobramycin concentrations have a longer PAE than those ex-posed to exponentially decreasing tobramycin concentrations at similar AUCs

Aminoglycosides 135

above the MIC These studies suggest that conventional testing yields an overesti-mate of the PAE in comparison to the PAE presented in a clinical situation withcontinuously changing concentrations

Furthermore the duration of PAE significantly varies depending on thetesting environment important factors influencing the testing environment in-clude inoculum concentration temperature pH oxygen tension and free cation(Ca2 Mg2) content [53ndash57] The other obstacle to applying this in vitro PAEduration to clinical practice is that the PAE does not consider host immunityHowever some effort has been undertaken to include host immunity by usingother terminology such as postantibiotic leukocyte enhancement (PALE) andpostantibiotic sub-MIC effect PALE describes the phenomenon that pathogensin the PAE phase are more susceptible to the antimicrobial effect of human leuko-cytes than non-PAE controls The postantibiotic sub-MIC effect illustrates theadditive effects of PAE and the bactericidal effects when the drug is still presentonly in sub-MIC levels

Despite some of the limitations involved in predicting the exact durationof PAE the general consensus is that PAE is an important factor to be consideredin the development of a drug regimen The precise mechanisms of the PAE arelargely unknown However several hypotheses have been suggested They in-clude limited persistence of antibiotic at the site of action recovery from nonle-thal damage to cell structures and the time required for synthesis of new proteinsor enzymes before growth Drug-induced nonlethal damage due to the irreversiblebinding to bacterial ribosomes represents a feasible mechanism of the PAE ofaminoglycoside [4259] In a study measuring the rate of [3H]-adenosine incorpo-ration Gottfredsson et al [60] showed that DNA synthesis by P aeruginosa afterexposure to tobramycin was markedly affected during the PAE phase Howeverin a study that used cumulative radiolabeled nucleoside precursor uptake in aclinical strain of E coli Barmada et al [61] showed that DNA and RNA synthesisresumed almost immediately after exposure to tobramycin whereas protein syn-thesis did not recover until 4 h later Therefore the duration of PAE producedby aminoglycosides against E coli seems to be better correlated with inhibitionof protein synthesis than with inhibition of DNA or RNA synthesis [61] Evenif the rationale for this difference is unknown it may be due to a discrepancy inthe mechanisms of action of two species Theoretically provided that a certainthreshold of growth suppression to restrain DNA synthesis is attained greateraccumulation or entrapment of intracellular tobramycin in P aeruginosa mayaccount for this disagreement [6061]

Although numerous data are available on the in vitro PAE there is less invivo information Six animal models have been developed to evaluate the in vivoPAE thigh infection in mice pneumonia in mice infected subcutaneous threadsin mice meningitis in rabbits infected tissue cages in rabbits and endocarditisin rats [36] Among these models the mice thigh infection model is commonlyused to evaluate the PAE of aminoglycosides although the pneumonia model is

136 Kim and Nicolau

also adopted for them [62ndash65] The endocarditis rat model has been used toevaluate the in vivo PAEs of aminoglycosides when they are added to penicillinor imipenem [4066] In these models antibiotic is administered to achieve aconcentration that exceeds the MIC during the first 1ndash2 h Next bacterial loadsfrom tissue are counted at various time points and drug concentrations of plasmaare measured simultaneously After graphing the bacterial growth curve in vivoPAE can be calculated by the equation

PAE T C M

where M is the length of time serum concentration exceeds the MIC T is thetime required for the counts of CFU in tissue to increase by 1 log10 above thecount at the time closest to but not less than time M and C is the time requiredfor the counts of CFU in tissue of untreated control to increase by 1 log10 abovethe count at time zero [36]

Major factors affecting the in vivo PAE include the infection site typeof organism type of antimicrobial agent the drug dose simulation of humanpharmacokinetics and the presence of leukocytes [3637] For example the invivo PAEs for 15 clinical isolates of Enterobacteriaceae following administrationof gentamicin (8 mgkg) ranged from 14 to 73 h [67] Like the in vitro PAEhigher doses of drugs are correlated with longer in vivo PAEs [36] The PAEsof single doses of 4 12 and 20 mgkg tobramycin in the thighs of neutropenicmice infected with P aeruginosa were 22 h 48 h and 73 h respectively Gener-ally the combinations of aminoglycoside and β-lactam lengthened the PAE forS aureus and P aeruginosa by 10ndash33 h compared to the longest PAE of theindividual drugs However no difference was observed against E coli and Kpneumoniae [65] The adoption of a different infection model also may influencethe duration of the in vivo PAE PAEs with amikacin against Klebsiella pneumon-iae in the mouse pneumonia model was roughly 15ndash25 times as long as thatobserved in the mouse thigh model at the corresponding dose [65] Furthermoreother environmental conditions also influence the in vivo PAE [67]

The in vivo PAE can be used to incorporate the effect of host immunityin conjunction with the PAE The duration of the in vivo PAE of aminoglycosideswas prolonged 19ndash27-fold by the presence of leukocytes [67] In addition neu-trophils are also proven to prolong the in vivo PAEs for aminoglycosides againsta standard strain of K pneumoniae [68] The other benefit of the in vivo PAEis that the half-life of some antimicrobials can be prolonged to simulate humanpharmacokinetics by inducing transient renal impairment in mice with uranylnitrite The in vivo PAE in the renally impaired mice was approximately 7 hlonger than that observed in normal mice with large doses inducing a similareffect This difference is likely due to sub-MIC levels that persist for a longertime with renal impairment than with normal renal function [36]

Aminoglycosides 137

In spite of the lack of an ideal method to apply the PAE to clinical practicethe PAE has a major impact on antimicrobial dosing regimens For antibioticswith longer PAEs dosing may be less frequent than that of antibiotics with shorterPAEs Therefore PAE may be one of the rationales for the implementation ofonce-daily aminoglycoside dosing

7 RESISTANCE AND SYNERGY

Although this chapter primarily concerns the pharmacodynamic profile of theaminoglycosides and its implications for clinical practice it is important to realizethat the development of antimicrobial resistance is often the rate-limiting stepfor a compoundrsquos clinical utility Not unlike other antimicrobials the aminogly-cosides face similar issues regarding resistance Although this topic is beyondthe scope of this chapter it should be noted that at least three mechanisms conferresistance to the aminoglycosides impaired drug uptake mutations of the ribo-some and enzymatic modification of the drug Intrinsic resistance is often dueto impaired uptake whereas acquired resistance usually results from acquisitionof transposon- and plasmid-encoded modifying enzymes [69] To this end thepharmacodynamic implications regarding resistance are that one should select aregimen that maximizes the rate and extent of killing If this approach is univer-sally endorsed it will likely minimize the development of resistance in vivobecause the pharmacodynamic optimization of aminoglycosides has been shownto have this effect in vitro [2070] Additionally adaptive resistance and refracto-riness to aminoglycosides has been demonstrated in vitro and in a neutropenicmurine model by exposing Pseudomonas aeruginosa to concentrations below orat the MIC of the organism [71ndash73] Exposure to an aminoglycoside without adrug-free period leads to decreased bacterial killing Therefore longer dosingintervals which can be achieved with the pharmacodynamically based once-dailyaminoglycoside dosing approach allow for a drug-free period in which the bacte-ria are not exposed to an aminoglycoside yet they still preserve the antibacterialactivity of these agents after multiple doses

Aminoglycosides exhibit synergistic bactericidal activity when given incombination with cell-wall-active agents such as β-lactams and vancomycin[7475] For example enterococcal endocarditis should be treated with a combi-nation of an aminoglycoside plus a penicillin or vancomycin because by itselfneither agent is sufficiently bactericidal However when combination therapy isadvocated to achieve synergy for gram-negative organisms maximally effectivedoses of both agents should be maintained because synergy does not occur uni-versally for all pathogens to all β-lactamndashaminoglycoside combinations [7476]It should also be noted that combination exposure may also prolong the in vitroand in vivo PAE observed with the aminoglycosides (see Sect 6) although theclinical relevance of this effect is not fully understood

138 Kim and Nicolau

8 TOXICODYNAMICS

Since the introduction of aminoglycosides into clinical practice a variety of ad-verse events have been reported during aminoglycoside therapy however most(eg gastrointestinal) are mild and resolve when the drug is discontinued Theaminoglycosides rarely produce hypersensitivity reactions and despite direct in-jection into the central nervous system and the eye local adverse events (ieseizures hypersensitivity reactions) are generally not observed Although infre-quent in contemporary clinical practice the aminoglycosides have the potentialto cause or exacerbate neuromuscular blockade Despite the concern for increasedrisk with the administration of the high doses routinely used in once-daily dosingprotocols this adverse event has not been observed [7778] However althoughthey are generally well tolerated the major obstacle that has curtailed the use ofaminoglycosides is the potential for ototoxicity and nephrotoxicity

Although ototoxicity has long been recognized as a potential complicationof aminoglycoside therapy questions still remain regarding the full delineationof risk factors and a universally accepted definition As a result of discrepanciesin both the definition of ototoxicity and the sensitivity of testing the reportedincidence of ototoxicity has spanned a wide range (2ndash25) Two distinct formsof ototoxicitymdashauditory and vestibularmdashhave been reported and may occuralone or simultaneously The precise mechanism of injury remains elusive how-ever ototoxicity is believed to result from the destruction of the sensory haircells in the cochlea and the vestibular labyrinth [79]

Auditory toxicity often occurs at frequencies that are higher than that re-quired for conversation and thus patient complaints that usually manifest as tinni-tus or a feeling of fullness in the ear are usually voiced after considerable auditorydamage has already been done [80] Like the progression of auditory loss theinitial symptoms of vestibular toxicity often go unrecognized due to the nonspe-cific nature of its initial presentation (ie nausea vomiting cold sweats nystag-mus vertigo and dizziness) [81] Although considered to be less frequent thanauditory toxicity these vestibular effects are by and large irreversible and there-fore may have a profound impact on the daily function status Owing to the lackof well controlled comparative trials with sufficient power to detect differences inototoxicity among aminoglycosides and the generally poor risk factors analysis itis difficult if not impossible to substantiate that a particular agent may preferen-tially result in one form of ototoxicity rather than another

Although serum concentration data may be useful to ensure an adequatepharmacodynamic profile these data cannot accurately predict the developmentof ototoxicity Recently acquired data suggest that toxicity is related to drug accu-mulation within the ear not peak concentrations which supports the concept ofsaturable transport and reinforces the belief that higher peak concentrationsshould not result in increased ototoxicity [82] For these reasons the once-daily

Aminoglycosides 139

administration techniques may minimize drug accumulation and therefore drug-related toxicity [8384]

Although nephrotoxicity has been reported in more than half of patientsreceiving aminoglycoside therapy the broad range of definitions and the poor riskfactor assessment of the affected patient population often make the true incidencedifficult if not impossible to determine Considered by many to be a noteworthyevent toxicity is nevertheless generally mild and reversible and few patientshave progressive toxicity severe enough to warrant dialysis [85] At present it isthought that this toxicity is due to aminoglycoside accumulation in the lysosomesof the renal proximal tubule cells which results in necrosis of the tubular cellsand the clinical presentation of acute tubular necrosis manifested by nonoliguricrenal failure within a week [86]

Several investigators have reported that advanced age pre-existing renaldysfunction hypovolemia shock liver dysfunction obesity duration of therapyuse of concurrent nephrotoxic agents and elevated peaktrough aminoglycosideconcentrations are risk factors for development of nephrotoxicity [87ndash90] Addi-tionally in the last-cited study [90] multiple logistic regression analysis alsorevealed that trough concentration duration of therapy advanced age leukemiamale gender decreased albumin ascites and concurrent clindamycin vancomy-cin piperacillin or cephalosporins were independent risk factors for nephrotoxic-ity Similar risk factors were identified in patients receiving once-daily aminogly-cosides [91]

Similar to that previously described in the inner ear a saturable aminogly-coside transport system has been used to describe the uptake of drug in the kidneyTherefore less frequent single daily dose administration may minimize accumu-lation and nephrotoxicity [1092] In this regard once-daily regimens have beenreported to lessen the incidence of nephrotoxicity [788493]

Although the risk of these toxicities cannot be completely eliminated rec-ognition of risk factors and the implementation of regimens that minimize drugaccumulation will lead to optimal therapeutic outcomes and minimize toxicity

9 CLINICAL USE AND APPLICATION

OF PHARMACODYNAMICS

The parenteral aminoglycosides particularly gentamicin tobramycin and ami-kacin have long been used empirically for treatment of the febrile neutropenicpatient or of patients with serious nosocomial infection Although aminoglyco-side utilization has generally been declining owing to the introduction of paren-teral fluoroquinolones emergence of fluoroquinolone-resistant P aeruginosa willlikely result in resurgence in clinical use of the aminoglycosides To this pointit is also apparent that the antipseudomonal β-lactams should not be given aloneto treat systemic pseudomonal infections because this organism often develops

140 Kim and Nicolau

resistance under therapy Thus the aminoglycosides play an important role incombination therapy for gram-negative infections

As discussed earlier the aminoglycosides are also commonly used with acell-wall-active agent for synergistic purposes for gram-positive infections Inthis situation gentamicin is frequently administered to provide synergy in thetreatment of serious infections due to Staphylococci Enterococci and Viridansstreptococci

In the current era of aminoglycoside utilization two predominant intrave-nous administration techniques are employed in clinical practice The older ofthe two approaches is the administration of multiple doses usually 17ndash2 mgkg every 8 h for gentamicin and tobramycin whereas amikacin was frequentlydosed using regimens of 5 mgkg every 8 h or 75 mgkg every 12 h (Fig 3)Using this technique maintenance of concentrations within the therapeutic rangefor patients with alterations in elimination or volume of distribution was achievedwith the use of a nomogram or by individualized pharmacokinetic dosing meth-ods based on the patient-specific aminoglycoside disposition Of the nomogram-based methods the scheme of Sarubbi and Hull [94] appears to have gained thewidest acceptance By this method a loading dose of 1ndash2 mgkg gentamicin ortobramycin and of 5ndash75 mgkg amikacin based on ideal body weight was givento adults with renal impairment After the loading dose subsequent doses were

FIGURE 3 Concentrationndashtime profile comparison of () conventional q8h in-termittent dosing versus () the once-daily daily administration technique

Aminoglycosides 141

selected as a percentage of the chosen loading dose according to the desireddosing interval and the estimated creatinine clearance of the patient (Table 1)Alternatively one-half the loading dose may be given at intervals equal to thatof the estimated half-life Although the nomogram approach has been utilizedfrequently the preferred method of dosage adjustment is to individualize theregimen by using the standard pharmacokinetic dosing principles when aminogly-coside concentrations are available [9596]

The second method has been referred to as the once-daily single-daily orextended interval dosing method (Fig 3) Although the potential benefits of thissecond method were not well described until the early 1990s this administrationtechnique has now become the standard of practice in the United States threeof every four hospitals surveyed in 1998 used it [9798] For this reason and thewide availability of tertiary text references concerning the dosing of aminoglyco-sides using the more frequent intermittent approach the remainder of this sectionwill focus on the once-daily dosing methodology

When considering the pharmacodynamic profile of aminoglycosides as de-scribed earlier in this chapter four distinct advantages of using extended dosing

TABLE 1 Selection of Aminoglycoside MaintenanceDosing Using the Method of Sarubbi and Hull

Dose intervalCreatinine clearance Half-life(mLmin) (hs) 8 h 12 h 24 h

90 31 84 mdash mdash80 34 80 91 mdash70 39 76 88 mdash60 45 71 84 mdash50 53 65 79 mdash40 65 57 72 9230 84 48 63 8625 99 43 57 8120 119 37 50 7517 136 33 46 7015 151 31 42 6712 179 27 37 6110 204 24 34 56

7 259 19 28 475 315 16 23 412 468 11 16 300 693 8 11 21

Source Ref 94

142 Kim and Nicolau

intervals are readily apparent [99] As stated previously giving aminoglycosidesas a single daily dose as opposed to conventional strategies provides the opportu-nity to maximize the peak concentrationMIC ratio and the resultant bactericidalactivity (Fig 3) Second this administration technique should minimize drugaccumulation within the inner ear and kidney and therefore minimize the potentialfor toxic effects to these organs Third the PAE may also allow for longer periodsof bacterial suppression during the dosing interval Finally this aminoglycosidedosing approach may prevent the development of bacterial resistance

Once-daily aminoglycoside therapy has been evaluated in several largeclinical studies with a total study population of 100 or more patients [100ndash109]Compared with multidose aminoglycoside regimens the once-daily regimen wasshown to be as efficacious as or superior to traditional dosing for the treatmentof a wide variety of infections Toxicity evaluations showed that there were nodifferences between the two dosing methods for either nephrotoxicity or ototoxi-city These toxicity data have been supported by other recent observations frominvestigators in Detroit [8493] In addition clinical experience at our own institu-tion in a large patient population who received 7 mgkg of either gentamicin ortobramycin indicates a reduced potential for nephrotoxicity [78]

Studies have also been conducted in pediatrics [110111] and pregnant pop-ulations [112] for determination of serum concentrations as well as clinical effi-cacy Several recently published meta-analyses evaluating once-daily dosing withstandard dosing regimens also demonstrate that increased bacterial killing andtrends for decreased toxicity are actually borne out in clinical practice when theextended interval dosing is used [113ndash121]

At present the strategy for once-daily dosing has not been consistent inthe literature as doses for gentamicin tobramycin and netilmicin have rangedfrom 3 to 7 mgkg whereas the usual amikacin dose is 15ndash20 mgkg Dosingregimens that use doses of less than 6 mgkg for gentamicin tobramycin andnetilmicin have arrived at the dose by converting the conventional mgkg doseto a dose that is then administered once daily At present there appear to be fourcommonly advocated methods for the once-daily administration of aminoglyco-sides Although these approaches differ somewhat with regard to dose andorinterval all reflect the need for dosage modification in the patient with renaldisease As of yet no method has been shown to be superior to any of the othersConcerns about the extended intervals and possible risk of increased toxicity inpatients with reduced drug clearance should be mentioned but they should beno greater than those encountered with conventional dosing based on our currentunderstanding of aminoglycoside-induced toxicity

The first method of once-daily dosage determination was proposed andimplemented on the basis of the pharmacokinetic and pharmacodynamic profilesof these agents This method which was developed at our institution is intendedto optimize the peakMIC ratio in the majority of clinical situations by adminis-

Aminoglycosides 143

tering a dose of 7 mgkg of either gentamicin or tobramycin [78] Like conven-tional regimens once-daily protocols require modification for patients with renaldysfunction in order to minimize drug accumulation In the Hartford Hospitalprogram this is accomplished by administering a fixed dose with dosing intervaladjustments for patients with impaired renal function [78] Due to the high peakconcentrations obtained and the drug-free period at the end of the dosing intervalit is no longer necessary to draw standard peak and trough samples rather asingle random blood sample is obtained between 6 and 14 h after the start of theaminoglycoside infusion This serum concentration is used to determine the dos-ing interval based on a nomogram for once-daily dosing (Fig 4) Although Dem-czar et al [122] suggested that the nomogram may be inappropriate for the moni-toring of therapy based on their assessment of aminoglycoside distribution in11 healthy subjects a subsequent population pharmacokinetic analysis using dataderived from more than 300 patients receiving 7 mgkg of tobramycin furthersupports the clinical utility of the original nomogram [123]

As a result of low toxicity the short duration of therapy and the excellentrenal function of most patients criteria have been developed to withhold theinitial random concentration (which is obtained after the first or second dose) inpatients (1) receiving 24 h dosing (2) without concurrently administered nephro-toxic agents (eg amphotericin cyclosporine vancomycin) (3) without exposure

FIGURE 4 Once-daily aminoglycoside nomogram for the assessment of dos-ing interval using a 7 mgkg dose of gentamicin or tobramycin (From Ref 78)

144 Kim and Nicolau

to contrast media (4) neither quadriplegic nor amputee (5) not in the intensivecare unit and (6) less than 60 years of age [78] Even though the initial randomconcentration may be withheld in eligible patients monitoring of the serum creat-inine should continue to occur at 2ndash3 day intervals throughout the course oftherapy For patients who continue on the once-daily regimen for 5 or more daysa random concentration is obtained on the fifth day and weekly thereafter Eventhough an initial random concentration may no longer be necessary in many pa-tients for those experiencing rapidly changing creatinine clearances or those inwhom the creatinine clearance is significantly reduced (ie 30 mLmin) it maybe necessary to obtain several samples to adequately structure the administrationschedule to maximize efficacy and minimize toxicity The 7 mgkg dosage regi-men has also been advocated by other investigators to rapidly obtain sufficientaminoglycoside exposures [34124]

The second method proposed by Gilbert uses a 5 mgkg gentamicin ortobramycin dose in patients without renal dysfunction [99125] If dosage adjust-ment is required to compensate for diminished renal function the dose andordosing interval may be modified to optimize therapy and minimize drug accumu-lation (Table 2) A similar scheme for dosage modification was advocated by

TABLE 2 Suggested Once-Daily Dosage Requirements for Patients withAltered Renal Function

Creatinine clearance Dosage DoseAminoglycoside (mlmin) interval (h) (mgkg)

Gentamicintobramycin 80 24 5070 24 4060 24 4050 24 3540 24 2530 24 2520 48 4010 48 30

Hemodialysisa 48 20Amikacin 80 24 15

70 24 1250 24 7530 24 4020 48 7510 48 40

Hemodialysisa 48 50

a Administered post-hemodialysisSource Adapted from Ref 125

Aminoglycosides 145

Prin et al [126] for patients with renal dysfunction Finally Begg et al [127]suggested two methods to optimize once-daily dosing The first suggested forpatients with normal renal function uses a graphical approach with target AUCvalues The second method for patients with renal dysfunction uses two amino-glycoside serum concentrations and a target AUC value based on the 24 h AUCthat would result with multiple-dose regimens for dosage modifications

Although all the above-noted once-daily methodologies have used fixeddoses subsequent dosage adjustments may be guided by individualized pharma-cokinetic methods similar to that used for the conventional multiple-dose ap-proach On the other hand although individualization of therapy can be accom-plished no data are available to support the assumption that these manipulationswill improve outcomes or minimize toxicity further than the fixed dose methodol-ogies

At the time of once-daily implementation the methodology was introducedinto clinical practice to further optimize the clinical outcomes of patients receiv-ing these agents for serious infections However in addition to meeting this goaland reducing the incidence of drug-induced adverse events this approach hasalso substantially reduced expenditures associated with the initiation of aminogly-coside therapy compared to traditional dosing techniques [128ndash130]

10 SUMMARY

The pharmacodynamic profile of aminoglycosides is maximized when high doseextended interval aminoglycoside therapy is employed The use of this aminogly-coside administration technique has considerable in vitro and in vivo scientificsupport which justifies its wide-scale use within this country The implementa-tion of such programs should maximize the probability of clinical cure and mini-mize toxicity and may help to avoid the development of resistance Althoughsuch dosing is not appropriate for all patients this strategy appears to be useful inthe majority of patients requiring aminoglycoside therapy and can be successfullyemployed as a hospital-wide program

REFERENCES

1 Cundliffe E Antibiotics and prokaryotic ribosomes Action interaction and resis-tance In WE Hill A Dahlberg RA Garrett PB Moore D Schlessinger JR Warnereds Ribosomes Am Soc Microbiol Washington DC 1990479ndash490

2 Davies JE Resistance to aminoglycosides Mechanisms and frequency Rev InfectDis 19835S261ndashS266

3 Hancock REW Aminoglycoside uptake and mode of action With special referenceto streptomycin and gentamicin J Antimicrob Chemother 19818249ndash276

4 Hancock REW Farmer SW Li Z Poole K Interaction of aminoglycosides with

146 Kim and Nicolau

the outer membranes and purified lipopolysaccharide and OmpF porin of Esche-richia coli Antimicrob Agents Chemother 1991351309ndash1314

5 Gale EF Cundliffe E Reynolds PE Richmond MH Waring MJ The MolecularBasis of Antibiotic Action Wiley London 1981

6 Davis BD Tai PC Wallace BJ Complex interactions of antibiotics with the ribo-some In M Nomura A Tissieres P Lengyel eds Ribosomes Cold Spring HarborLaboratory Cold Spring Harbor NY 1974771ndash789

7 Gorini L Streptomycin and misreading of the genetic code In M Nomura A Tis-sieres P Lengyel eds Ribosomes Cold Spring Harbor Laboratory Cold SpringHarbor NY 1974791ndash803

8 Ruusala T Kurland CG Streptomycin preferentially perturbs ribosomal proofread-ing Mol Gen 1984198100ndash104

9 Matsunaga K Yamaki H Nishimura T Tanaka N Inhibition of DNA replicationinitiation by aminoglycoside antibiotics Antimicrob Agents Chemother 198630468ndash474

10 De Broe ME Verbist L Verpooten GA Influence of dosage schedule on renalcortical accumulation of amikacin and tobramycin in man J Antimicrob Chemother199127(suppl C)41ndash47

11 Santre C Georges H Jacquier JM Leroy O Beuscart C Buguin D Beaucaire GAmikacin levels in bronchial secretions of 10 pneumonia patients with respiratorysupport treated once daily versus twice daily Antimicrob Agents Chemother 199539264ndash267

12 Valcke YJ Vogelaers DP Colardyn FA Pauwels RA Penetration of netilmicinin the lower respiratory tract after once-daily dosing Chest 19921011028ndash1032

13 Zarowitz BJ Robert S Peterson EL Prediction of glomerular filtration rate usingaminoglycoside clearance in critically ill medical patients Ann Pharmacother 1992261205ndash1210

14 Begg EJ Peddie BA Chambers ST Boswell DR Comparison of gentamicin dosingregimens using an in-vitro model J Antimicrob Chemother 199229427ndash433

15 Craig WA Ebert SC Killing and regrowth of bacteria in vitro A review ScandJ Infect Dis 1991suppl 7463ndash70

16 Drusano GL Johnson DE Rosen M Standiford HC Pharmacodynamics of a flu-oroquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sep-sis Antimicrob Agents Chemother 199337483ndash490

17 Dudley MN Pharmacodynamics and pharmacokinetics of antibiotics with specialreference to the fluoroquinolones Am J Med 199191(suppl 6A)45Sndash50S

18 Davis BD Mechanism of the bactericidal action of the aminoglycosides MicrobiolRev 198751341ndash350

19 Ebert SC Craig WA Pharmacodynamic properties of antibiotic Application todrug monitoring and dosage regimen design Infect Control Hosp Epidemiol 199011319ndash326

20 Blaser J Stone B Groner M et al Comparative study with enoxacin and netilmicinin a pharmacodynamic model to determine importance of ratio of antibiotic peakconcentration to MIC for bactericidal activity and emergence of resistance Antimi-crob Agents Chemother 1987311054ndash1060

21 Leggett JE Ebert S Fantin B Craig WA Comparative dose-effect relationships

Aminoglycosides 147

at several dosing intervals for β-lactam aminoglycoside and quinolone antibioticsagainst gram-negative bacilli in murine thigh-infection and pneumonitis modelsScan J Infect Dis 199174179ndash184

22 Moore RD Smith CR Lietman PS Association of aminoglycoside plasma levelswith therapeutic outcome in gram-negative pneumonia Am J Med 198477657ndash662

23 Moore RD Smith CR Lietman PS The association of aminoglycoside plasma lev-els with mortality in patients with gram-negative bacteremia J Infect Dis 1984149443ndash448

24 Moore RD Lietman PS Smith CR Clinical response to aminoglycoside therapyImportance of the ratio of peak concentration to minimal inhibitory concentrationJ Infect Dis 198715593ndash99

25 Deziel-Evans L Murphy J Job M Correlation of pharmacokinetic indices withtherapeutic outcomes in patients receiving aminoglycoside Clin Pharm 19865319ndash324

26 Keating MF Bodey GP Valdivieso M Rodriguez V A randomized comparativetrial of three aminoglycosides Comparison of continuous infusions of gentamicinamikacin and sisomicin combined with carbenicillin in the treatment of infectionsin neutropenic patients with malignancies Medicine 197958159ndash170

27 Williams PJ Hull JH Sarubbi FA Rogers JF Wargin WA Factors associatedwith nephrotoxicity and clinical outcome in patients receiving amikacin J ClinPharmacol 19862679ndash86

28 Fiala M Chatterjee SN Antibiotic blood concentrations in patients successfullytreated with tobramycin Postgrad Med 198157548ndash551

29 Berirtzoglou E Golegou S Savvaidis I Bezirtzoglou C Beris A Xenakis T Arelationship between serum gentamicin concentrations and minimal inhibitor con-centration Drugs Exp Clin Res 19962257ndash60

30 Binder L Schiel X Binder C Menke CF Schuttrumpf S Armstrong VW UnterhaltM Erichsen N Hiddemann W Oellerich M Clinical outcome and economic impactof aminoglycoside peak concentrations in febrile immuocompromised patients withhematologic malignancies Clin Chem 199844408ndash414

31 Anderson ET Young LS Hewitt WL Simultaneous antibiotic levels in lsquolsquobreak-throughrsquorsquo gram-negative rod bacteremia Am J Med 197661493ndash497

32 Noone P Parsons TMC Pattison JR Slack RCB Garfield-Davies D Hughes KExperience in monitoring gentamicin therapy during treatment of serious gram-negative sepsis Br Med J 19741477-481

33 Reymann MT Bradac JA Cobbs CG Dismukes WE Correlation of aminoglyco-side dosage with serum concentrations during therapy of serious gram-negativebacillary disease Antimicrob Agents Chemother 197916353ndash361

34 Kashuba ADM Bertino JS Jr Nafziger AN Dosing of aminoglycosides to rapidlyattain pharmacodynamic goals and hasten therapeutic response by using individual-ized pharmacokinetic monitoring of patients with pneumonia caused by gram-negative organism Antimicrob Agents Chemother 1998421842ndash1844

35 Kashuba ADM Nafziger AN Drusano GL Bertino JS Jr Optimizing aminoglyco-side therapy for nosocomial pneumonia caused by gram-negative bacteria Antimi-crob Agents Chemother 1999431623ndash1629

148 Kim and Nicolau

36 Craig W Gunmundsson S Postantibiotic effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams amp Wilkins Baltimore 1987296ndash329

37 Zhanel G Hoban D Harding G The postantibiotic effect a review of in vitro andin vivo data Ann Pharmacother 199125153ndash163

38 Bundtzen R Gerber A Cohn D et al Postantibiotic suppression of bacterialgrowth Rev Infect Dis 1981328ndash37

39 Vogelman B Gudmundsson S Turnidge J et al In vivo postantibiotic effect in athigh infection in neutropenic mice J Infect Dis 1988157287ndash298

40 Hessen M Pitsakis P Levison M Postantibiotic effect of penicillin plus gentamicinversus Enterococcus faecalis in vitro and in vivo Antimicrob Agents Chemother198933608ndash611

41 McGrath B Marchbanks C Gilbert D et al In vitro postantibiotic effect followingexposure to imipenem temafloxacin and tobramycin Antimicrob Agents Chemo-ther 1993371723ndash1725

42 Vogelman B Craig W Postantibiotic effects J Antimicrob Chemother 198515(suppl A)37ndash46

43 Odenholt-Tornqvist I Pharmacodynamics of beta-lactam antibiotics Studies on theparadoxical effect and postantibiotic effects in vitro and in an animal model ScandJ Infect Dis 198958(suppl)1ndash55

44 Craig W Leggett K Totsuka K et al Key pharmacokinetic parameters of antibioticefficacy in experimental animal infections J Drug Dev 19981(suppl 3)7ndash15

45 Dornbusch K Henning C Linden E In-vitro activity of the new penems FCE 22101and FCE 24362 alone or in combination with aminoglycosides against streptococciisolated from patients with endocarditis J Antimicrob Chemother 198923(supplC)109ndash117

46 Fuursted K Comparative killing activity and postantibiotic effect of streptomycincombined with ampicillin ciprofloxacin imipenem piperacillin or vancomycinagainst strains of Streptococcus faecalis and Streptococcus faecium Chemotherapy198834229ndash234

47 Winstanley T Hastings J Penicillin-aminoglycosides synergy and post-antibioticeffect for enterococci J Antimicrob Chemother 198923189ndash199

48 Gudmundsson S Erlendsdottir H Gottfredsson M et al The postantibiotic effectinduced by antimicrobial combinations Scand J Infect Dis 19917480ndash93

49 Karlowsky J Zhanel G Davidson R et al Postantibiotic effect in Pseudomonasaeruginosa following single and multiple aminoglycoside exposures in vitro J Anti-microb Chemother 199433937ndash947

50 Karlowsky J Zhanel G Davidson R et al In vitro postantibiotic effects followingmultiple exposures of cefotaxime ciprofloxacin and gentamicin against Esche-richia coli in pooled human cerebrospinal fluid and Mueller-Hinton broth Antimi-crob Agents Chemother 1993371154ndash1157

51 McGrath B Marchbanks C Gilbert D et al In vitro postantibiotic effect followingrepeated exposure to imipenem temafloxacin and tobramycin Antimicrob AgentsChemother 1993371723ndash1725

52 Li R Zhu Z Lee S et al Antibiotic exposure and its relationship to postantibioticeffect and bactericidal activity Constant versus exponentially decreasing tobra-

Aminoglycosides 149

mycin concentrations against Pseudomonas aeruginosa Antimicrob Agents Che-mother 1997411808ndash1811

53 Rescott D Nix D Holden P et al Comparison of two methods for determiningin vitro postantibiotic effects of three antibiotics on Escherichia coli AntimicrobAgents Chemother 199832450ndash453

54 Fuursted K Postexposure factors influencing the duration of postantibiotic effectSignificance of temperature pH cations and oxygen tension Antimicrob AgentsChemother 1997411693ndash1696

55 Gudmundsson A Erlendsdottir H Gottfredsson M et al The impact of pH andcationic supplementation on in vitro postantibiotic effect Antimicrob Agents Che-mother 1991352617ndash2624

56 Park MK Myers R Marzella L Hyperoxia and prolongation of aminoglycoside-induced postantibiotic effect in Pseudomonas aeruginosa Role of reactive oxygenspecies Antimicrob Agents Chemother 199337120ndash122

57 Hanberger H Nilsson L Maller R et al Pharmacodynamics of daptomycin andvancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated bystudies of initial killing and postantibiotic effect and influence of Ca2 and albuminon these drugs Antimicrob Agents Chemother 1991351710ndash1716

58 Cars O Odenholt-Tornqvist I The postantibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 199331(suppl D)159ndash166

59 Craig W Vogelman B The postantibiotic effect Ann Intern Med 1987106900ndash902

60 Gottfredsson M Erlendsdottir H Gudmundsson et al Different patterns of bacte-rial DNA synthesis during the postantibiotic effect Antimicrob Agents Chemother1995391314ndash1319

61 Barmada S Kohlhepp S Leggett J et al Correlation of tobramycin-induced inhibi-tion of protein synthesis with postantibiotic effect in Escherichia coli AntimicrobAgents Chemother 1993372678ndash2683

62 Minguez F Izquierdo J Caminero M et al In vivo postantibiotic effect of isepa-micin and other aminoglycosides in a thigh infection model in neutropenic miceChemotherapy 199238179ndash184


64 Gudmundsson S Einarsson S Erlendsdottir H The post-antibiotic effect of antimi-crobial combinations in a neutropenic murine thigh infection model J AntimicrobChemother 199331(suppl D)177ndash191

65 Craig W Redington J Ebert S Pharmacodynamics of amikacin in vitro and inmouse thigh and lung infections J Antimicrob Chemother 199127(suppl C)29ndash40

66 Hessen M Pitsakis P Levison M Absence of a postantibiotic effect in experimentalPseudomonas endocarditis treated with imipenem with or without gentamicin JInfect Dis 1998158542ndash548

67 Craig W Post-antibiotic effects in experimental infection model relationship toin-vitro phenomena and to treatment of infection in man J Antimicrob Chemother199331(suppl D)149ndash158

150 Kim and Nicolau

68 Fantin B Craig W Factors affecting duration of in vivo postantibiotic effect foraminoglycosides against gram-negative bacilli Antimicrob Agents Chemother199127829ndash836

69 Mingeot-Leclercq MP Glupczynski Y Tulkens PM Aminoglycosides Activityand resistance Antimicrob Agents Chemother 199943727ndash737

70 Karlowsky JA Zhanel GG Davidson RJ Hoban DJ Once-daily aminoglycosidedosing assessed by MIC reversion time with Pseudomonas aeruginosa AntimicrobAgents Chemother 1994381165ndash1168

71 Daikos GL Jackson GG Lolans V Livermore DM Adaptive resistance to amino-glycoside antibiotics from first-exposure down-regulation J Infect Dis 1990162414ndash420

72 Daikos GL Lolans VT Jackson GG First-exposure adaptive resistance to amino-glycoside antibiotics in vivo with meaning for optimal clinical use AntimicrobAgents Chemother 199135117ndash123

73 Gerber AU Vastola AP Brandel J Craig WA Selection of aminoglycoside-resistant variants of Pseudomonas aeruginosa in an in vivo model J Infect Dis1982146691ndash697

74 Owens RC Jr Banevicius MA Nicolau DP Nightingale CH Quintiliani R In vitrosynergistic activities of tobramycin and selected β-lactams against 75 gram-nega-tive clinical isolates Antimicrob Agents Chemother 1997412586ndash2588

75 Marangos MN Nicolau DP Quintiliani R Nightingale CH Influence of gentamicindosing interval on the efficacy of penicillin containing regimens in experimentalEnterococcus faecalis endocarditis J Antimicrob Chemother 199739519ndash522

76 Hallander HO Donrbusch K Gezelius L Jacobson K Karlsson I Synergism be-tween aminoglycosides and cephalosporins with anti-pseudomonal activity Inter-action index and killing curve method Antimicrob Agents Chemother 198222743ndash752

77 Gilbert DN Once-daily aminoglycoside therapy Antimicrob Agents Chemother199135399ndash405

78 Nicolau DP Freeman CD Belliveau PP Nightingale CH Ross JW Quintiliani RExperience with a once-daily aminoglycoside program administered to 2184 adultpatients Antimicrob Agents Chemother 199539650ndash655

79 Hutchin T Cortopassi G Proposed molecular and cellular mechanism for amino-glycoside ototoxicity Antimicrob Agents Chemother 1994382517ndash2520

80 Fausti SA Henry JA Scheffer HI Olson DJ Frey RH McDonald WJ High-fre-quency audiometric monitoring for early detection of aminoglycoside ototoxicityJ Infect Dis 19921651026ndash1032

81 Federspil P Drug-induced sudden hearing loss and vestibular disturbances AdvOtorhinolaryngol 198127144ndash158

82 Beaubien AR Ormsby E Bayne A Carrier K Crossfield G Downes M Henri RHodgen M Evidence that amikacin ototoxicity is related to total perilymph areaunder the concentration-time curve regardless of concentration Antimicrob AgentsChemother 1991351070ndash1074

83 Proctor L Petty B Lietman P Thakor R Glackin R Shimizu H A study of poten-tial vestibulotoxicity effects of once daily versus thrice daily administration of to-bramycin Laryngoscope 1987971443ndash1449

Aminoglycosides 151

84 Rybak MJ Abate BJ Kang SL Ruffing MJ Lerner SA Drusano GL Prospectiveevaluation of the effect of an aminoglycoside dosing regimen on rates of observednephrotoxicity and ototoxicity Antimicrob Agents Chemother 1999431549ndash1555

85 Garrison MW Zaske DE Rotschafer JC Aminoglycosides Another perspectiveDICP Ann Pharmacother 199024267ndash272

86 Mingeot-Leclercq MP Tulkens PM Aminoglycosides Nephrotoxicity AntimicrobAgents Chemother 1999431003ndash1012

87 Moore RD Smith CR Lietman PS Risk factors for nephrotoxicity in patientstreated with aminoglycosides Ann Intern Med 1984100352ndash357

88 Sawyers CL Moore RD Lerner SA Smith CR A model for predicting nephrotox-icity with aminoglycosides J Infect Dis 19861531062ndash1068

89 Whelton A Therapeutic initiatives for avoidance of aminoglycoside toxicity J ClinPharmacol 19852567ndash81

90 Bertino JS Jr Booker LA Franck PA Jenkins PL Nafziger AN Incidence andsignificant risk factors for aminoglycoside-associated nephrotoxicity in patientsdosed by using individualized pharmacokinetic monitoring J Infect Dis 1993167173ndash179

91 Nodoushani M Nicolau DP Hitt CH Quintiliani R Nightingale CH Evaluation ofnephrotoxicity associated with once-daily aminoglycoside administration J PharmTech 199713258ndash262

92 Verpooten GA Giuliano RA Verbist L Eestermans G De Broe ME Once dailydosing decreases the accumulation of gentamicin and netilmicin Clin PharmacolTher 19894522ndash27

93 Murray KR McKinnon PS Mitrzyk B Rybak MJ Pharmacodynamic characteriza-tion of nephrotoxicity associated with once-daily aminoglycoside Pharmacother-apy 1999191252ndash1260

94 Sarubbi FA Jr Hull JH Amikacin serum concentrations Prediction of levels anddosage guidelines Ann Intern Med 197889612ndash618

95 Sawchuk RJ Zaske DE Pharmacokinetics of dosing regimens which utilize multi-ple intravenous infusions Gentamicin in burn patients J Pharmacokinet Biopharm19764183ndash195

96 Sawchuk RJ Zaske DE Cipolle RJ Wargin WA Strate RG Kinetic model forgentamicin dosing with the use of individual patient parameters Clin PharmacolTher 197721362ndash369

97 Schumock GT Raber SR Crawford SY Naderer OJ Rodvold KA National surveyof once-daily dosing of aminoglycoside antibiotics Pharmacotherapy 199515201ndash209

98 Chuck SK Raber SR Rodvold KA Areff D National survey of extended-intervalaminoglycoside dosing Clin Infect Dis 200030433ndash439


100 DeVries PJ Verkooyen RP Leguit P Verbrugh HA Prospective randomized studyof once-daily versus thrice-daily netilmicin regimens in patients with intraabdomi-nal infections Eur J Clin Microbiol Infect Dis 19909161ndash168

101 Mauracher EH Lau WY Kartowisastro H et al Comparison of once-daily and

152 Kim and Nicolau

thrice-daily netilmicin regimens in serious systemic infections A multicenter studyin six asian countries Clin Ther 198911604ndash613

102 Maller R Ahrne H Eilard T et al Efficacy and safety of amikacin in systemicinfections when given as a single daily dose or in two divided doses J AntimicrobChemother 199127(suppl C)121ndash128

103 Maller R Ahrne H Holmen C et al Once- versus twice-daily amikacin regimenefficacy and safety in systemic gram-negative infections J Antimicrob Chemother199331939ndash948

104 Prins JM Buller HR Kuijper EJ Tange RA Speelman P Once versus thrice dailygentamicin in patients with serious infections Lancet 1993341335ndash339

105 Rozdzinski E Kern WV Reichle A et al Once-daily versus thrice-daily dosing ofnetilmicin in combination with β-lactam antibiotics as empirical therapy for febrileneutropenic patients J Antimicrob Chemother 199331585ndash598

106 TerBraak EW DeVries PJ Bouter KP et al Once-daily dosing regimen for amino-glycoside plus β-lactam combination therapy of serious bacterial infections Com-parative trial with netilmicin plus ceftriaxone Am J Med 19908958ndash66

107 International Antimicrobial Therapy Cooperative Group of the EORTC Efficacyand toxicity of single daily doses of amikacin and ceftriaxone versus multiple dailydoses of amikacin and ceftazidime for infection in patients with cancer and granulo-cytopenia Ann Intern Med 1993119584ndash593

108 Beaucaire G Leroy O Beuscart C et al Clinical and bacteriological efficacy andpractical aspects of amikacin given once daily for severe infections J AntimicrobChemother 199127(suppl C)91ndash103

109 Prins JM Buller HR Kuijper EJ Tange RA Speelman P Once-daily gentamicinversus once-daily netilmicin in patients with serious infections A randomized clini-cal trial J Antimicrob Chemother 199433823ndash835

110 Marik PE Lipman J Kobilski S Scribante J A prospective randomized study com-paring once- versus twice-daily amikacin dosing in critically ill adult and paediatricpatients J Antimicrob Chemother 199128753ndash764

111 Nicolau DP Quintiliani R Nightingale CH Once-a-day aminoglycoside therapyRep Ped Infect Dis 1997728

112 Bourget P Fernandez H Delouis C Taburet AM Pharmacokinetics of tobramycinin pregnant women Safety and efficacy of a once-daily dose regimen J Clin PharmTher 199116167ndash176

113 Galoe AM Graudal N Christensen HR Kampmann JP Aminoglycosides Singleor multiple daily dosing A meta-analysis on efficacy and safety Eur J Clin Phar-macol 19954839ndash43

114 Freeman CD Strayer AH Mega-analysis of meta-analysis An examination ofmeta-analysis with an emphasis on once-daily aminoglycoside comparative trialsPharmacotherapy 1996161093ndash102

115 Hatala R Dinh T Cook D Once-daily aminoglycoside dosing in immunocompe-tent adults A meta-analysis Ann Intern Med 1996124717ndash725

116 Barza M Ioannidis JP Cappelleri JC Lau J Single or multiple doses of aminogly-cosides A meta-analysis Br Med J 1996312338ndash345

117 Munckhof WJ Grayson JL Turnidge JD A meta-analysis of studies on the safety

Aminoglycosides 153

and efficacy of aminoglycosides given with once daily or as divided doses J Anti-microb Chemother 199637645ndash663

118 Ferriols-Lisart R Alos-Alminana M Effectiveness and safety of once-daily amino-glycosides A meta-analysis Am J Health-Syst Pharm 1996531141ndash1150

119 Bailey TC Little JR Littenberg B Reichley RM Dunagan WC A meta-analysisof extended-interval dosing versus multiple daily dosing of aminoglycosides ClinInfect Dis 199724786ndash795


121 Hatala R Dinh TT Cook DJ Single daily dosing of aminoglycosides in immuno-compromised adults A systematic review Clin Infect Dis 199724810ndash815

122 Demczar DJ Nafziger AN Bertino JS Jr Pharmacokinetics of gentamicin at tradi-tional versus high doses Implications for once-daily aminoglycoside dosing Anti-microb Agents Chemother 1997411115ndash1119

123 Xuan D Lu JF Nicolau DP Nightingale CH Population pharmacokinetics studyof tobramycin after once-daily dosing in hospitalized patients Int J AntimicrobAgents 200015185ndash191

124 Konrad F Wagner R Neumeister B Rommel H Georgieff M Studies on drugmonitoring in thrice and once daily treatment with aminoglycosides Intensive CareMed 199319215ndash220

125 Gilbert DN Bennett WM Use of antimicrobial agents in renal failure Infect DisClin North Am 19893517ndash531

126 Prins JM Koopmans RP Buller HR Kuijper EJ Speelman P Easier monitoringof aminoglycoside therapy with once-daily dosing schedules Eur J Clin MicrobiolInfect Dis 199514531ndash535

127 Begg EJ Barclay ML Duffull SB A suggested approach to once-daily aminoglyco-side dosing Br J Clin Pharmacol 199539605ndash609

128 Nicolau DP Wu AHB Finocchiaro S Udeh E Chow MSS Quintiliani R Nightin-gale CH Once-daily aminoglycoside dosing Impact on requests for therapeuticdrug monitoring Thera Drug Mon 199618263ndash266

129 Hitt CM Klepser ME Nightingale CH Quintiliani R Nicolau DP Pharmacoeco-nomic impact of a once-daily aminoglycoside administration Pharmacotherapy199717810ndash17814

130 Parker SE Davey PG Once-daily aminoglycoside administration in gram-negativesepsis Economic and practical aspects PharmacoEconomics 19957393ndash402

7

Pharmacodynamics of Quinolones

Robert C Owens Jr

Maine Medical Center Portland Maine and University of Vermont Collegeof Medicine Burlington Vermont

Paul G Ambrose

Cognigen Corporation Buffalo New York

1 INTRODUCTION

lsquolsquoWe know everything about antibiotics except how much to giversquorsquo MaxwellFinland once stated With the proliferation of pharmacodynamics as a sciencewe are finally addressing the question of how much to give We are watchingthe pendulum swing from an era of more-or-less arbitrary dosage selection towardthe present-day science that integrates both pharmacokinetic and microbiologicdata to determine optimal dosing strategies and to set perhaps more clinicallymeaningful breakpoints It is conceivable that body-site-specific susceptibilitybreakpoints based on the pharmacodynamic profiles of antimicrobial agents willexist in the not-too-distant future replacing the one breakpoint fits all approachthat we have historically endured Pharmacodynamics is central to the idea ofoptimizing antimicrobial therapy Figure 1 illustrates the relationship betweenpharmacodynamics and the optimal selection dosing and duration of antimicro-bial therapy A comprehensive review by Ron Polk [1] provides in great detailthe various aspects of the optimal use of antibiotics

155

156 Owens and Ambrose

FIGURE 1 Pharmacodynamics is central to the optimization of antimicrobialtherapy (Courtesy of Robert Owens PharmD)

During the three decades since nalidixic acid was first introduced thou-sands of related quinolone compounds have been synthesized and most havebeen abandoned prior to development for a variety of reasons A better under-standing of structurendashactivity and structurendashtoxicity relationships has allowedchemists to modify the adaptable basic quinolone structure to enhance or limitthe extent of antimicrobial activity and to improve various other product attri-butes such as the pharmacokinetic profile tolerability drug interaction potentialand toxicity of each new agent The quinolones have undergone a lsquolsquostructuralevolutionrsquorsquo that began in 1962 upon the inadvertent discovery of nalidixic acidand the pilgrimage continues today with the search for the perfect compound[2]

For some time now clinicians have been familiar with antibiotic classescategorized by generations (eg cephalosporins quinolones macrolides) Cer-tain schemes have been based upon pharmacodynamics [2ndash5] (Table 1) ratherthan relying on simply microbiological susceptibility data or merely the date acompound was licensed for use Classifying quinolones by generations allowsone to realize the pharmacokinetic and microbiological evolution of the quino-lones as a direct result of structural changes To this day the fluoroquinolonesremain the prototypical class of antibiotics because they are available in bothoral and parenteral dosage forms have excellent oral bioavailability are activeagainst a wide range of bacteria achieve therapeutic concentrations both intracel-lularly and extracellularly distribute widely into organ tissue and secretions andare rapidly bactericidal It is for these reasons that the fluoroquinolones haverevolutionized transitional therapy as well as other aspects of the treatment ofmodern infectious diseases

Pharmacodynamics of Quinolones 157

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2 PHARMACODYNAMIC CONCEPTS

Since the dawn of the antibiotic era in the late 1930s controversy has existedas to the most appropriate method to administer and dose antibiotics to maximizethe killing of microorganisms while minimizing toxicity Harry Eagle a half-century ago pioneered the first pharmacodynamic studies using penicillin instreptococcal and syphylitic animal models of infection [6ndash8] It was at this timethat dosing methods (eg continuous infusion versus intermittent injection) aswell as the dose of penicillin employed in relation to the minimum inhibitoryconcentration (MIC) were evaluated for their ability to impact in vivo outcomeFollowing a long respite the science known as pharmacodynamics reemergedas Shah et al [9] classified antimicrobial agents on the basis of their patternsof bactericidal activity One of the two patterns described was a concentration-dependent killing effect where an increase in the rate and extent of bacterialkilling occurred with increasing drug concentrations in relation to the MIC of thebacteria Agents that demonstrated concentration-dependent bactericidal activityincluded the aminoglycosides (and now include the fluoroquinolones and mostlikely metronidazole) (see Fig 2)

The second pattern now known as time-dependent killing exhibited a satu-rable concentration-dependent increase in the rate and extent of bacterial killingoccurring at approximately 2ndash4 times the MIC of the pathogen In essence theseagents proceeded to kill bacteria most efficiently when the concentration of drugremained in excess of the MIC of the pathogen for a specified length of timerather than when doses were employed that provided high serum concentrations

FIGURE 2 Concentration-dependent versus time-dependent bactericidal ac-tivity (From Ref 20)


(relative to the MIC) because the latter did not result in enhanced bacterial kill-ing β-Lactams (eg penicillins cephalosporins monobactams carbapenems)lincosamides (eg clindamycin) macrolides (eg erythromycin clarithro-mycin) and oxazolidinones (eg linezolid) best fit this pattern of bactericidalactivity For the purposes of this chapter we will focus on the quinolones andconcentration-dependent bacterial killing

Historically the MIC has been used by the practicing clinician to selectdrug therapy for a particular infection The lower the MIC the better the drugmust be and the choice has been traditionally made accordingly and sometimesstill is today Unfortunately the MIC has many shortcomings and therefore can-not be used alone to predict the outcome for a given infection The MIC is ameasure of a drugrsquos potency against a particular organism It should be remem-bered that the MIC is an artificial value and is further subject to a one-tubedilution standard error if broth dilution techniques are used This can translateinto a considerable difference in actual MIC values (eg is the MIC 8 microgmLor 16 microgmL or somewhere in between) The MIC does not describe the rateand extent of bacterial killing or persistent antimicrobial effects such as the post-antibiotic effect (PAE) or the more clinically relevant sub-MIC effect (SME)The PAE and SME are used to describe the continued suppression of bacterialgrowth despite either complete removal of the antimicrobial agent from the organ-ismrsquos milieu or concentrations of the agent that are below the MIC value for theparticular organism respectively Also the MIC does not describe the impactof increasing drug concentrations or increased time of exposure of the drug onbacteriological outcome MIC testing does not account for protein binding al-though somewhat controversial this has historically misled clinicians to choosetherapies with confidence based solely on in vitro data For example the use ofoxacillin for the treatment of enterococcal endocarditis (despite MIC values inthe susceptible range) has led to clinical and microbiological failures in humanssimilarly the use of cefonicid and teicoplanin for the treatment of staphylococcalinfections has led to treatment failures some so extensive that clinical trials wereterminated sooner than planned [10ndash12] Fortunately these weaknesses can beovercome or a drug selection can be modified if the appropriate pharmacokineticaspects of the antimicrobial agent are also factored into the equation

Bacterial killing can be characterized mathematically For example theproduct of concentration and time (C t) may be reflected by the pharmacoki-netic term lsquolsquoarea under the concentrationndashtime curversquorsquo (AUC) Hence bacterialkilling is a function of a drugrsquos AUC when it is indexed to the MIC The 24 hAUCMIC ratio is the pharmacodynamic correlate that can be used to describethe time course of antimicrobial activity and to predict clinical or microbiologicaloutcome and perhaps the development of resistance

Under certain circumstances one of the terms of the product (either concen-tration or time) makes a small or negligible contribution to the killing process


and can therefore be ignored The pharmacodynamic parameter can be simplifiedto the peak concentration (peak)MIC ratio or the length of time the serum con-centration remains above the MIC (t MIC) How this simplification occursdepends on the pattern of bactericidal activity demonstrated by the antimicrobialagent in question (eg concentration-dependent killing or time-dependent kill-ing) For concentration-dependent killing agents (eg aminoglycosides quino-lones) the 24 h AUCMIC ratio and peakMIC ratio have been used to correlatein vivo outcome For the time-dependent killing agents (eg β-lactams) t MIC has best correlated with efficacy

3 CLINICAL PHARMACODYNAMIC TARGETS

For the quinolones as one might expect both the peakMIC ratio and the 24 hAUCMIC ratio have been correlated with outcome The magnitude of the peakMIC ratio that has been associated with improved outcomes is 10ndash12 [13] Inaddition the peakMIC ratio has been suggested to be the parameter of choicewhen resistant subpopulations of bacteria exist [26] Unfortunately increasingthe amount of drug given in excess of standard doses for many agents in thisclass also increases the probability of unwanted adverse events so unless thepathogen is relatively susceptible this ratio may not always be the best parameterto optimize

When a peakMIC ratio of at least 101 is not possible one can no longerignore the contribution of the time of exposure (Fig 3) Under these circum-

FIGURE 3 Pharmacodynamic relationships of (a) peakMIC ratio and (b) 24 hAUCMIC ratio (Courtesy of Robert Owens Pharm D)


stances the parameter that best correlates with efficacy defaults to the 24 h AUCMIC ratio

The optimal pharmacodynamic targets are pathogen-specific Data obtainedfrom animal models of sepsis in vitro pharmacodynamic experiments and clinicaloutcome studies indicate that the magnitude of the 24 h AUCMIC ratio can beused to predict clinical response Forrest et al [14] demonstrated that a 24 hAUCMIC ratio of 125 was associated with the best clinical cure rates in thetreatment of infections caused by gram-negative enteric pathogens and P aerugi-nosa However for gram-positive bacteria data collected in humans by Drusanoand colleagues [13] and Ambrose et al [15] suggest that the 24 h AUCMICratio can be appreciably lower For infections caused by anaerobic pathogensthe optimal pharmacodynamic correlate for efficacy remains to be determinedHowever the likely 24 h AUCMIC ratio target will again be below 125 The24 h AUCMIC ratio for trovafloxacin ranged from 50 to 100 [16] similar tothat of metronidazole and was successful within this range One must also re-member that free drug concentrations should be considered so taking into ac-count that trovafloxacin is 75 protein-bound a free drug 24 h AUCMIC ratiorequired to predict success against anaerobic pathogens may in fact be as low as15ndash25 Pharmacodynamic studies evaluating quinolones against anaerobic patho-gens are needed considering that most newer agents (eg gatifloxacin moxiflox-acin gemifloxacin sitafloxacin) have in vitro activity against these organisms

4 PHARMACOKINETICS OF FLUOROQUINOLONES

The pharmacokinetic profiles for the newer and some older fluoroquinolones arelisted in Table 2 Once a standard of care for most infections the appeal ofparenteral therapy has lost significant ground to newer equally potent and phar-macokinetically equivalent oral dosing formulations Although bacteria have be-come quite sophisticated they are still unable to discriminate between the routesby which an antibiotic is delivered to the infection site The fluoroquinoloneshave revolutionized the treatment of modern infectious diseases for a variety ofreasons none perhaps more important than their pharmacokinetic characteristicsWhether administered intravenously or orally similar pharmacodynamic rela-tionships can be achieved because of their high degree of bioavailability whichis consistent among the newer generation agents Protein binding is one character-istic that is highly variable among the quinolones The degree of protein bindingnot only influences penetration into tissues but also determines the amount ofdrug that is capable of interacting with bacteria at the site of infection [17] Theexact role of protein binding is often more controversial than not but clearlythe pharmacokinetic parameter used in pharmacodynamic analyses should reflectfree drug (active drug) rather than total drug concentrations


TA

BLE

2P

har

mac

oki

net

icP

rofi

les

of

Var

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uo

roq

uin

olo

nes

Par

amet

erN

orfl

oxa

cin

Cip

rofl

oxa

cin

Levo

flo

xaci

nS

par

flo

xaci

nG

atifl

oxa

cin

Mo

xifl

oxa

cin

Tro

vafl

oxa

cin

Gem

iflo

xaci

n

Do

se(m

g)

400

750

500

200

400

400

200

320

Bio

avai

lab

ility

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5070

9992

9690

8880

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ou

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148

Free

pea

k(micro

gm

L)1

32

64

20

83

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00

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6A

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(0ndash2

4)(micro

gsdoth

mL)

136

6447

517

751

348

312

93

Free

AU

C(0

ndash24)(micro

gsdoth

mL)

116

4833

310

641

021

67

83

8T

12

33

46

209

1212

74

R

enal

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(act

ive

dru

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6095

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206

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Co

urt

esy

of

Ro

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sP

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D


5 OUTCOME DATA

51 Animal Models and In-Vitro Models of Infection

In 1991 Leggett et al [18] published data evaluating ciprofloxacin in neutropenicmurine thigh and pulmonary infection models using strains of P aeruginosa andK pneumoniae respectively The manipulation of the dosing intervals in thesestudies minimally affected the extent of bacterial killing suggesting that theAUCMIC ratio may be most closely associated with this endpoint

Drusano et al [26] evaluated lomefloxacin in the neutropenic rat model ofPseudomonas sepsis Doses were administered in a variety of regimens to studythe effect of different pharmacodynamic indices associated with outcome ThepeakMIC ratio was significantly associated with reduced mortality comparedwith the AUCMIC ratio and the time MIC The doses used in this studyprovided peakMIC ratios of approximately 201 and 101 The reason for theclear association between the index of the peakMIC ratio and survival was statedto be the increased inoculum size used (1 109) in the study These findingsdemonstrated that with a higher burden of organisms one is more likely to en-counter resistant subpopulations and in such settings the peakMIC ratio be-comes the more appropriate pharmacodynamic index that predicts outcome

Several studies have been recently published or presented evaluating thepharmacodynamic relationships of quinolones against gram-positive pathogensparticularly S pneumoniae Onyeji et al [19] compared the efficacy of ciproflox-acin and levofloxacin in a murine model of peritoneal sepsis Clinical isolates ofS pneumoniae were used that displayed a variety of susceptibilities to penicillinand MIC values for ciprofloxacin (1 and 2 microgmL) and levofloxacin (1 and 2microgmL) were reflective of those also seen clinically Dosing regimens were variedsuch that concentrations of drug measured in the animals simulated those in hu-mans Five-day survival rates between ciprofloxacin (2ndash6) and levofloxacin(7ndash9) groups were negligible (p 005) perhaps because the 24 h AUCMICratios were not very different from those seen in humans For ciprofloxacin 24h AUCMIC ratios ranged between 175 and 35 and for levofloxacin valuesranged between 22 and 44 Neither ciprofloxacin nor levofloxacin achieved adesirable peakMIC ratio of at least 10 as expected

Studies evaluating the impact of protein binding have been conductedCraig and Andes [20] studied six fluoroquinolones in the classic nonneutropenicmurine thigh infection model in an effort to determine optimal AUCMIC ratiosin terms of survival and bactericidal activity using both free and total drug con-centrations After being infected with S pneumoniae mice were treated withciprofloxacin levofloxacin sitafloxacin moxifloxacin gemifloxacin or gati-floxacin Serum concentrations and protein binding were determined using micro-biological assay and ultrafiltration respectively The Emax model was used to cor-relate 24 h AUCMIC values using both free and total drug concentrations with


colony forming units in the thigh on day 1 and survival on day 5 Results indicatedthat the 24 h AUCMIC ratio correlated with survival on day 5 and the extentof bacterial killing on day 1 (p 0001) Additionally free drug better correlatedwith survival on day 5 than did total drug concentrations (R 2 82 and 74respectively) (see Fig 4) Survival (90 of animals) was predicted with an 24 hAUCMIC ratio of 34 4 and a similar value predicted a 25 log10 kill afterone day of treatment Again agreement existed among all quinolones tested asto the pharmacodynamic breakpoint required for endpoints of survival and theextent of bacterial killing (24 h AUCMIC somewhere between 25 and 35) andunbound drug was better correlated with these effects than total drug concentra-tions

Similarly for S pneumoniae in vitro models of infection have demon-strated that for levofloxacin and ciprofloxacin a 24 h AUCMIC ratio of approxi-mately 30 was associated with a 4 log kill whereas values less than 30 wereassociated with a significantly reduced extent of bacterial killing and in someinstances bacterial regrowth [2122] These observations are supported by datafrom nonneutropenic animal models of infection where maximal survival wasassociated with a 24 h AUCMIC ratio of 25 against the pneumococcus [23]Clinically there have been a significant number of treatment failures and superin-fections involving meningeal seeding from S pneumoniae in patients receivingciprofloxacin where the 24 h AUCMIC ratio is approximately 12 [24] Con-versely similar treatment failures or superinfections have not occurred with quin-

FIGURE 4 Correlation between survival and (a) free-drug concentration (R 2 74) and (b) total drug concentrations (R 2 82) (From Ref 20)


olones for which the 24 h AUCMIC ratios against this bacterium are greaterthan 30ndash40 [15]

52 Human Studies

Some of the first data to correlate pharmacodynamics and response in humanswere published by Peloquin et al [25] Intravenous ciprofloxacin was evaluatedin the treatment of lower respiratory tract infections in seriously ill patients hospi-talized in the intensive care unit A relationship between time of exposure andoutcome was established Interestingly against P aeruginosa a low peakMICratio (101) was observed and resulted in the development of resistance in 10of 13 pathogens Although the researchers concluded that the time duration ofexposure was the important determinant of outcome against difficult to treat andless susceptible pathogens (eg P aeruginosa) failure to obtain an optimal peakMIC ratio resulted in the emergence of resistance This is consistent with findingsfrom the animal model of infection reported by Drusano et al [26] further em-phasizing the importance of the peakMIC ratio when dealing with resistant sub-populations Eventually after the addition of 24 new patients Forrest et al [14]in 1993 published their reanalyzed data from the 1989 publication (see Fig 5)Multivariate analysis and logistic regression showed that the 24 h AUCMIC ratiopredicted outcome best rather than the time of exposure as previously reported

A relationship also exists between the 24 h AUCMIC ratio and the likli-hood of developing resistance while on therapy Thomas et al [27] describedthis relationship in 107 hospitalized patients being treated for bacterial pneumo-nia Patients were treated with a variety of dosing regimens involving a fluoro-quinolone (ciprofloxacin 200 mg q12h to 400 mg q8h) or a cephalosporin (cef-menoxime 1ndash2 g q4-6h or ceftazidime 1ndash2 g q8-12h) providing a wide rangeof exposures Gram-negative pathogens predominated as expected in nosocomialpneumonia accounting for over 90 of the organisms isolated Gram-positivepathogens such as S aureus and S pneumoniae were infrequently isolated Withthe exception of Bush group 1 β-lactamase elaborating organisms (eg Entero-bacter spp) treated with a β-lactam a pharmacodynamic breakpoint was estab-lished If a 24 h AUCMIC ratio of at least 100 was achieved the potential todevelop resistance was 10 In contrast the probability of developing a resis-tant strain during therapy was greater than 80 if the 24 h AUCMIC was 100Because the majority of pathogens encountered were gram-negative bacilli mostof which were P aeruginosa it is difficult to generalize this identified pharmaco-dynamic relationship to gram-positive pathogens More information is clearlyneeded here

Ciprofloxacin a second-generation fluoroquinolone remains the most po-tent antipseudomonal quinolone in terms of in vitro microbiological activity For


FIGURE 5 Pharmacodynamic correlates of efficacy for gram-negative patho-gens (From Ref 14)

instance ciprofloxacin is consistently 4ndash8 times as active against Pseudomonasaeruginosa in vitro as trovafloxacin and levofloxacin Along these lines ci-profloxacin is also pharmacodynamically superior to other available fluoroquino-lones against P aeruginosa in terms of an achievable 24 h AUCMIC ratio whendosed appropriately Unfortunately none of the currently available fluoroquino-lones routinely achieve a target 24 h AUCMIC ratio against P aeruginosa ofat least 125 which is one reason combination therapy is always recommendedfor the treatment of infection outside of the lower urinary tract when a quinoloneis employed

One must consider however that ciprofloxacinrsquos strength against P aeru-ginosa is counterbalanced by its poor pharmacodynamic profile against manygram-positive microorganisms For instance ciprofloxacin even when dosed at750 mg every 12 h does not reach a pharmacodynamic goal of approximately30 for S pneumoniae This poor pharmacodynamic profile is consistent withnumerous reports of failures and superinfections when ciprofloxacin has beenused in community-acquired infection due to S pneumoniae [2428ndash33]

With the advent of newer generation fluoroquinolones (eg temafloxacin)with significantly greater potency against the pneumococcus in the early 1990s


and the recent observations of increasing β-lactam resistance among pneumo-cocci it is not surprising that Daniel Musher in 1992 stated lsquolsquoThose of us whoa few years ago scoffed at the notion of using a quinolone for treatment ofpneumococcal pneumonia may be just doing that in the not-too-distant futurersquorsquo

Newer generation fluoroquinolones are filling the ever-widening niche be-ing created by pneumococci that are now demonstrating widespread resistanceto multiple drug classes including the β-lactams macrolides and azalides sulfon-amides and tetracyclines In fact with respect to the treatment of community-acquired pneumonia Marvin Turck has widely been quoted as saying lsquolsquoa fluoro-quinolone for your mother or a β-lactam for your mother-in-lawrsquorsquo Despite theirhigh degree of activity against most strains of S pneumoniae the fluoroquino-lones particularly less active agents such as ciprofloxacin ofloxacin and lev-ofloxacin are showing elevated rates (albeit small percentages) of resistance[3435] Some newer generation fluoroquinolones (eg moxifloxacin gatifloxa-cin gemifloxacin) are also affected but to a lesser degree [3436] The fluoroqui-nolones do have the potential for overuse because of their convenience and agent-specific low side effect profiles Appropriate use of these compounds and theselection of those agents with optimized pharmacodynamic profiles will mostlikely reduce the selective pressure being placed on pneumococci

Preston et al [13] evaluated the association between levofloxacinrsquos pharma-codynamic profile and clinical as well as microbiological outcomes In this studyconcentrations of levofloxacin in serum were obtained from patients being treatedfor urinary tract pulmonary and skinsoft-tissue infections after they had beengiven standard doses appropriate for the site of infection Of the initial 313 pa-tients 116 had sufficient pharmacokinetic microbiological and outcome data forevaluation Patients in whom a peakMIC ratio of 122 was achieved had a100 chance of a eradicating the infecting organism from the site of infectionConversely if the peakMIC ratio was 122 the liklihood of successful eradica-tion was 808 Although there was significant covariation between the peakMIC and 24 h AUCMIC indices on outcome predictability the peakMIC ratioreached statistical significance for all pathogens and infection sites A recent re-analysis of the infections caused by S pneumoniae however revealed that a 24h AUCMIC ratio of 30 was associated with favorable outcomes [37] Thesedata are consistent with the previous work conducted by Drusanorsquos group whichdemonstrated that when a peakMIC ratio of at least 101 could not be achievedthe index most closely associated with outcome was the 24 h AUCMIC ratio

Most recently Ambrose and Grasela [15] reported a correlation betweenthe free-drug 24 h AUCMIC ratio and microbiological eradication of S pneu-moniae in patients enrolled in phase III double-blinded randomized trials inNorth America Of the initial 778 patients enrolled in these studies involvingcommunity-acquired pneumonia and acute bacterial exacerbations of chronicbronchitis 376 patients had sufficient clinical and microbiological data for evalu-


FIGURE 6 Pharmacodynamic correlates of efficacy for Streptococcus pneu-moniae (Courtesy of Paul Ambrose)

ation Of these patients 58 were infected with S pneumoniae that was isolatedfrom either blood or sputum These data established that for gatifloxacin andlevofloxacin a 24 h AUCMIC ratio of at least 33 correlated with the eradicationof S pneumoniae in patients being treated for pneumococcal pulmonary infec-tions (Fig 6) Although not every patient with an 24 h AUCMIC ratio that didnot reach this pharmacodynamic breakpoint failed therapy these patients cer-tainly had a high probability of failure

In the clinic we are now seeing what has already been predicted pharmaco-dynamically [38] As MICs continue to increase gradually for older fluoroquino-lones the pharmacokinetics of these agents can no longer compensate for thisdecrease in activity Davidson et al [39] reported recently well documented clini-cal failures that could be traced back to the inability of a less active fluoroquino-lone to eradicate S pneumoniae When pulse field gel electropheresis and PCRwere performed on the pretherapy and post-therapy isolates it was evident thatthe organisms were in fact the same and resistance had developed during therapyPretherapy isolates in both cases were susceptible to the agent and after receivinglevofloxacin for the treatment of community-acquired pneumococcal pneumoniathe strains had acquired resistance via gyrA and parC mutations A likely expla-nation for these findings are the propensity to select for resistant strains secondaryto reduced concentrations at the end of the dosing interval There are differencesamong the newer generation fluoroquinolones in their ability to select for resistantstrains The rank order in terms of most likely to least likely to select for resis-


tance among pneumococci is as follows ciprofloxacin levofloxacin gati-floxacin moxifloxacin [40ndash43]

Finally various pharmacodynamic targets for predicting higher probabili-ties for successul outcomes in humans now exist For quinolones against gram-negative enteric bacilli and P aeruginosa a 24 h AUCMIC ratio of 125 maybe targeted for the treatment of infections by these pathogens For preventing theemergence of resistance primarily for non-Bush group 1 β-lactamase-elaboratinggram-negative bacilli a 24 h AUCMIC ratio of at least 100 has been suggestedAnd for gram-positive pathogens such as S pneumoniae an AUCMIC ratio ofat least 30 has been associated with higher probabilities of successful eradicationof these organisms More data are clearly needed to assess the effect of thesepharmacodynamic indices on newly emerging mechanisms of resistance and foranaerobic pathogens

6 PHARMACODYNAMIC ANALYSES

Once data have become available demonstrating an association between a phar-macodynamic relationship and outcome one can predict the efficacy of certaincompounds using available pharmacokinetic and microbiological data individual-ized toward specific patient and pathogen populations This can be quite useful tothe clinician in the assessment of newer agents in the formulary decision processparticularly when they are being compared to other newer agents as well as moretraditional anti-infectives The methodology of such analyses should be viewedwith a certain amount of skepticism as these analyses have now become popularamong the marketing departments of the pharmaceutical industry Moreover cli-nicians should be careful when performing these analyses to ensure that the dataare generalized to appropriate patient populations Ultimately this approachwould be most useful when performed individually at the bedside using patient-specific data in an effort to optimize treatment in real time

61 Single-Point Analyses

Because of the recent interest in pharmacodynamics and its ability to distinguishanti-infective agents it has become popular to perform so-called single pointpharmacodynamic analyses These analyses are popular because they are bothconvenient and easy to perform They rely primarily on the selection of meanpharmacokinetic values (eg 24 h AUC) collected in healthy volunteers whichcan be readily gleaned from product package inserts In addition to the meanpharmacokinetic parameters MIC90 values are obtained most often from largenational surveillance studies By integrating these data one can calculate 24 hAUCMIC ratios for various quinolonendashpathogen combinations very quickly At


best these evaluations convey what is possible rather than what is probable Atworst they are meaningless to the practicing clinician [44] Unfortunately manyhazards exist with these evaluations For instance significant variability existsin both pharmacokinetic data and MIC values local resistance trends are notrepresented when national susceptibility data are used and significant bias isoften introduced into the analysis

Considerable variability exists when fixed doses of drug are administeredto populations of individuals with varying weights heights and organ functionAdditionally healthy volunteers must be discriminated from clinically sick pa-tients Mean 24 h AUC values cannot adequately reflect the range of possibilities

Similarly the MIC90 value of a given drug versus a specific organism al-though perhaps useful for comparing susceptibility data from different locationscannot possibly be used alone to simulate the range of potential susceptibilitiesencountered clinically In fact most strains encountered would have MIC valuesfar less than the MIC90 value they are labeled with By the same token how dowe accommodate for the 10 of organisms whose MICs are in excess of theMIC90 value

Because of this rather arbitrary method for conducting a pharmacodynamicanalysis one can also foresee the introduction of bias With the availability ofa variety of surveillance studies one is left to decide which MIC value to selectA twofold variance in MIC can lead to the resulting 24 h AUCMIC ratio beingeither below or above the desired pharmacodynamic target with the agent beingdeemed either unfavorable or favorable respectively As an example for lev-ofloxacin if one selects an MIC90 of 10 or 20 microgmL keeping the mean 24 hAUC value fixed (500 mg dose administered orally 24 h AUC 475 microg sdot hmL) the conclusions are dramatically different If an MIC90 of 10 microgmL isused the resulting 24 h AUCMIC ratio is 475 whereas if 20 microgmL is usedthe 24 h AUCMIC ratio is 2375 Keeping in mind that the 24 h AUCMICratio pharmacodynamic target of 30 is desirable for S pneumoniae the seeminglyminute one-dilution difference in MIC resulted in two discordant conclusionsOne can clearly see that although this approach might seem convenient the re-sults may equate to lsquolsquogarbage in garbage outrsquorsquo

62 Monte Carlo Analysis

A departure from a past paradigm involves a novel method for conducting phar-macodynamic analyses The use of a certain methodology has received consider-able attention lately This methodology termed Monte Carlo analysis accountsfor individual variability across a wide range of input variables The results ofMonte Carlo analyses estimate what is probable rather than defining what ispossible [45ndash48] Monte Carlo simulations are sampling experiments for estimat-ing the distribution of an outcome that is dependent on multiple probabilisticinput variables For example MIC values obtained from an institution or region


FIGURE 7 Pharmacodynamics and the use of Monte Carlo methodology In-dividual AUC values obtained from patients (clinical trials) Individual MICvalues derived from local or national surveillance data

and the 24 h AUC values from patients are considered as input variables Randomvalues across input variables that conform to their probabilities are generatedand then an output is calculated (eg AUCMIC ratio) (see Fig 7) Each individ-ual output that is calculated is then plotted in a probability chart Monte Carlomethodology demonstrates the range of possible outcomes and the probabilityassociated with each

In a recent study we characterized the pharmacodynamics of levofloxacingatifloxacin and moxifloxacin using pharmacokinetic parameters and local clini-cal isolates of Streptococcus pneumoniae [47] Clinical isolates of Streptococcuspneumoniae were collected consecutively over the 1999ndash2000 respiratory infec-tion season by our laboratory (n 100) Pharmacokinetic data were obtainedfrom patients enrolled in a variety of trials for levofloxacin (172 acutely ill adultpatients with community-acquired infections enrolled in multicenter clinical trialstreated intravenously [mean AUC SD 508 358 microg(mL sdot h)]) [13]gatifloxacin (64 acutely ill adult patients with community-acquired infections en-rolled in a multicenter trial treated intravenously [mean AUC SD 41 163 microgmL sdot h)]) [46] and moxifloxacin (286 adult volunteers in phase I andII trials that received oral moxifloxacin were used [mean AUC SD 185 45 microgmL sdot h)]) [49] Twenty-four-hour AUC values were corrected for proteinbinding based on the following levofloxacin 30 protein-bound gatifloxacin20 and moxifloxacin 50 protein bound Monte Carlo simulation (1000 sub-jects 3) was used to estimate the probability of attaining a free-drug 24 h


AUCMIC ratio of at least 30 Streptococcus pneumoniae susceptibility testingresults were as follows gatifloxacin MIC5090 025025 range 0125ndash120 levo-floxacin MIC5090 07510 range 0125 to 32 moxifloxacin MIC5090 0125019 range 0023 to 60 The probabilities (mean SD) of achieving a 24 hAUCMIC ratio of at least 30 against S pneumoniae isolates were as followsgatifloxacin 988 023 moxifloxacin 979 075 and levofloxacin 811 144

Our results indicated that in our region of the country gatifloxacin andmoxifloxacin were associated with high probabilities of reaching a target 24 hAUCMIC ratio of 30 over a wide range of actual AUCs and MICs comparedwith levofloxacin The results of our regional pharmacodynamic analysis weresimilar to those previously presented using similar pharmacokinetic data and thenational SENTRY database of 1977 isolates of S pneumoniae [46] A limitationof our analysis is that the 24 h AUC values for moxifloxacin were obtained fromindividuals enrolled in phase I and II trials rather than from infected patientsHowever because moxifloxacin is considered to have a dual route of elimination(eg urinary hepatobiliary elimination) the AUC values would not be expectedto be dramatically influenced by moderate renal or hepatic impairment [49] Also24 h AUC values for gatifloxacin and levofloxacin were measured after intrave-nous administration whereas moxifloxacin AUC values were obtained after oraldosing Nevertheless because of the high degree of bioavailability of all threecompounds it is not likely that this fact would change the results of the analysissignificantly [49]

The implications of more sophisticated pharmacodynamic analyses arewide ranging but from a practical viewpoint we have been able to use these datafor formulary evaluation of new antimicrobial agents where sufficient data existto correlate findings with clinical outcome Furthermore clinicians practicing atinstitutions that perform such analyses will have an opportunity to select agentsthat demonstrate optimal pharmacodynamic profiles against specific pathogenssuch as in this case the pneumococci

7 SUMMARY

Over the last several years an amalgam of information has become available toclinicians and researchers alike illustrating the importance of pharmacodynamicsand its role in optimizing drug selection and dosing Once of putative valuerecent developments in pharmacodynamics have provided insight into far morethan the basic understanding of how antimicrobial agents actually kill bacteriamore efficiently In terms of the fluoroquinolones new pharmacodynamicbreakpoints have been discovered and validated in humans Twenty-four-hourAUCMIC ratios of 125 and around 25ndash35 have been established for many gram-


negative and gram-positive pathogens respectively More data are needed to as-sess anaerobic breakpoints in patients to clarify outcome determinants

Pharmacodynamic data have provided insight into the determination ofnew clinically meaningful breakpoints germane to patient care for other drugclasses as well One day clinicians may not ask why their patients with pulmo-nary infections caused by penicillin-resistant S pneumoniae still in fact respondto penicillin Pharmacodynamics will provide the backbone of such a monumen-tal shift in both theory and practice

REFERENCES

1 Polk R Optimal use of modern antibiotics Emerging trends Clin Infect Dis 199929264ndash274

2 Owens RC Jr Ambrose PG Clinical use of the fluoroquinolones Med Clin N Am2000841447ndash1469

3 Ambrose PG Owens RC Jr Quintiliani R Nightingale CH New generation quino-lones Conn Med 199761269ndash272

4 Owens RC Jr Ambrose PG Quintiliani R Nightingale CH Classifying quinoloneanti-infective agents by generation A pharmacodynamic approach to rational drugselection Antibio Clin 1997170ndash74

5 Ambrose PG Owens RC Jr New antibiotics in pulmonary and critical care medicineFocus on advanced generation quinolones and cephalosporins Semin Respir CritCare Med 2000 2119ndash32

6 Eagle H Fleischman R Levy M Continuous vs discontinuous therapy with penicil-lin N Engl J Med 1953238481ndash486

7 Eagle H Effect of schedule of administration on therapeutic efficacy of penicillinImportance of aggregate time penicillin remains at effectively bactericidal levelsAm J Med 19509280ndash299

8 Eagle H Fleischman R Mussleman AD Effective concentrations of penicillin invitro and in vivo for streptococci and pneumococci and Treponema J Bacteriol 195059625ndash643

9 Shah PM Junghanns W Stille W Dosis-Wirkungs-Beziehung der Bakterizidie beiE coli K pneumoniae und Staphylococcus aureus Deut Med Wochenschr 1976101325ndash328

10 Eliopoulos GM Moellering RC Jr Antibiotic synergism and antimicrobial combina-tions in clinical infections Rev Infect Dis 19824282ndash293

11 Calain P Krause K-H Vadaux P et al Early termination of a prospective random-ized trial comparing teicoplanin and flucloxacillin for treating severe staphylococcalinfections J Infect Dis 1987155187ndash191

12 Chambers HF Mills J Drake TA Sande MA Failure of a once-daily regimen ofcefonicid for treatment of endocarditis due to Staphylococcus aureus Rev Infect Dis19846(suppl 4)S870ndashS874

13 Preston SL Drusano GL Berman AL Fowler CL Chow AT Dornseif B ReichlV Natarajan J Corrado M Pharmacodynamics of levofloxacin J Am Med Assoc1998279125ndash129


14 Forrest A Nix DE Ballow CH Schentag J Pharmacodynamics of intravenous ci-profloxacin in seriously ill patients Antimicrob Agents Chemother 1993371073ndash1081

15 Ambrose PG Grasela DM Grasela TH et al Pharmacodynamics of fluoroquino-lones against Streptococcus pneumonia Analysis of phase III clinical trials In Pro-gram and Abstracts of the 40th Interscience Conference on Antimicrobial Agentsand Chemotherapy Sept 17ndash21 2000 Toronto Canada

16 Quintiliani R Owens RC Jr Grant ED Clinical role of fluoroquinolones in patientswith respiratory tract infections Infect Dis Clin Pract 19998(suppl 1)S28ndashS41

17 Craig WA Ebert SC Protein binding and its significance in antibacterial therapyInfect Dis Clin N Am 19893407ndash414

18 Leggett JE Ebert S Fantin B et al Comparative dose-effect relations several dosingintervals for β-lactam aminoglycoside and quinolone antibiotics against gram-nega-tive bacilli in murine thigh-infection and pneumonitis models Scand J Infect Dis199174179ndash184

19 Onyeji CO Bui KQ Owens RC Jr Nicolau DP Quintiliani R Nightingale CHComparative efficacies of levofloxacin and ciprofloxacin against Streptococcuspneumoniae in a mouse model of experimental septicemia Int J Antimicrob Agents199912107ndash114

20 Craig WA Andes DR Correlation of the magnitude of the AUC24MIC for 6 fluoro-quinolones against Streptococcus pneumoniae with survival and bactericidal activityin an animal model In Program and Abstracts of the 40th Interscience Conferenceon Antimicrobial Agents and Chemotherapy Sept 17ndash21 2000 Toronto Canada

21 Lacy MK Lu W Xu X Nicolau DP Quintiliani R Nightingale CH Pharmacody-namic comparison of levofloxacin ciprofloxacin and ampicillin against Streptococ-cus pneumoniae in an in vitro model of infection Antimicrob Agents Chemother199943672ndash677

22 Lister PD Sanders CC Pharmacodynamics of levofloxacin and ciprofloxacin againstStreptococcus pneumoniae J Antimicrob Chemother 19994379ndash86

23 Vesga O Craig WA Activity of levofloxacin against penicillin-resistant Streptococ-cus pneumoniae in normal and neutropenic mice In Program and Abstracts of the36th Annual Interscience Conference on Antimicrobial Agents and ChemotherapyNew Orleans LA September 1996

24 Lee BL Padula AM Kimbrough RC Infectious complications with respiratorypathogens despite ciprofloxacin therapy N Engl J Med 1991325520ndash521

25 Peloquin CA Cumbo TJ Nix DE et al Evaluation of intravenous ciprofloxacin inpatients with nosocomial lower respiratory tract infections Arch Intern Med 19891492269ndash2273

26 Drusano GL Johnson DE Rosen M et al Pharmacodynamics of a fluoroquinoloneantimicrobial agent in a neutropenic rat model of Pseudomonas sepsis AntimicrobAgents Chemother 199337483ndash490

27 Thomas JK Forrest A Bhavnani SM et al Pharmacodynamic evaluation of factorsassociated with the development of bacterial resistance in acutely ill patients duringtherapy Antimicrob Agents Chemother 199842521ndash527

28 Gordon JJ Kauffman CA Superinfections with Streptococcus pneumoniae duringtherapy with ciprofloxacin Am J Med 199089383ndash384


29 Perez-Trallero E Garcia-Arenzana JM Jimenez JA et al Therapeutic failure andselection of resistance to quinolones in a case of pneumococcal pneumonia treatedwith ciprofloxacin Eur J Clin Microbiol Infect Dis 19909905ndash906

30 Righter J Pneumococcal meningitis during intravenous ciprofloxacin therapy AmJ Med 199088548

31 Frieden TR Mangi RJ Inappropriate use of oral ciprofloxacin J Am Med Assoc19902641438ndash1440

32 Giamarrellou H Activity of quinolones against gram-positive cocci Clinical fea-tures Drugs 199549(suppl 2)58ndash66

33 Cooper B Lawlor M Pneumococcal bacteremia during ciprofloxacin therapy forpneumococcal pneumonia Am J Med 198987475

34 Chen DK McGeer A DE Azavedo JC et al Decreased susceptibility of Streptococ-cus pneumoniae to fluoroquinolones in Canada N Engl J Med 1999341233ndash239

35 Linares J De La Campa AG Palleres R Fluoroquinolone resistance in Streptococcuspneumoniae N Engl J Med 19993411546ndash1547

36 Applebaum PC Microbiological and pharmacodynamic considerations in the treat-ment of infection due to antimicrobial-resistant Streptococcus pneumoniae Clin In-fect Dis 200031(suppl 2)S29ndashS34

37 Woodnut G Pharmacodynamics to combat resistance J Antimicrob Chemother20004625ndash31

38 Fishman NO Suh B Weigel LM Lorber B Gelone S Truant AL Gootz TD Chris-tie JE Edelstein PH Three levofloxacin treatment failures of pneumococcal respira-tory tract infections In Program and Abstracts of the 39th Interscience Conferenceon Antimicrobial Agents and Chemotherapy San Francisco CA September 1999

39 Davidson RJ De Azavedo J Bast D et al Levofloxacin treatment failure of pneumo-coccal pneumonia and development of resistance during therapy In Programs andAbstracts of the 40th International Conference on Antimicrobial Agents and Chemo-therapy Toronto Canada 2000

40 Hooper DC Mechanisms of action and resistance of older and newer fluoroquino-lones Clin Infect Dis 200031(suppl 2)S24ndashS28

41 Pestova E Millichap JJ Noskin GA Peterson LR Intracellular targets of moxiflox-acin A comparison with other fluoroquinolones J Antimicrob Chemother 200045583ndash590

42 Fukuda J Hiramatsu K Primary targets of fluoroquinolones in Streptococcus pneu-moniae Antimicrob Agents Chemother 199943410ndash412

43 Zhao X Xu C Domagala J Drlica K DNA topoisomerase targets of the fluoroqui-nolones A strategy for avoiding bacterial resistance Proc Natl Acad Sci USA 19979413991ndash13996

44 Ambrose PG Quintiliani R Limitations of single point pharmacodynamic analysesPediatr Infect Dis J 200019769

45 Drusano GL Preston SL Hardalo CJ et al A model using population pharmacoki-netics of ziracin with Monte Carlo simulation for preliminary MIC breakpoint selec-tion and dose selection decision support In Program and Abstracts of the 39th An-nual Interscience Conference on Antimicrobial Agents and Chemotherapy Sept 26ndash29 1999 San Francisco CA

46 Ambrose PG Grasela DM The use of Monte Carlo simulation to examine pharma-


codynamic variance of drugs fluoroquinolone pharmacodynamics against Strepto-coccus pneumoniae Diagn Microbiol Infect Dis 2000 in press

47 Owens RC Jr Ambrose PG Piper D Thomas S Pharmacodynamic comparison ofnew fluoroquinolones against Streptococcus pneumoniae using Monte Carlo analy-sis In Program and Abstracts of the 40th Interscience Conference on AntimicrobialAgents and Chemotherapy Sept 17ndash21 2000 Toronto Canada

48 Dudley MN Ambrose PG Pharmacodynamics in the study of drug resistance andestablishing in vitro susceptibility breakpoints Ready for prime time Curr OpinionMicrobiol 20003515ndash521

49 Stass H Kubitza D Pharmacokinetics and elimination of moxifloxacin after oraland intravenous administration in man J Antimicrob Chemother 199943(suppl B)83ndash90

8

Glycopeptide Pharmacodynamics

Gigi H Ross and David H Wright

Ortho-McNeil Pharmaceutical Raritan New Jersey

John C Rotschafer and Khalid H Ibrahim

University of Minnesota Minneapolis Minnesota

1 INTRODUCTION

Pharmacodynamics represents a blending of pharmacokinetic parameters with ameasure of bacterial susceptibility the minimum inhibiting concentration (MIC)As such there is a prerequisite that the pharmacokinetic parameters of the antibi-otic be adequately defined prior to exploring the drugrsquos pharmacodynamic proper-ties This in itself has not been an easy task with a drug such as vancomycinwhich has undergone several different formulation changes to remove impuritiesand increase the drugrsquos purity

Measuring vancomycin concentrations by any method other than microbio-logical assay was not possible until the late 1970s when a radioimmunoassay wasintroduced Microbiological assays were technically challenging were accurate atbest to 10 [1] and often could not be performed if patients were receivingother antibiotics

177

178 Ross et al

Pharmacokinetically vancomycin the only commercially available gly-copeptide in the United States has been characterized using one- two- andthree-compartment models as well as noncompartmentally As a result there ismodel-dependent variability in the reporting of vancomycin pharmacokineticparameters Thus getting to a point where clinically applicable pharmacodynamicparameters could be identified and quantified has not been easy Even todaythere are extremely limited in vitro animal and human data characterizing vanco-mycinrsquos performance against only a few bacteria Clearly the characterizationand quantification of vancomycin pharmacodynamics remains a work in progressThe purpose of this review is to examine the microbiology pharmacology andpharmacokinetics of vancomycin so as to build on the data presently availablefor describing the pharmacodynamics of the drug

11 History of Vancomycin

Vancomycin was first introduced in 1956 with widespread clinical use by 1958[2] Originally the drug was isolated from the actinomycete Streptomyces orien-talis however its structure and molecular weight were not identified until 1978The compound consists of a seven-membered peptide chain and two chlorinatedβ-hydroxytyrosine moieties with a molecular weight of 1449 [2] Clinical use ofthe drug was highly prevalent in the late 1950s due to the emergence of penicil-linase-producing strains of staphylococcus but it soon lost favor with the intro-duction of methicillin Impurities in early vancomycin formulations led to anunacceptable incidence of infusion-related reactions Subsequently for 20 yearsvancomycin was used exclusively for the treatment of serious staphylococcalinfections in patients with severe penicillin allergies The current Eli Lilly formu-lation marketed in 1986 is estimated to be 93 pure factor B (vancomycin) andis the result of several production changes and improved separation techniques[2] With the enhancement in purity and the heightened frequency of methicillin-resistant staphylococci and ampicillin-resistant enterococci clinical use of vanco-mycin has significantly increased Today approximately 800000 patients receivevancomycin each year accounting for 14000 kg of drug worldwide [3]

12 Antimicrobial Spectrum

Vancomycin is primarily effective against gram-positive cocci including staphy-lococcus streptococcus and enterococcus and is considered to be bactericidal(MBCMIC 4) against most gram-positive pathogens with the exception toenterococci limited numbers of tolerant (MBCMIC 32) S pneumoniae andtolerant staphylococci The National Committee for Clinical Laboratory Stan-dards has established minimum inhibitory concentration (MIC) standards of sus-ceptibility for vancomycin against staphylococci and enterococci [4] Sensitivestrains have MICs of 4 mgL intermediate isolates have MICs of 8ndash16 mg

Glycopeptide Pharmacodynamics 179

L and resistant strains have MICs 32 mgL Staphylococcus aureus and Staphy-lococcus epidermidis including both methicillin-susceptible and methicillin-re-sistant strains are usually sensitive with MIC90 values of 2 mgL [5] All strainsof Streptococcus are sensitive to vancomycin regardless of penicillin susceptibil-ity with MIC90 values less than 1 mgL [4] A recent report however claimsthat approximately 2 of S pneumoniae isolates have developed tolerance tovancomycin [6] Enterococcus faecalis organisms are typically susceptible tovancomycin with MIC50 1 mgL whereas Entercoccus faecium are generallynonsusceptible with MIC50 16 mgL [5] Vancomycin is also effective againstother Streptococcus spp Listeria monocytogenes Bacillus spp Corynebacteriaand anaerobes such as diphtheroids and Clostridium spp including C per-fringens and C difficile Vancomycin has no activity against gram-negative or-ganisms atypical pathogens fungi or viruses

2 PHARMACOLOGY

Vancomycin has multiple mechanisms of action preventing the synthesis andassembly of a growing bacterial cell wall altering the permeability of the bacte-rial cytoplasmic membrane and selectively inhibiting bacterial RNA synthesis[7] Vancomycin prevents polymerization of the phosphodisaccharidendashpentapep-tidendashlipid complex of the growing cell wall at the D-alanyl-D-alanine end of thepeptidoglycan precursor during the latter portion of biosynthesis [7ndash8] By tightlybinding the free carboxyl end of the cross-linking peptide vancomycin stericallyprevents binding to the enzyme peptidoglycan synthetase This activity occursat an earlier point and at a separate site from that of penicillins and cephalosporins[8] Therefore no cross resistance or competition of binding sites occurs betweenthe classes Vancomycin like β-lactams does require actively growing bacteriain order to exert its bactericidal effect However vancomycinrsquos bactericidal activ-ity is restricted to gram-positive organisms because the molecule is too large tocross the outer cell membrane of gram-negative species

Many factors appear to impede vancomycinrsquos bactericidal activity the ab-sence of environmental oxygen the size of the bacterial inoculum and the phaseof bacterial growth The antibiotic appears to kill bacteria more effectively underaerobic conditions than under anaerobic conditions [9] The fact that many gram-positive pathogens including streptococcus and staphylococcus can grow underaerobic and anaerobic conditions could prove problematic in clinical situationsVancomycin activity was reduced by 19 and 99 with increases in inoculumsize from 106 CFUmL to 107 and 108 CFUmL respectively [10ndash11] Whenvancomycin was evaluated against growing and nongrowing Staphylococcusepidermidis cells the drug was found to be effective only against actively grow-ing cultures [12] Finally activity is relatively unaffected by extremes in pH butis maximal at pH 65ndash80 [101113]

180 Ross et al

3 PHARMACOKINETICS

The pharmacokinetics of vancomycin are highly dependent upon the modelingmethod used to characterize the parameters Data can be found in the literaturethat characterize vancomycin using one- two- three-compartment and noncom-partmental pharmacokinetic models that employ different serum samplingschemes and vary in the duration of study As a result the literature varies in thereporting of vancomycin pharmacokinetic parameters

Absorption is complete only when the drug is given intravenously becauseoral absorption is poor and intramuscular administration is both erratic and pain-ful Vancomycin is readily absorbed after intraperitoneal administration also [14]

The distribution of vancomycin is a complex process and is best character-ized by using a multicompartmental approach Vancomycin has a large volumeof distribution varying from 04 to 06 Lkg in patients with normal renal functionand up to 09 Lkg in patients with end stage renal disease [131516] Distributionincludes ascitic pericardial synovial and pleural fluids as well as bone and kid-ney Penetration into bile however is generally considered poor Cerebral spinalfluid concentrations are minimal unless sufficient inflammation is present where10ndash15 of serum concentrations can be obtained [1315] Approximately 10ndash50 of vancomycin is protein-bound primarily to albumin providing a relativelyhigh free fraction of active drug [1317] Studies attempting to measure the effectof other serum proteins have reported virtually no binding to the reactive proteinα-1 glycoprotein but have noted binding to IgA [17]

Drug elimination is almost exclusively via glomerular filtration with 80ndash90 of the vancomycin dose appearing unchanged in the urine within 24 h inpatients with normal renal function [131516] The remainder of the dose is elim-inated via biliary and hepatic means Vancomycin when taken orally is excretedprimarily in the feces Vancomycin is not significantly removed by conventionalhemodialysis or peritoneal dialysis owing to its large molecular weight (2000)however high-flux dialyzers can remove vancomycin and other molecules withmolecular weights of less than 20000 [18]

The elimination of vancomycin is multicompartmental with an alpha ordistribution half-life of 06ndash3 h and a beta or elimination half-life of 4ndash8 hwith normal renal function [1516] Renal insufficiency can prolong the terminalhalf-life to as much as 7ndash12 days Due to the complexity of this biexponentialdecay attempts to utilize various modeling techniques are difficult A one-com-partment model inappropriately characterizes the distribution phase by formulat-ing a regression line that is a hybrid of the alpha and beta phases The pharmacoki-netic parameters produced are accordingly mythical values that may or may notrelate to the actual parameters The extrapolated peak concentration and the half-life can be greatly underestimated depending upon the sampling scheme usedGenerally pairing a serum concentration obtained early in the distribution phase


with a serum concentration late in the elimination phase results in the greatesterror Because one compartment modeling also underestimates the area under theserum concentrationndashtime curve this error is passed along in the calculation ofboth distribution volume and drug clearance

For a concentration-independent or time-dependent antibiotic vancomycinhas an almost ideal pharmacokinetic profile The drug has a large volume ofdistribution low serum protein binding and a long terminal half-life Addition-ally due to modest hepatic metabolism vancomycin-drug interactions are lim-ited As such vancomycin can be used effectively and conveniently to treat infec-tions in most body sites

4 GLYCOPEPTIDE RESISTANCE

Vancomycin has been in clinical use for over 40 years without the emergenceof resistance The multiple modes of action of vancomycin necessitate significantalterations in bacterial wall synthesis in order for the intrinsically susceptibleorganisms to develop resistance Thus the rarity of acquired vancomycin resis-tance led to predictions that such resistance is unlikely to occur on any significantscale [1920]

The first reports of vancomycin-resistant enterococci however began toappear in Europe in the mid-1980s [19] How the enterococci were able to de-velop resistance to vancomycin is unclear However several hypotheses havebeen elucidated ranging from the overuse of antibiotics to the incorporation ofglycopeptide antibiotics into animal feed

Enterococci are normal gut flora and the emergence of resistance has beenlinked to vancomycin overuse in the treatment of Clostridium difficile enterocoli-tis [20] Additionally the parenteral use of vancomycin has steadily increasedsince the late 1970s and may have played a role in the development of vancomy-cin-resistant enterococci (VRE) [21] The agricultural use of avoparcin a relatedglycopeptide may have been important in Europe but this drug has not beenused in the United States In any case the enterococci were the first class oforganisms to acquire vancomycin resistance and vancomycin resistance are nowproblematic in both Europe and the United States [20]

The genetic basis for glycopeptide resistance in enterococci is complexand is characterized by several different phenotypes Resistance-conferring genesencode a group of enzymes that enable the enterococci to synthesize cell wallprecursors generally ending in D-alanine-D-lactate rather than the usual D-ala-nine D-alanine vancomycin binding site [22ndash23] The affinity of vancomycin andteicoplanin for D-alanine-D-lactate is 1000-fold less than that for D-alanine-D-alanine [20]

The most frequently encountered resistance phenotype vanA consists ofhigh level vancomycin resistance (MIC 32 mgL) accompanied by high level

182 Ross et al

resistance to teicoplanin [22] The resistance found on vanA strains is vancomy-cin- andor teicoplanin-inducible The genes encoding vanA resistance are rela-tively easily transferred to other enterococcal species via conjugation [2223]Significant concern has been expressed in both the lay and professional literaturethat this plasmid mediated form of resistance could be passed on not only toother enterococci but also to gram-positive organisms such as staphylococciwhich could lead to catastrophic consequences worldwide Although this eventhas not been realized naturally the vanA plasmid has been successfully intro-duced into staphylococci in the laboratory raising concerns that given enoughtime vancomycin-resistant staphylococci will eventually become a clinical prob-lem [24]

Enterococci with vanB phenotypic resistance have variable levels of vanco-mycin resistance and are susceptible to teicoplanin The vanB phenotype is induc-ible by vancomycin but not teicoplanin and vancomycin exposure produces tei-coplanin resistance Genes that encode VanB are more commonly chromosomalbut can be transferred by conjugation [2225]

The vanC resistance phenotype consists of relatively low levels of vanco-mycin resistance (MIC 8ndash16 mgL) and is devoid of teicoplanin resistanceResistance to vanC is chromosomally produced by encoded genes found in allstrains of Enterococcus flavescens Enterococcus casseliflavus and Entercoccusgallinarum Genes encoded with vanC are not transferable [20] In 1996 Perichonet al [26] described a fourth phenotype vanD similar to vanB found in a rarestrain of Enterococcus faecium [26]

Following a steady increase of VRE prevalence in the United States overthe past 10 years almost 15 of enterococci in hospital intensive care units(participating in the National Nosocomial Infections Surveillance surveys) ex-hibit vancomycin resistance [2327] Similarly rapid increases in VRE prevalencehave also been observed outside the intensive care units in US hospitals [23]Approximately 70 of VRE found in the United States exhibit the vanA resis-tance phenotype with the remaining 25 mostly constituted by the vanB resis-tance phenotype [28]

Evidence exists for both clonal dissemination of resistant strains and rapidtransfer of vancomycin resistance genes among species of hospital enterococci[29ndash30] With the transfer of resistance genes multiple different enterococcalsubtypes carry the same vancomycin resistance genes suggesting a possiblelsquolsquoplasmid or transposon VRE epidemicrsquorsquo [20] Considerable heterogeneity in thegenetic sequence of vancomycin resistance genes found in the United States fur-ther suggest that these genes are being modified as they spread among the variousenterococcal strains [31]

The greatest threat VRE pose is the potential that they could transfer theirresistance encoding genes to other more pathogenic gram-positive bacteria Van-comycin resistance has been transferred from enterococci to streptococci listeria


and S aureus in vitro [2432] Also the recent description of a naturally occurringvancomycin-resistant strain of Streptococcus bovis harboring the vanB resistancephenotype is of significant concern [33]

Low-level vancomycin resistance was reported in clinical isolates of coag-ulase-negative staphylococci in the late 1980s and early 1990s [34ndash36] Al-though troubling these reports were not terribly feared due to the relative lack ofvirulence associated with the coagulase-negative staphylococci In vitro studieshowever demonstrated that both coagulase-negative staphylococci and Saureus isolates when exposed to increasing levels of glycopeptides demon-strated the ability to select for resistant subpopulations [3738] Given thesefindings and the spread of VRE for which excessive use of vancomycin wasidentified as an important control measure the prudent use of vancomycin wassuggested by the CDC as critical to prevent the emergence of resistance amongstaphylococci [39]

In May 1996 a methicillin-resistant Staphylococcus aureus (MRSA) clini-cal isolate that had reduced susceptibility to vancomycin (MIC 8 mgL) wasisolated from a 4 month-old boy with a sternal surgical incision site [4041] Thisisolate has been referred to as Mu50 by the investigators who isolated the organ-ism By current NCCLS standards this S aureus clinical isolate is classified ashaving intermediate resistance to vancomycin In August 1997 the first MRSAisolate intermediately susceptible to vancomycin was reported in Michigan andNew Jersey [4243] Since these reports the organism has been identified in NewYork and England The two US isolates exhibited different antimicrobial suscep-tibility patterns suggesting that these strains are developing de novo secondaryto vancomycin exposure All of these decreased susceptibility strains were iso-lated from patients who had received multiple extended courses of vancomycintherapy

The exact mechanism of resistance for these glycopeptide intermediate sus-ceptibility S aureus (GISA) strains remains largely unknown None of the GISAstrains isolated to date have carried the vanA or vanB genes as judged by PCRDNA amplification Changes in the GISA cell wall structure have been notedhowever and may be in part responsible for the decreased sensitivity to vancomy-cin This is inferred from three findings The cell wall appeared twice as thickas the wall of control strains on electron microscopy there was a three foldincrease in cell wall murein precursor production compared with vancomycin-susceptible MRSA strains and there was a threefold increase in the productionof penicillin-binding protein (PBP) 2 and PBP2prime [4041]

To date there is no evidence that vancomycin resistance genes have beennaturally transferred to the staphylococci or pneumococci however that does notpreclude this event from happening in the future If such a transfer of vancomycinresistance were to occur particularly if the S aureus strain is already methicillin-resistant the result would be an especially terrifying pathogen

184 Ross et al

5 PHARMACODYNAMICS

51 Introduction to Basic Principles

Evaluations of serum peakMIC ratios the ratio of the area under the serumconcentrationndashtime curve for 24 h to the MIC (AUCMIC24) and the length oftime for which antibiotic concentration exceeds the MIC of the infecting organ-ism (T MIC) have been employed as surrogate markers of the bactericidaleffects of antibiotics Pharmacodynamic indices for vancomycin have beenpoorly characterized and therefore most dosing strategies have been based onextrapolations from aminoglycoside studies By modifying aminoglycoside dos-ing models specific peak and trough concentrations have been proposed withthe assumption that similar clinical outcomes will be produced high peak concen-trations being essential for bacterial killing and definitive trough concentrationranges minimizing drug-related toxicity

On the basis of limited in vitro studies T MIC appears to most closelypredict efficacy of vancomycin Therefore the length of time the antibiotic con-centration exceeds the MIC of the offending organism and not the height of thepeak above the MIC as in aminoglycosides should be considered the goal ofthe dosing of vancomycin Although higher serum concentrations of vancomycinmay be helpful in driving the drug to relatively inaccessible sites of infectionsuch as endocardial vegetation or cerebrospinal fluid they are unlikely to improvethe rate of bacterial kill Attempting to push the dose of vancomycin for seriousbut relatively accessible infections will likely only expose patients to an increasedrisk of adverse reactions it is unlikely this approach will alter bacterial response

Investigations of other pharmacodynamic parameters including postantibi-otic effect (PAE) sub-MIC effect (SME) and postantibiotic sub-MIC effect (PASME) have also been undertaken to create a more informative depiction of van-comycin bactericidal activity than MICs allow alone The PAE or the continuedsuppression of microbial growth after limited antibiotic exposure of vancomycinagainst gram-positive bacteria can persist for several hours depending on theorganism and the initial antibiotic concentration [4445] This effect may inhibitregrowth when antibiotic concentrations fall below the MIC of the infecting or-ganism and may be important to consider when dosing vancomycin because ofthe extended half-life and prolonged dosing intervals The postantibiotic effectof vancomycin was evaluated against Staphylococcus epidermidis by Svenssonet al [12] The PAE was dependent upon concentration as drug concentrationincreased from 05 to 8 times the MIC of the organism the PAE increased from02 h to 19 h Another study found PAEs ranging from 06ndash20 h for S aureusto 43ndash65 h for S epidermidis [46]

Because patients receiving antibiotics will always have some amount ofdrug remaining in the body after dosing and elimination PAEs are typically stud-


ied in vitro SMEs and PA SMEs are parameters studied in vivo Generally allof these effects are longer when measured in vivo than when mesaured in vitroSMEs characterize the inhibition of bacterial regrowth following initial sub-MICconcentrations of antibiotic [46] Postantibiotic SMEs on the other hand illus-trate microbial suppression following bacterial exposure to supra-MIC concentra-tions that have declined below the MIC This phenomenon is important clinicallywhere patients given intermittent boluses will experience gradually lowered se-rum and tissue levels that will expose bacteria to both supra- and sub-MICs duringthe dosing interval [46]

52 In Vitro Studies

In vitro investigations have demonstrated that like β-lactam antibiotics vanco-mycin is a concentration-independent or time-dependent killer of gram-positiveorganisms and exhibits minimal concentration-dependent killing In vitro studieshowever can be limiting for several reasons [47]

1 One compartment models represent only concentrations that would ex-ist in the central compartment and not necessarily those that wouldexist at the site of infection

2 Typically only bacteria in log phase growth at standard inocula (105

or 106 CFUmL) are used3 The effects of the immune system or protein binding are generally not

considered

Despite the limitations in vitro studies appear to correlate well with animal andhuman studies and therefore provide useful information for optimal dosing strate-gies in clinical situations

Several investigators demonstrated the concentration-independent killingof vancomycin by exposing various bacteria to increasing amounts of the drugVancomycinrsquos killing effect against Staphylococcus aureus was investigated invitro by Flandrois et al [48] The early portion of the timendashkill curve was thefocus of the study to characterize the bactericidal activity in the initial phases ofthe dosing interval A decrease in CFU of only 1 log was obtained at the end ofthe 8 h study at concentrations of 1 25 5 and 10 times the MIC indicating aconcentration-independent slow rate of kill The killing phase occurred betweenhours 2 and 4 with the CFUmL being held constant for the remainder of thecurve Ackerman et al generated mono- and biexponential killing curves forvancomycin over a 2ndash50 microgmL concentration range to evaluate the relationshipbetween concentration and pharmacodynamic response against Staphylococcusaureus and coagulase-negative Staphylococcus species For all organisms tested

186 Ross et al

killing rates did not change with increasing concentrations of vancomycin andmaximum killing appears to be achieved once concentrations of 4ndash5 times theMIC of the pathogen are obtained

Because the pharmacokinetics of vancomycin involve at minimum biex-ponential decay further studies attempting to simulate this elimination and anyeffects on bacterial killing were investigated Utilizing an in vitro model simulat-ing mono- or biexponential decay Larrson et al [9] found no statistically signifi-cant difference in either the rate or extent of bacterial killing of Staphylococcusaureus Again varying concentrations did not induce a change in bactericidalactivity thereby demonstrating that the high drug concentrations achieved duringthe distribution phase did not enhance the bactericidal activity attained duringthe elimination phase

With the understanding that vancomycin killed staphylococci in a concen-tration-independent fashion the need to select a pharmacodynamic index thatbest predicts efficacy was warranted Duffull et al [47] used four different vanco-mycin regimens against S aureus in an in vitro dynamic model47 Three dosingschedules with different peak concentrations but the same AUC and a fourthdosing regimen with a smaller AUC were compared for efficacy The authorsfound that killing was independent of both peak concentrations and total exposureto drug (AUC) In addition maintaining a constant concentration above the MICwas equally effective even with an AUC that was half of that obtained by theother three dosing regimens This investigation thus supported T MIC as theoptimal parameter for efficacy

Greenberg and Benes [50] produced time-kill curves from experiments per-formed in a static environment with 50 bovine serum and constant antibioticconcentrations They reported a significantly increased rate and extent of killingof Staphylococcus aureus when the concentration of vancomycin increased from20 to 80 mgL even though free drug concentrations for all regimens exceededthe MIC by at least three fold This experiment is one of a few that demonstratedsignificant concentration-dependent killing with vancomycin alone with concen-trations beyond the MIC of the organism

Vancomycin in combination with other antimicrobials has also been evalu-ated Houlihan et al [51] investigated the pharmacodynamics of vancomycinalone and in combination with gentamicin at various dosing intervals againstStaphylococcus aureusndashinfected fibrin-clots in an in vitro dynamic model Van-comycin monotherapy simulations included continuous infusion 500 mg every6 h 1 g every 12 h and 2 g every 24 h all of which produced varying peaks andtroughs While all regimens produced concentrations above the MIC for 100of the dosing intervals no difference in kill was seen with higher peak concentra-tions The investigators also discovered that vancomycin killing was significantlyenhanced by the addition of gentamicin whether it was given every 12 or 24 hand in fact it killed in a concentration-dependent fashion The 2 g dosing scheme


of vancomycin significantly reduced bacterial counts to a greater extent than anyother combination regimen Whether this finding is due to augmented penetrationinto the fibrin clots in the presence of gentamicin is unknown

The vast majority of pharmacodynamic investigations with vancomycininclude the use of Staphylococcus aureus few studies involve other gram-positiveor anaerobic organisms Levett [52] demonstrated time-dependent killing of Clos-tridium difficile by vancomycin in vitro Vancomycin was sub inhibitory at con-centrations below the MIC of the organism Once concentrations at the MIC wereobtained no difference in kill was seen whether 4 mgL (at the MIC) or 1000mgL (250 MIC) was utilized Therefore as for other organisms vancomycinkills C difficile in a concentration-dependent manner until the MIC is achievedbeyond which time-dependent killing is observed

Odenholt-Tornqvist Lowdin and Cars have been the primary source ofinvestigations on the SMEs and PA SMEs of vancomycin In an initial studywith Streptococcus pyogenes and Streptococcus pneumoniae the investigatorsfound that the PA SME with concentrations as low as 03 the MIC preventedregrowth of both Steptococcus species for 24 h [53] In a recent in vitro investiga-tion of the pharmacodynamic properties of vancomycin against Staphylococcusaureus and Staphylococcus epidermidis the same authors detected no concentra-tion-dependent killing [46] Low killing rates were demonstrated by time to 3log kill (T3K) at 24 h with all strains the exception being a methicillin-sensitivestrain of Staphylococcus epidermidis (MSSE) that attained T3K at 9 h Regrowthoccurred between 12 and 24 h when drug concentration had declined to the MICPA SME SME and post-MIC effect (PME) were also evaluated in this studyLong PA-SMEs (23 to 20 h) were found with all strains while SMEs wereshorter (00ndash158 h) Both PA-SMEs and SMEs increased with increasing multi-ples of the MIC Interestingly longer PMEs lsquolsquothe difference in time for the num-bers of CFU to increase 1 logmL from the values obtained at the time when theantibiotic concentration has declined to the MIC compared with the correspond-ing time for a antibiotic-free growth controlrsquorsquo [46] were found with shorter half-lives Other investigations have suggested that the regrowth of bacteria can occurif insufficiently inhibited bacteria are allowed to synthesize new peptidoglycanto overcome the antimicrobialrsquos bactericidal effect [54] The authors assumedthat the PAE PA SME and PME would emulate the time for which the amountof peptidoglycan is kept below a critical level needed for bacterial growth [46]Subsequently the investigators postulated that longer PMEs may occur withshorter half-lives due to the fact that the MIC is obtained faster thereby notallowing adequate peptidoglycan production to initiate regrowth Converselyshorter PMEs were found with longer half-lives With a slower decline to theMIC and a longer period of time at the MIC sufficient peptidoglycan could beproduced to allow regrowth How PA-SMEs SMEs and PMEs will influencedosing schedules is unknown and further investigations are needed

188 Ross et al

53 Animal Studies

Animal studies focusing on pharmacodynamic predictors of efficacy for vanco-mycin are quite limited Peetermans et al [10] with a granulocytopenic mousethigh infection model showed concentration-dependent killing of staphylococcusfor concentrations at or below the MIC Once concentrations exceeded that valuehowever no further kill was seen with increasing doses

The activity of vancomycin was again evaluated against penicillin-resistantpneumococci using a mouse peritonitis model [55] In comparing variouspharmacokineticpharmacodynamic parameters at the ED50 values investigatorsconcluded that both T MIC and Cmax were important predictors of efficacyin their model These parameters were deemed best predictors because they variedthe least Also of significance with this study was the discovery that vancomycinactivity was not influenced by the penicillin susceptibility of the organism

Cantoni et al [56] in an attempt to compare the efficacy of amoxicillinndashclavulanic acid against methicillin-sensitive and methicillin-resistant Staphylo-coccus aureus (MSSA and MRSA respectively) versus vancomycin in a ratmodel of infection found vancomycin activity to be dependent upon strainAgainst the MSSA strain vancomycin at 30 mgkg given every 6 h was moreeffective than the same dose every 12 h Against the MRSA strain the fourtimes daily regimen only marginally improved outcome compared to the twice-daily regimen In that vancomycin concentrations were undetectable after 6 hof therapy the four times daily regimen was the only therapy that allowedconcentrations to remain above the MIC for a majority of the dosing intervalThis finding further supports the dependence of vancomycin activity upon the T MIC

54 Human Studies

In vivo serum bactericidal titers (SBTs) have been evaluated to determine antimi-crobial efficacy An SBT of 18 with vancomycin has been associated with clini-cal cure in patients with staphylococcal infections [57ndash58] This SBT was associ-ated with serum concentrations greater than 12 mgL James et al [59] conducteda prospective randomized crossover study to compare conventional dosing ofvancomycin versus continuous infusions in patients with suspected or docu-mented gram-positive infections In that the most effective concentration of van-comycin against staphylococcus is not known the investigators chose a targetconcentration of 15 microgmL via continuous infusion and peak and trough concen-trations of 25ndash35 and 5ndash10 microgmL respectively with conventional dosing of 1g every 12 h Despite variability in actual concentrations obtained continuousinfusion produced SBTs of 116 whereas conventional dosing produced troughSBTs of 18 which was not found to be statistically insignificant Concentrationsremained above the MIC throughout the entire dosing intervals for all patients


whether they received conventional dosing or continuous infusion and thereforethe authors concluded that both methods of intravenous administration demon-strated equivalent pharmacodynamic activities Although continuous infusiontherapy was more likely than conventional dosing to produce SBTs of 18 orgreater this study did not attempt to evaluate clinical efficacy associated withsuch values Therefore it is unknown whether improved patient outcome wasobtained

Klepser et al [60] in a preliminary report of a multicenter study of patientswith gram-positive infections receiving vancomycin therapy found increasedrates of bactericidal activity with vancomycin trough concentrations greater than10 mgL [60] Bacterial eradication was also correlated with trough SBTs of 18 or greater Patients that failed therapy had pathogen MICs of 1 mgL Hyattet al [61] suggest that the area under the inhibitory serum concentrationndashtimecurve (AUIC) as well as the organismrsquos MIC were associated with clinical out-come By performing a retrospective analysis of 84 patients receiving vancomy-cin therapy for gram-positive infections these authors found that therapy thatproduced AUIC 125 and pathogens with MICs 1 mgL had a higher likeli-hood of failure Therefore these two studies propose that not only T MIC butalso trough values may be important for maximum clinical efficacy

In summary vancomycin demonstrates concentration-independent killingof gram-positive bacteria and peak concentrations do not appear to correlate withrate or extent of kill Maximum killing is achieved at serum concentrations of4ndash5 times the MIC of the infecting pathogen and sustaining concentrations ator above these levels for the entire dosing interval will likely produce the bestantimicrobial effect Dosing strategies should therefore be aimed at maximizingthe time in which concentration at the site of infection remains above the MICof the pathogen Whether the most efficient killing is obtained by continuousinfusion of vancomycin or by intermittent bolus is controversial Several studiesrevealed that no difference in killing is seen between the two methods of adminis-tration [515962] however such benefits as predictable serum concentrationsand ease of administration might be advantageous [62] Conversely due to vanco-mycinrsquos long half-life and the perceived better tolerability associated with inter-mittent bolus injections continuous infusion of this drug may not be needed andis often discouraged [62]

6 CLINICAL APPLICATION

61 Clinical Uses

Vancomycin is available as vancomycin hydrochloride (Vancocin LyphocinVancoled and others) for intravenous use as powder for oral solution and ascapsules for oral use (Vancocin Pulvules) The indications for vancomycin use

190 Ross et al

are limited in relation to its strong gram-positive spectrum Although vancomycinis bactericidal against most gram-positive cocci and bacilli the intravenous prepa-ration should be reserved for serious gram-positive infections not treatable withβ-lactams or other traditional options The use of vancomycin should not precedetherapy with β-lactams for susceptible organisms Clinical outcomes in bothstaphylococci and enterococci show vancomycin inferiority as compared to naf-cillin and ampicillin regarding bactericidal rate and rapidity of blood sterility[63ndash67]

Vancomycin is the drug of choice for serious staphylococcal infections thatcannot be treated with β-lactams due to bacterial resistance [methicillin-resistantStaphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epid-ermidis (MRSE)] or to the patientrsquos inability to receive these medications [68ndash70] Staphylococcal infections include bacteremia endocarditis skin and softtissue infections pneumonia and septic arthritis Dialysis peritonitis due to staph-ylococci may also be treated with IV vancomycin Although vancomycin is indi-cated for S aureus osteomyelitis bone penetrations are extremely variable espe-cially between published studies and treatment with other options could provemore effective [71ndash75] Vancomycin is also indicated for infections due to coagu-lase-negative staphylococci including catheter-associated bacteremia prostheticvalve endocarditis vascular graft infections prosthetic joint infections centralnervous system shunt infections and other infections associated with indwellingmedical devices [68ndash70] Complete cure of most medical-device-related infec-tions usually requires the removal of the device due to the biofilm secreted bythe S epidermidis Staphylococcal treatment with vancomycin may require upto 1 week or longer for clinical response in serious infections such as MRSA[70] Courses of vancomycin that fail to cure serious staphylococcal infectionsmay require the addition of gentamicin rifampin or both [697076]

Two significant clinical issues surround the use of vancomycin for the treat-ment of staphylococcal endocarditis First controversy exists as to whether theaddition of rifampin is synergistic or antagonistic Although certain studies haveproven the combination to be more efficacious than single therapy with vancomy-cin [77ndash79] other more recent publications site the combination as antagonistic[65] Additionally clinical experience with the combination has been inconsistent[80]

The second issue that surrounds vancomycin use for staphylococcal endo-carditis is the potentially better outcome with β-lactams In addition to the invitro data that suggest that vancomycin is less rapidly bactericidal than nafcillinclinical data exist to support this conclusion [63ndash67] Although no large-scalecomparison studies exist to evaluate the efficacy of vancomycin versus β-lactamsin staphylococcal endocarditis assumptions can be formulated from publishedstudies In a study by Korzeniowski and Sande [67] the duration of bacteremiadue to S aureus endocarditis lasted a median of 34 days after treatment with


nafcillin whereas bacteremia lasted a median of 7 days for patients treated withvancomycin in a study conducted by Levine et al [65] The patients in the Levinestudy were infected with methicillin-resistant S aureus in comparison to themethicillin-sensitive organisms from the Korzeniowski study yet in general themorbidity and mortality of bacteremic infections due to MSSA and MRSA arecomparable [66] In a small study that compared vancomycin to nafcillin in Saureus endocarditis the investigators found that patients treated with nafcillinplus tobramycin had a cure rate of 94 whereas only 33 of patients treatedwith vancomycin plus tobramycin were cured [64] Worth mentioning howeveris the fact that while the nafcillin plus tobramycin group consisted of 50 patientsonly three patients received vancomycin plus tobramycin due to β-lactam allergySmall and Chambers [63] performed another study that evaluated the use of van-comycin in 13 patients with staphylococcal endocarditis five of whom failedtherapy The reason for vancomycin ineffectiveness in these cases may be theneed for prolonged high levels of a bactericidal antibiotic however with longerdurations of bacteremia and poorer clinical outcomes serious consideration needsto be given to whether vancomycin should be considered at all in patients withMSSA endocarditis who can tolerate β-lactam therapy

Streptococcal infections not treatable with β-lactams or other traditionaloptions are also proper indications for vancomycin [68ndash70] Endocarditis due toβ-lactam-resistant S viridans or S bovis is a common use of vancomycin al-though organisms with elevated MIC values may require that it be combinedwith an aminoglycoside Vancomycin is the drug of choice for pneumococcalinfections showing high-level resistance to penicillin [68ndash70] Cefotaxime orceftriaxone plus rifampin may be needed to adequately cover S pneumoniae men-ingitis due to vancomycinrsquos poor penetration in the central nervous system[81ndash82] Although penetration is enhanced while meninges are inflamed as inmeningitis and shunt infections certain cases may require intrathecal or intraven-tricular administration to obtain therapeutic levels

As for enterococcal infections vancomycin represents the treatment ofchoice for ampicillin-resistant enterococcus [68ndash70] Enterococcus endocarditisand other infections may require the addition of an aminoglycoside such as genta-micin Vancomycin is also the treatment of choice for corynebacterial infections[68ndash70]

Empirically vancomycin should be used only in limited situations Vanco-mycin can be considered for febrile neutropenic patients presenting with clinicalsigns and symptoms of gram-positive infections in areas of high MRSA preva-lence [39] Other indications for empirical use of vancomycin in neutropenicpatients with fever include the presence of severe mucositis colonization withMRSA or penicillin-resistant Streptococcus pneumoniae prophylaxis with quino-lone antibiotics or obvious catheter-related infection [83] Vancomycin shouldbe discontinued after 4ndash5 days if no infection is identified or if initial cultures

192 Ross et al

for gram-positive organisms are negative after 24ndash48 h For prophylaxis vanco-mycin may be used perioperatively with prosthesis implantation only in severelyβ-lactam allergic patients [39] Vancomycin is also used for endocarditis prophy-laxis for β-lactam allergic patients

Orally vancomycin is indicated for metronidazole-refractory antibiotic-associated colitis caused by Clostridium difficile [3968ndash70] Intravenous admin-istration of vancomycin typically does not achieve adequate levels in the colonlumen to successfully treat antibiotic-associated colitis however there are rarereports of success with this route cited in the literature84 Administration via na-sogastric tube enema ileostomy colostomy or rectal catheter may be neededif the patient presents with severe ileus Oral vancomycin has also been usedprophylactically to prevent endogenous infections in cancer and leukemia pa-tients This regimen seems to decrease the C difficile associated with the chemo-therapy [85ndash87]

62 Inappropriate Uses

Although vancomycin is an effective option for most gram-positive infectionsthe drug needs to be judiciously used to prevent the emergence and spread ofresistance Vancomycin should not be used when other drug options such as β-lactams are viable Microbial susceptibilities need to be treated to determine theappropriateness of vancomycin therapy and the antibiotic should be changed ifthe organism is susceptible to a different agent

The CDC has published guidelines for the appropriate use of vancomycin(Tables 1 and Table 2) [39] however vancomycin misuse around the nation iswidespread A retrospective study from May 1993 to April 1994 identified 61 ofvancomycin usage as inappropriate according to the CDC criteria [88] A similarevaluation published in 1997 found that only 47 of vancomycin orders pre-scribed for 7147 patients were appropriate [89] According to this study inade-

TABLE 1 Appropriate Use of Vancomycin

Treatment of serious infections due to β-lactam-resistant gram-positivepathogens

Treatment of gram-positive infections in patients with serious β-lactamallergies

Antibiotic-associated colitis failure to metronidazoleEndocarditis prophylaxis per American Heart Association

recommendationsAntibiotic prophylaxis for implantation of prosthetic devices at institutions

with a high rate of infections due to methicillin-resistant staphylococci

Source Ref 37


TABLE 2 Inappropriate Use of Vancomycin

Routine surgical prophylaxisEmpirical treatment for febrile neutropenic patients without strong

evidence of gram-positive infection and high prevalence of β-lactamresistant organisms in the institution

Treatment in response to a single positive blood culture for coagulase-negative staphylococci when other blood cultures taken appropriately inthe same time frame are negative

Continued empirical use without positive culture for β-lactam-resistantgram-positive pathogen

Systemic or local prophylaxis for central or peripheral catheterSelective gut decontaminationEradication of methicillin-resistant Staphylococcus aureus colonizationPrimary treatment of antibiotic-associated colitisRouting prophylaxis for patients on chronic ambulatory peritoneal dialysisRoutine prophylaxis for very low birthweight infantsTopical application or irrigation

Source Ref 37

quate use and inappropriate control patterns were similar whether large teachingcenters or small rural hospitals were evaluated As such alternative methods ofvancomycin control need to be implemented to ensure adequate use and limitresistance

63 Toxicity and Adverse Drug Reactions

A variety of adverse reactions have been associated with vancomycin includingfever rash phlebitis neutropenia nephrotoxicity auditory toxicity interstitialnephritis and infusion-related reactions Many of the infusion-related reactionswere likely due to impurities in the initial formulations and have been signifi-cantly reduced with the newer formulations The red man or red neck syndromeis an anaphylactoid reaction related to rapid infusion of large doses typically12 mg(kg sdot h) [1369ndash70] The reaction begins 10 min after infusion and gener-ally resolves within 15ndash20 min after stopping the dose Patients may experiencetachycardia chest pain dyspnea urticaria and swelling of the face lips andeyelids Additionally patients may experience a hypotensive episode with a 25ndash50 reduction in systolic blood pressure Interestingly volunteers receiving van-comycin infusions have a higher propensity toward the reaction than patients [62]The reason is unknown Symptoms of red nan syndrome appear to be histamine-mediated however investigations are inconclusive Extending the administrationof vancomycin to 1 h or a maximum of 15 mgmin should prevent most infusion-related reactions

194 Ross et al

Vancomycin toxicity was retrospectively studied by Farber and Moellering[90] in 98 patients They noted a 13 incidence of phlebitis a 3 incidence offever and rash and a 2 incidence of neutropenia However this report mayoverestimate true adverse reactions because of the inclusion of many potentiallyhigh-risk patients Interestingly whereas other studies have shown that concomi-tant aminoglycosides are not a risk factor for nephrotoxicity [91] patients receiv-ing both vancomycin and an aminoglycoside experienced a 35 incidence ofreversible nephrotoxicity which is more than expected from either antibioticalone Only 5 of patients receiving vancomycin alone experienced nephrotoxic-ity The authors also found that patients with nephrotoxicity had trough concen-trations of 20ndash30 mgL

Vancomycin ototoxicity has been reported with peak serum concentrationsof 80ndash100 mgL [92] Geraci [92] identified two patients with vancomycin-induced ototoxicity one of whom had a history of renal disease an elevatedblood urea nitrogen on admission and a recorded diastolic blood pressure ofzero Serum concentrations determined 3ndash6 h after the dose was administeredranged from 80 to 95 mgL Due to the biexponential nature of the vancomycinserum concentrationndashtime curve the true vancomycin peak was likely near 200ndash300 mgL Farber and Moellering [90] also reported the occurrence of ototoxicityin a patient who at 1 h postinfusion had serum concentrations of 50 mgLhowever the true peak was likely in the toxic range as defined by Geraci [92]

In summary the incidence of adverse reactions associated with vancomycinare relatively infrequent Only approximately 40 cases of oto- and nephrotoxicitywere reported in the medical literature in the years 1956ndash1984 despite incessantuse Most of these cases were complicated by concomitant aminoglycoside ther-apy and pre-existing renal problems as well as investigator discrepancies in inter-preting serum levels

64 Dosing and Therapeutic Monitoring

Medical literature abounds that questions the need to therapeutically monitor van-comycin concentrations Cantu et al [93] suggest that monitoring vancomycinconcentrations is unnecessary in that no correlation has been demonstrated be-tween drug levels toxicity and clinical response Opponents propose that vanco-mycin can be dosed using published nomograms based on the the patientrsquos ageweight and estimated creatinine clearance Conversely Moellering et al [94]argue that therapeutic vancomycin monitoring would in fact be prudent for opti-mal clinical response and restriction of toxicity in such situations as patients onhemodialysis patients with rapidly changing renal function and patients receiv-ing high dose vancomycin or concomitant aminoglycoside therapy

Numerous strategies do exist for empirically dosing vancomycin Adminis-tering 500 mg every 6 h 1 g every 12 h or 20ndash40 mgkg body weightday are


commonly employed In addition nomograms exist such as those established byMatzke et al [95] Moellering et al [94] Lake and Peterson [96] and Nielsenet al [97] Serious faults lie in the dependence of these nomograms on efficacioususe of vancomycin however because the authors assume rather than prove thattheir method of pharmacokinetically modeling the data was appropriate Mostempirical regimens were designed to provide peak concentrations of 20ndash40 mgL and trough concentrations of 5ndash10 mgL (or approximately 5 times the MICof the infecting pathogen) however such practices place only 3ndash23 of patientsin this therapeutic range according to one published study [98] Unfortunatelyalthough such goals in serum levels are set no solid data are available to supportthis therapeutic range and accordingly serum peak and trough concentrationshave been selected somewhat arbitrarily based on speculations from retrospec-tive studies case reports and personal opinions Peak concentrations appear toplay little to no role in the efficacy of the drug and appear to have limited involve-ment in toxicity unless exceedingly large peak values are obtained On the otherhand trough concentrations may be useful monitoring parameters Because van-comycin is a concentration-independent killer the goal of therapy should be tomaintain the unbound concentration above the microbial MIC for a significantportion of the dosing interval because regrowth of most organisms will beginshortly after drug concentrations fall below the MIC A depiction of predictedvancomycin pharmacodynamic indices obtained from a typical intravenous doseusing various pathogen MICs is presented in Table 3

The role of vancomycin degradation products also needs to be consideredwhen interpreting levels in patients with renal failure where half-lives are signifi-cantly extended [99ndash100] In vitro and in vivo vancomycin breaks down overtime to form crystalline degradation products Antibodies in commercial assayssuch as TDx fluorescence polarization immunoassay cross react with major and

TABLE 3 Estimated Vancomycin PharmacodynamicRatios for Various MIC Valuesa

MIC (mgL) CpmaxMIC T MIC (h) AUC24MIC

025 140 12 78405 70 12 39210 35 12 19620 175 12 9840 875 12 4980 438 11 245

a Calculations based on a 1 g dose given every 12 h to a 70 kgpatient with normal renal function

196 Ross et al

minor degradation products thereby overstating factor B (active drug) content inthe level This can result in an overstated vancomycin concentration of 20ndash50

In summary trough concentrations of 5ndash10 mgL appear to be reasonablegoals for vancomycin therapy in that MICs of most gram-positive pathogens are1 mgL Such concentrations would allow the unbound concentrations to re-main above the MIC of the organism for the entire dosing interval Administering10ndash15 mgkg per dose and adjusting the dosing interval per renal function basedupon numerous published nomograms is not likely to produce lsquolsquotoxicrsquorsquo peak con-centrations and should allow lsquolsquotherapeuticrsquorsquo concentrations throughout the dosinginterval in the majority of patients with normal renal function Loading dosesare not typically needed because transiently high distribution phase concentra-tions are unlikely to enhance bacterial killing However loading doses may bereasonable in patients in whom the site of infection is distal to the central compart-ment or poorly accessible Until a relationship among clinical efficacy toxicityand vancomycin concentration is established vancomycin therapy will inevitablycontinue to be monitored in an attempt to improve patient outcome Whethertherapeutic monitoring of vancomycin should be a standard of practice or is nec-essary only in patients receiving high dose therapy patients on concomitantaminoglycoside therapy or patients with renal insufficiency or failure on dialysisis likely to remain a personal preference until further studies establish guidelinesHowever if the CDC guidelines for appropriate vancomycin usage were strin-gently followed at least half of vancomycin use could be eliminated leaving theremaining patients to be monitored

7 OTHER GLYCOPEPTIDES

71 Teicoplanin

Teicoplanin like vancomycin binds to the terminal D-alanyl-D-alanine portionof the peptidoglycan cell wall of actively growing gram-positive bacteria to exertits bactericidal activity [101] Currently available only in Europe teicoplanin canbe used to treat infections caused by both methicillin-sensitive and -resistantstrains of Staphylococcus aureus S epidermidis streptococci and enterococciClinical trials have demonstrated teicoplanin to be a safe well tolerated agentwith reports of side effects occurring in 6ndash13 of recipients [101] The mostprevalent adverse reactions reported are pain at the injection site and skin rashNephro- and ototoxicity are uncommon even when the drug is used concomitantlywith other nephro- and ototoxic drugs Pharmacokinetically teicoplanin differsfrom vancomycin The half-life is considerably longer (47 h) and the percentprotein-bound nears 90 [101] Also teicoplanin can be administered by eitherthe intravenous or intramuscular route as opposed to vancomycin which is lim-ited parenterally to the intravenous route Pharmacodynamic evaluations virtually


duplicate those of vancomycin once the heightened protein binding of teicoplaninand subsequent lower active free concentrations are accounted for [102] Furtherreviews of teicoplanin can be found elsewhere [101103]

72 LY333328

LY333328 (Eli Lilly and Company) is a synthetic glycopeptide that is currentlybeing developed to treat gram-positive bacterial infections including those resis-tant to vancomycin Because it is still in the early stages of development littleis known about the antibiotic The drug acts on the same molecular target asvancomycin and other glycopeptide antibiotics [104] however LY333328 ap-pears to display concentration-dependent bactericidal activity against gram-positive pathogens [102ndash106] The half-life is long approaching 105 dayswhich may allow for infrequent dosing [107] Pharmacodynamic investigationsand clinical efficacy trials are needed prior to drug approval and utilization

8 CONCLUSION

With years of clinical experience vancomycin has proven to be a safe and effica-cious agent against gram-positive pathogens including many multidrug-resistantstrains Despite this history to date the therapeutic range has not been rigorouslydefined however going beyond the currently suggested therapeutic range is notlikely to improve antibiotic performance The accumulation of in vitro and invivo studies suggests that vancomycin is a concentration-independent killer ofgram-positive organisms with maximum killing occurring at serum concentra-tions of 4ndash5 times the MIC of the infecting organism High peak concentrationsare not associated with an improved rate or extent of kill and therefore therapyshould be targeted toward sustaining serum concentrations above the MIC for alarge portion of the dosing interval With the high level of vancomycin use thedevelopment and spread of vancomycin-resistant organisms is a formidable andpredictable occurrence At a time when we are attempting to be more prudentand judicious in the use of vancomycin we also find ourselves more dependenton the drug Unfortunately this combination of factors may drive bacterial resis-tance and ultimately nullify a drug that has been a gold standard product for ahalf a century

REFERENCES

1 KB Crossley JC Rotschafer MM Chern KE Mead DE Zaske Comparison of aradioimmunoassay and a microbiological assay for measurement of serum vanco-mycin concentrations Antimicrob Agents Chemother 198017654ndash657

2 GL Cooper DB Given The development of vancomycin In GL Cooper and DB

198 Ross et al

Given eds Vancomycin A Comprehensive Review of 30 Years of Clinical Re-search Park Row Publ 19861ndash6

3 HA Kirst DG Thompson TI Nicas Historical yearly usage of vancomycin Antimi-crob Agents Chemother 1998421303ndash1304

4 National Committee for Clinical Laboratory Standards Performance standards forantimicrobial susceptibility testing 9th Informational Supplement M100-S9 1999Vol 19(1) National Committee for Clinical Laboratory Standards Wayne PA

5 RN Jones CH Ballow DJ Biedenbach JA Deinhart JJ Schentag Antimicrobialactivity of quinupristindalfopristin (RP 59500 Synercid) tested against over28000 recent clinical isolates from 200 medical centers in the United States andCanada Diagn Microbiol Infect Dis 199831437ndash451

6 Nature 1999399524ndash526590ndash5937 PE Reynolds Structure biochemistry and mechanism of action of glycopeptide

antibiotics Eur J Clin Microbiol Infect Dis 19898943ndash9508 PE Reynolds EA Somner Comparison of the target sites and mechanisms of glyco-

peptide and lipoglycodepsipeptide antibiotics Drugs Under Exp Clin Res 199016385ndash389

9 AJ Larsson KJ Walker JK Raddatz JC Rotschafer The concentration-independenteffect of monoexponential and biexponential decay in vancomycin concentrationon the killing of Staphylococcus aureus under aerobic and anaerobic conditionsJ Antimicrob Chemother 199638589ndash597

10 WE Peetermans JJ Hoogeterp AM Hazekamp-VanDokkum P Van Den BroekH Mattie Antistaphylococcal activities of teicoplanin and vancomycin in vitro andin an experimental infection Antimicrob Agents Chemother 1990341869ndash1874

11 KC Lamp MJ Rybak EM Bailey GW Kaatz In vitro pharmacodynamic effectsof concentration pH and growth phase on serum bactericidal activities of dapto-mycin and vancomycin Antimicrob Agents Chemother 1992362709ndash2714

12 E Svensson H Hanberger LE Nilsson Pharmacodynamic effects of antibiotics andantibiotic combinations on growing and nongrowing Staphylococcus epidermidiscells Antimicrob Agents Chemother 199741107ndash111

13 TS Lundstrom JD Sobel Vancomycin trimethoprim-sulfamethoxazole and rifam-pin Infect Dis Clin N Am 19959747ndash767

14 GD Morse MA Apicella JJ Walshe Absorption of intraperitoneal antibiotics DrugIntell Clin Pharm 19982258ndash61

15 RC Moellering Pharmacokinetics of vancomycin J Antimicrob Chemother 198414(suppl D)43ndash52

16 JC Rotschafer K Crossley DE Zaske K Mead RJ Sawchuk LD Solem Pharmaco-kinetics of vancomycin Observation in 28 patients and dosage recommendationsAntimicrob Agents Chemother 198222391ndash394

17 H Sun EG Maderazo AR Krusell Serum protein-binding characteristics of vanco-mycin Antimicrob Agents Chemother 1993371132ndash1136

18 GR Matzke RF Frye Drug therapy individualization for patients with renal insuf-ficiency In JT DiPiro RL Talbert GC Yee GR Matzke BG Wells LM Poseyeds Pharmacotherapy A Physiologic Approach 3rd ed Stamford CT Appleton ampLange 19971083ndash1103


19 N Woodford AP Johnson D Morrison DCE Speller Current perspectives on gly-copeptide resistance Clin Microbiol Rev 19958585ndash615

20 RC Moellering Vancomycin-resistant enterococci Clin Infect Dis 1998261196ndash1199

21 J Ena RW Dick R Jones RP Wenzel The epidemiology of intravenous vancomy-cin usage in a university hospital J Am Med Assoc 1993269598ndash602

22 M Arthur PE Reynolds F Depardieu et al Mechanisms of glycopeptide resistancein enterococci J Infect Dis 19963211ndash16

23 HS Gold RC Moellering Jr Drug therapy Antimicrobial-drug resistance N EnglJ Med 19963351445ndash1454

24 WC Noble Z Virani RGA Cree Co-transfer of vancomycin and other resistancegenes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus FEMSMicrobiol Lett 199293195ndash198

25 R Quintiliani S Evers P Courvalin The vanB gene confers various levels of self-transferable resistance to vancomycin in enterococci J Infect Dis 1993161220ndash1223

26 B Perichon PE Reynolds P Courvalin VanD-type glycopeptide-resistant Entero-coccus faecium (abst LB12) In Program and Abstracts of the 36th InterscienceConference on Antimicrobial Agents and Chemotherapy (New Orleans) Washing-ton DC Am Soc Microbiol 19965

27 Centers for Disease Control and Prevention Nosocomial enterococci resistant tovancomycinmdashUnited States 1989ndash1993 MMWR Morb Mortal Wkly Rep 199342597ndash599

28 NC Clark RC Cooksey BC Hill JM Swenson FC Tenover Characterization ofglycopeptide-resistant enterococci from US hospitals Antimicrob Agents Chemo-ther 199342597ndash599

29 LG Rubin V Tucci E Cercenado GM Eliopoulos HD Isenberg Vancomycin resis-tant Enterococcus faecium in hospitalized children Infect Control Hosp Epidemiol199213700ndash705

30 JF Boyle SA Saumakis A Rendo et al Epidemiologic analysis and genotypiccharacteristic of a nosocomial outbreak of vancomycin-resistant enterococci J ClinMicrobiol 1993311280ndash1285

31 S Handwerger J Skoble LF Discotto MJ Pucci Heterogeneity of the vanA genecluster in clinical isolates of enterococci from the northeastern United States Anti-microb Agents Chemother 199539362ndash368

32 F Biavasco E Giovanetti A Miele C Vignaroli B Facinelli PE Varalso In vitroconjugative transfer of vanA vancomycin resistance between enterococci and lister-iae of different species Eur J Clin Microbiol Infect Dis 19961550ndash59

33 C Poyart C Pierre G Quesne et al Emergence of vancomycin resistance in thegenus Streptococcus Characterization of a vanB transferable determinant in Strep-tococcus bovis Antimicrob Agents Chemother 19974124ndash29

34 RS Schwalbe JT Stapleton PH Gilligan Emergence of vancomycin resistance incoagulase-negative staphylococci N Engl J Med 1987316927ndash931

35 LA Veach MA Pfaller M Barrett FP Koontz RP Wenzel Vancomycin resistancein Staphylococcus haemolyticus causing colonization and bloodstream infectionJ Clin Microbiol 1990282064ndash2068

200 Ross et al

36 D Sanyal AP Johnson RC George BD Cookson AJ Williams Peritonitis dueto vancomycin-resistant Staphylococcus epidermidis Lancet 199133754

37 RS Schwalbe WJ Ritz PR Verma EA Barranco PH Gilligan Selection for van-comcyin resistance in clinical isolates of Staphylococcus haemolyticus J Infect Dis199016145ndash51

38 RS Daum S Gupta R Sabbagh WM Milewski Characterization of Staphylococcusaureus isolates with decreases in susceptibility to vancomycin and teicoplanin Iso-lation and purification of a constitutively produced protein associated with de-creased susceptibility J Infect Dis 19921661066ndash1072

39 Hospital Infection Control Practices Advisory Committee Recommendations forpreventing the spread of vancomycin resistance Recommendations of the HospitalInfection Control Practices Advisory Committee (HICPAC) MMWR Morb MortalWkly Rep 199544(12)

40 K Hiramatsu H Hanaki T Ino K Yabuta T Oguri FC Tenover Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin suscepti-bility J Antimicrob Chemother 199740135ndash136

41 Centers for Disease Control and Prevention Reduced susceptibility of Staphylococ-cus aureus to vancomycinmdashJapan 1996 MMWR Morb Mortal Wkly Rep 199746(27)624ndash628

42 Centers for Disease Control and Prevention Staphylococcus aureus with reducedsusceptibility to vancomycinmdashUnited States 1997 MMWR Morb Mortal WklyRep 199746(33)756ndash766

43 Centers for Disease Control and Prevention Update Staphylococcus aureus withreduced susceptibility to vancomycinmdashUnited States 1997 MMWR Morb MortalWkly Rep 199346(35)813ndash815

44 WA Craig B Vogelman The post-antibiotic effect Ann Intern Med 1987106900ndash902

45 MA Cooper YF Jin JP Ashby JM Andrews R Wise In vitro comparison of thepostantibiotic effect of vancomycin and teicoplanin J Antimicrob Chemother 199026203ndash207

46 E Lowdin I Odenholt O Cars In vitro studies of pharmacodynamic properties ofvancomycin against Staphylococcus aureus and Staphylococcus epidermidis Anti-microb Agents Chemother 1998422739ndash2744

47 SB Duffull EJ Begg ST Chambers ML Barclay Efficacies of different vancomy-cin dosing regimens against Staphylococcus aureus determined with a dynamic invitro model Antimicrob Agents Chemother 1994382480ndash2482

48 JP Flandrois G Fardel G Carret Early stages of in vitro killing curve of LY146032and vancomycin for Staphylococcus aureus Antimicrob Agents Chemother 198832454ndash457

49 Ackerman AM Vannier E Eudy Analysis of vancomycin time-kill studies withStaphylococcus species by using a curve stripping program to describe the relation-ship between concentration and pharmacodynamic response Antimicrob AgentsChemother 1992361766ndash1769

50 RN Greenberg CA Benes Time-kill studies with oxacillin vancomycin and tei-coplanin versus Staphylococcus aureus J Infect Dis 1611036ndash1037 1990

51 HH Houlihan RC Mercier MJ Rybak Pharmacodynamics of vancomycin alone


and in combination with gentamicin at various dosing intervals against methicillin-resistant Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infectionmodel Antimicrob Agents Chemother 1997412497ndash2501

52 PN Levett Time-dependent killing of Clostridium difficile by metronidazole andvancomycin J Antimicrob Chemother 19912755ndash62

53 I Odenholt-Tornqvist E Lowdin O Cars Postantibiotic sub-MIC effects of vanco-mycin roxithromycin sparfloxacin and amikacin Antimicrob Agents Chemother1992361852ndash1858

54 D Greenwood K Bidgood M Turner A comparison of the responses of staphylo-cocci to teicoplanin and vancomycin J Antmicrob Chemother 198720155ndash164

55 JD Knudsen K Fuursted F Espersen N Frimodt-Moller Activities of vancomycinand teicoplanin against penicillin-resistant pneumococci in vitro and in vivo andcorrelation to pharmacokinetic parameters in the mouse peritonitis model Antimi-crob Agents Chemother 1997411910ndash1915

56 L Cantoni A Wenger MP Glauser J Bille Comparative efficacy of amoxicillin-clavulanate cloxacillin and vancomycin against methicillin-sensitive and methicil-lin-resistant Staphylococcus aureus endocarditis in rats J Infect Dis 1989159989ndash993

57 UB Schadd GH McCracken JD Nelson Clinical pharmacology and efficacy ofvancomycin in pediatric patients J Pediatr 198096119ndash126

58 DB Louria T Kaminski J Buchman Vancomycin in severe staphylococcal infec-tions Arch Intern Med 1961107225ndash240

59 JK James SM Palmer DP Levine MJ Rybak Comparison of conventional dosingversus continuous-infusion vancomycin therapy for patients with suspected or doc-umented gram-positive infections Antimicrob Agents Chemother 199640696ndash700

60 ME Klepser SL Kang BJ McGrath et al Influence of vancomycin serum concen-tration on the outcome of gram-positive infections Presented at The American Col-lege of Clinical Pharmacy Annual Winter Meeting Feb 6ndash9 1994 San Diego

61 JM Hyatt PS McKinnon GS Zimmer JJ Schentag The importance ofpharmacokineticpharmacodynamic surrogate markers to outcome Clin PharmConcepts 199528143ndash160

62 ME Klepser KB Patel DP Nicolau R Quintiliani CH Nightingale Comparison ofbactericidal activities of intermittent and continuous infusion dosing of vancomycinagainst methicillin-resistant Staphylococcus aureus and Enterococcus faecalisPharmacotherapy 1998181069ndash1074

63 PM Small HF Chambers Vancomycin for Staphylococcus aureus endocarditis inintravenous drug users Antimicrob Agents Chemother 1990341227ndash1231

64 HF Chambers RT Miller MD Newman Right sided Staphylococcus aureus endo-carditis in intravenous drug abusers Two-week combination therapy Ann InternMed 1998109619ndash624

65 DP Levine BS Fromm BR Reddy Slow response to vancomycin or vancomycinplus rifampin in methicillin-resistant Staphylococcus aureus endocarditis Ann In-tern Med 1991115674ndash680

66 AW Karchmer Staphylococcus aureus and vancomycin The sequel Ann InternMed 1991115739ndash741

202 Ross et al

67 O Korzeniowski MA Sande National Collaborative Endocarditis Study GroupCombination antimicrobial therapy for Staphylococcus aureus endocarditis in pa-tients addicted to parenteral drugs and in nonaddicts Ann Intern Med 198297496ndash503

68 Anonymous The choice of antibacterial drugs Med Lett Drugs Ther 19984033ndash42

69 RH Glew MA Keroack Vancomycin and teicoplanin In SL Grobach JG BartlettNR Blacklow eds Infectious Diseases WB Saunders Philadelphia 1998260ndash269

70 R Fekety Vancomycin and teicoplanin In GL Mandell JE Bennett R Dolin Prin-ciples and Practice of Infectious Diseases 4th ed Churchill Livingstone NewYork 1995346ndash353

71 CW Norden K Niederreiter EM Shinners Treatment of experimental chronic os-teomyelitis due to staphylococcus aureus with vancomycin and rifampin J InfectDis 1983147352ndash357

72 C Martin M Alaya MN Mallet X Viviand K Ennabli R Said PD Micco Penetra-tion of vancomycin into mediastinal and cardiac tissues in humans AntimicrobAgents Chemother 199438396ndash399

73 L Massias C Dubois P de Lentdecker O Brodaty M Fischler R Farinotti Penetra-tion of vancomycin in uninfected sternal bone Antimicrob Agents Chemother1993362539ndash2541

74 AL Graziani LA Lawson GA Gibson MA Steinberg RR MacGregor Vancomy-cin concentrations in infected and noninfected human bone Antimicrob AgentsChemother 1998321320ndash1322

75 JR Torres CV Sanders AC Lewis Vancomycin concentrations in human tissuePreliminary report J Antimicrob Chemother 19795475

76 V Gopal AL Bisno FJ Silverblatt Failure of vancomycin treatment in Staphylococ-cus aureus endocarditis In vivo and in vitro observations J Am Med Assoc 19762361604ndash1606

77 AS Bayer K Lam Efficacy of vancomycin plus rifampin in experimental aortic-valve endocarditis due to methicillin-resistant Staphylococcus aureus In vitro-invivo correlations J Infect Dis 1985151157ndash165

78 RM Massanari ST Donta The efficacy of rifampin as adjunctive therapy in selectedcases of staphylococcal endocarditis Chest 197873371ndash375

79 RJ Faville DE Zaske EL Kaplan K Crossley LD Sabath PG Quie Staphylococ-cus aureus endocarditis Combined therapy with vancomycin and rifampin J AmMed Assoc 19782401963ndash1965

80 DP Levine RD Cushing J Ji WJ Brown Community-acquired methicillin-resistant Staphylococcus aureus endocarditis in the Detroit Medical Center AnnIntern Med 198297330

81 PF Viladrich F Gudiol J Linares R Pallares I Sabate G Rufi J Ariza Evaluationof vancomycin for therapy of adult pneumoccocal meningitis Antimicrob AgentsChemother 1991352467ndash2472

82 JS Bradley WM Scheld The challenge of penicillin-resistant Streptococcus pneu-moniae meningitis current antibiotic therapy in the 1990s Clin Infect Dis 199724(suppl 2)S213ndashS221


83 WT Hughes D Armstrong GP Bodey AE Brown JE Edwards R Feld P PizzoKVI Rolston JL Shenep LS Young 1997 guidelines for the use of antimicrobialagents in neutropenic patients with unexplained fever Clin Infect Dis 199725551ndash573

84 ST Donta GM Lamps RW Summers TD Wilkins Cephalosporin-associated coli-tis and Clostridium difficile Arch Intern Med 1980140574ndash576

85 JG Bartlett Antibiotic-associated colitis Disease-A-Month 1984301ndash5486 SD Miller HJ Koornhof Clostridium difficile colitis associated with the use of

antineoplastic agents Eur J Clin Microbiol 1984310ndash1387 MA Cudamore J Silva R Fekety MK Liepman KH Kim Clostridium difficile

colitis associated with cancer chemotherapy Arch Intern Med 1982142333ndash33588 SV Johnson LL Hoey K Vance-Bryan Inappropriate vancomycin prescribing

based on criteria from the Centers for Disease Control and Prevention Pharmaco-therapy 199515579ndash585

89 C Gentry Wide overuse of antibiotic cited in study Wall St J 19974B-190 BF Farber RC Moellering Retrospective study of the toxicity of preparations of

vancomycin from 1974 to 1981 Antimicrob Agents Chemother 19832313891 K Vance-Bryan JC Rotschafer SS Gilliland KA Rodvold CM Fitzgerald DR

Guay A comparative assessment of vancomycin-associated nephrotoxicity in theyoung versus the elderly hospitalized patient J Antimicrob Chemother 199433811ndash821

92 JE Geraci Vancomycin Mayo Clin Proc 19775263193 TG Cantu NA Yamanaka-Yuen PS Lietman Serum vancomycin concentrations

Reappraisal of their clinical value Clin Infect Dis 199418533ndash54394 RC Moellering DJ Krogstad DJ Greenblatt Vancomycin therapy in patients with

impaired renal function A nomogram for dosage Ann Intern Med 198194343ndash346

95 GR Matzke JM Kovarik MJ Rybak SC Boike Evaluation of the vancomycin-clearance Creatinine-clearance relationship for predicting vancomycin dosageClin Pharm 19854311ndash315

96 KD Lake CD Peterson A simplified dosing method for initiating vancomycin ther-apy Pharmacotherapy 19855340ndash344

97 HE Nielsen HE Hansen B Korsager PE Skov Renal excretion of vancomycin inkidney disease Acta Med Scand 1975197261ndash264

98 HZ Zokufa HA Rodvold RA Blum LJ Riff JH Fischer KB Crossley JCRotschafer Simulation of vancomycin peak and trough concentrations using fivedosing methods in 37 patients Pharmacotherapy 1989910ndash16

99 NJ Saunders SV Want DJ Adams Vancomycin monitoring in renal failure Varia-tion between assays In Program and Abstracts of the 34th Interscience Conferenceof Antimicrobial Agents and Chemotherapy 1994 Orlando FL Am Soc Microbi-ology Washington DC A-31

100 AL Somerville DH Wright JC Rotschafer Implications of vancomycin degrada-tion products on therapeutic drug monitoring in patients with end-stage renal dis-ease Pharmacotherapy 199919702ndash707

101 KW Shea BA Cunha Teicoplanin Med Clin N Am 199579833ndash 844102 H Lagast P Dodion J Klastersky Comparison of pharmacokinetics and bacteri-

204 Ross et al

cidal activity of teicoplanin and vancomycin J Antimicrob Chemother 198618513ndash520

103 AP MacGowan Pharmacodynamics pharmacokinetics and therapeutic drug moni-toring of glycopeptides Therap Drug Monit 199820473ndash477

104 NE Allen DL LeTourneau JN Hobbs Molecular interactions of a semisyntheticglycopeptide antibiotic with D-alanyl-D-alanine and D-alanyl-D-lactate residuesAntimicrob Agents Chemother 19974166ndash71

105 TI Nicas JE Flokowitsch DA Preston DL Mullen J Grissom-Arnold NJ Snyderet al Semisynthetic glycopeptides active against vancomycin-resistant enterococciActivity against staphylococci and streptococci in vitro and in vivo In Abstractsof the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy1995F-248

106 M Robbins D Felmingham Cidal activity of LY 333328 a new glycopeptideagainst Enterococcus spp In Program and Abstracts of the 20th International Con-ference of Chemotherapy Syndey Australia 19974292

107 J Chien S Allerheiligen D Phillips B Cerimele HR Thomasson Safety and phar-macokinetics of single intravenous doses of LY333328 diphosphate (glycopeptide)in healthy men In Programs and Abstracts of the 38th Interscience Conferenceon Antimicrobial Agents and Chemotherapy San Diego CA 1999A-55

9

Macrolide Azalide and KetolidePharmacodynamics



Holly M Mattoes

DesignWrite Incorporated Princeton New Jersey

1 INTRODUCTION

The macrolides and azalides have activity against gram-positive bacteria and arerelatively weakly active against many gram-negative bacteria These agents alsopenetrate well into mammalian tissue and achieve high concentrations in mamma-lian cells and are therefore very useful in the treatment of infections caused byintracellular pathogens Their spectrum of activity makes them a good choicefor the treatment of community acquired respiratory tract infections because theorganisms associated with such diseases usually involve Streptococcus pneumon-iae Haemophilus influenzae and Moraxella cattarhalis and frequently involveintracellular organisms (Table 1) [1ndash3] The macrolides and azalides (either asthe parent compound or in combination with a microbiologically active metabo-lite) have adequate activity against these pathogens and have emerged as usefuland popular agents for the treatment of milder forms of these diseases

205

206 Nightingale and Mattoes

TABLE 1 MIC90 Values of Organisms Commonly Associatedwith Community Acquired Respiratory Tract Infections

Organism Erythromycin Clarithromycin Azithromycin

S pneumoniae 003 0015 012S pyogenes 003 0015 012H influenzae 4 1(w14-OH) 05M catarrhalis 025 025 006M pneumoniae 001 003 0001C pneumoniae 006 0015 05Legionella sp 1 0125 0125

The macrolides and azalides bind to the 50S ribosome in the bacterial celland therefore interfere with the production of bacterial protein [45] The proteinis essential for the bacterial life cycle and because of the presence of the antibi-otic the bacteria cannot make the needed protein and eventually die or stop repro-ducing Although it is essential for bacterial life apparently existing stores ofthe protein must be depleted before effects of the antibiotic are noticeable As aresult many studies that use concentrations in the therapeutic range classify thesecompounds as bacteriostatic and pharmacodynamic studies have shown them tobe concentration independent (time-dependent) bacterial killers [6]

The first commercially available antibacterial agent in popular worldwideuse was erythromycin It is used as a single agent for the treatment of a varietyof community associated respiratory tract infections and as a second agent (dueto its effectiveness against intracellular pathogens) as part of a combination regi-men for hospitalized patients Although still popular as a less costly macrolideoption it does cause gastrointestinal side effects that many patients cannot toler-ate for a complete course of therapy This has implications for the selection ofmacrolide resistant organisms which is the topic of another chapter in this bookAdditionally its half-life is shorter than those of the newer agents requiring dailyfrequent multi-dosing The drug is also owing to its metabolic pathway of elimi-nation involved in a number of drug interactions [78] The popularity of erythro-mycin is decreasing and it is being replaced in clinical use with the newer macro-lide and azalide drugs (clarithromycin roxithromycin and azithromycin) As aresult less attention will be devoted to erythromycin in this chapter and the phar-macodynamics of the macrolidesazalides will be discussed with a focus on thenewer drugs

Information on the new ketolide class of antibiotics semisynthetic macro-lide characterized by the replacement of the clandinose moiety with a ketonegroup will also be discussed [9] The ketolides are effective in treating grampositive pathogens especially those exhibiting resistance to macrolides by utiliz-

Macrolide Azalide and Ketolide Pharmacodynamics 207

ing the same mechanism of action as the macrolide and azalide compounds [10]Due to the high achievable drug concentrations in white blood cells and broncho-pulmonary tissues the ketolides are especially effective against common respira-tory pathogens and intracellular organisms [11]

Because the purpose of this chapter is to discuss the pharmacodynamicsof these agents issues such as utility in clinical use side effect profiles and druginteractions will not be discussed The reader is referred to standard textbooksand primary literature references on these topics Additionally no attempt willbe made to provide every detail on pharmacodynamic properties of each drugRather there are certain principles that are important for this class of agents froma pharmacodynamic perspective and these principles will be discussed and illus-trated using some (but not all) of the newer agents

2 OVERVIEW OF MACROLIDE AND AZALIDE

PHARMACODYNAMICS

As discussed in an earlier chapter antibiotics can be classified as either bacterio-static or bactericidal drugs and further subcategorized as concentration dependentor time dependent bacterial killers In reality every antibiotic has all of theseproperties however the property that emerges and upon which the classificationis dependent is a function of the concentrations achieved after clinical dosingrelative to its activity against the pathogen For the macrolides the currentlyavailable data indicate that when the serum plasma concentrations are highenough the pharmacodynamic parameter that correlates best with bacterial eradi-cation is the time for which the organism is exposed to adequate concentrationsof these agents (clarithromycin roxithromycin) [12] Obviously if the concentra-tion of the drug is not high enough one cannot make the simplifying assumptionthat the concentration part of the AUC (Cp time) contributes little to the bacte-rial eradication process and can be ignored In such a case bacterial eradicationis related not just to the time of exposure of the bacteria to the drug (T MIC)but also to the concentration of drug to which the bacteria are exposed (AUC)Bacteria that have relatively high MICs may correlate clinical cure rates betterto the pharmacodynamic parameter AUCMIC This may become an issue as theMICs of pathogens increase owing to the emergence of resistant strains At thepresent time however most bacteria worldwide for which the macrolides havebeen useful agents still have MICs low enough such that the pharmacodynamicparameter of T MIC is appropriate This will probably change as resistanceincreases and may already have changed in some countries Although the exactduration of exposure that is adequate to cure or manage an infection in the clinicalsetting is not known animal experiments and in vitro data [12] suggest that agood working number is a time above the MIC of approximately 50 of thedosing interval for patients with a functioning host defense system [12ndash15] Ani-


mal studies in which neutropenia was induced indicate that superior animal sur-vival was associated with drug concentrations of 4ndash5 times the MIC [1213]This suggests that more appropriate pharmacodynamic criteria for neutropenicpatients might be a T MIC of 100 of the dosing interval

It has been reported that azithromycin has a prolonged post antibiotic effect(PAE) [1213] It is controversial whether this PAE is sufficiently long to signifi-cantly affect clinical outcome The usual regulatory agency approved doses ofdrugs are based on clinical studies that are derived after treatment with a particu-lar dose and dosing regimen This incorporates the total properties of the drugsand is then correlated to clinical outcome To state this in another way goodclinical outcomes are due to the dose and dosing regimen of the drug used Thistakes into account concentration issues and drug exposure issues which includeissues of protein binding and PAE It has been stated however that because ofthe long PAE of azithromycin the proper pharmacodynamic parameter wouldnot be the T MIC but rather the AUCMIC ratio Unfortunately there areinsufficient data to substantiate this assumption in humans Another assumptionalso unsubstantiated is that azithromycin like the macrolides eradicates bacteriaby having an adequate exposure time (T MIC) For the macrolides we sug-gested a T MIC of approximately 50 of the dosing interval Because of thePAE of azithromycin an appropriate number might be somewhat less than 50ie 35 or 40 This hypothesis is also possible however further investigationis needed to clarify this issue

3 TISSUE DISTRIBUTION

The macrolides and azalides share the common property of extensive distributioninto mammalian tissues Other drug classes such as the quinolones also have thisproperty but not to the extent exhibited by the macrolides and azalides Oneconsequence of this as noted above is that the latter particularly useful in thetreatment of infections caused by intracellular pathogens Another consequenceof this property is that it is responsible for the drugsrsquo longer serum half-life com-pared to erythromycin (Table 2) [1617] Examination of Table 2 reveals that allof the newer agents have a longer half-life than erythromycin The azithromycinhalf-life is particularly long (actually this is a working half-life the true half lifeis even longer than shown in this table) [41618ndash20] The reason for the longazithromycin half-life is not the fact that the normal excretory routes of the bodyhave difficulty in eliminating this drug Rather the body can only eliminate whatis available to it The rate of appearance to excretory organs is a function of itsrelease from tissue storage sites Kinetically we can calculate only the sloweststep in the elimination process which in the case of azithromycin is release fromtissue The long half-life of azithromycin therefore is really an estimate of thehalf-life of tissue elimination


TABLE 2 Important Parameters of the Pharmacokinetics of Macrolides

Erythromycin Clarithromycin Azithromycin RoxithromycinParameter (250 mg) (500 mg) (250 mg) (150 mg)

Half-life 2ndash4 3ndash4 68 8ndash15Cpmax

(microgmL) 025ndash05 2 024 66ndash79

Absorption 35ndash40 50ndash55 37 mdashAUC (microgmL) mdash 1214 21 726ndash81

Distribution into tissue sites has ramifications related to the clinical use ofthese agents This can be illustrated by referring to two studies of a similar nature[1213] In these studies human volunteers were dosed to steady state with clar-ithromycin and azithromycin Plasma samples were obtained and bronchial la-vage was performed allowing the calculation of epithelial lining fluid (ELF) drugconcentrations and determination of alveolar macrophage (AM) concentrationsof both drugs The results of both experiments demonstrate the same relationshipsbetween drug concentrations in these sites as they relate to the microbiologicalactivity of target pathogens

It is interesting to examine three figures that emanate from these studiesFigure 1 shows the plasma concentrations of both agents at steady state and in

FIGURE 1 Plasma concentrations of clarithromycin and azithromycin with Spneumoniae MIC90


this figure the typical MIC for S pneumoniae is superimposed upon the kineticcurves One can see that for clarithromycin concentrations in plasma at steadystate yield a T MIC of 100 of the dosing interval The azithromycin curveindicates that this drug stays above the MIC of this pathogen (when a 250 mgonce per day dose is used) for somewhat less than 50 of its dosing intervalOf course if all the pathogens are eradicated or if the remaining pathogens canbe eliminated by the patientrsquos host defense mechanisms then this drug would beexpected to be clinically useful against this organism However if any bacteriasurvive such a dose and dosing regimen they are exposed to subtherapeutic con-centrations of active azithromycin for long periods of time Theoretically this isa condition that can select for resistant organisms and exists because of the exten-sive tissue distribution properties of azithromycin [12] Resistance issues are dis-cussed more completely in another chapter of this book

As described in an earlier chapter the drug and bacteria need to be in thesame place at the same time in order for the antibiotic to function by interferingwith the bacterial life cycle The question of where the bacteria reside in relation-ship to the drug is an important and yet unresolved issue This affects the applica-tion of pharmacodynamics to any agent that has extensive tissue distribution andis a topic of discussion in the next section Assuming however that in a lunginfection the extracellular bacteria reside in some interstitial fluid such as theELF it is the concentration in the ELF that might be of clinical importanceFigure 2 shows the calculated ELF concentrations for each agent and again theMIC for S pneumoniae is superimposed on the kinetic curves In this case bothdrugs clearly show that the T MIC is 100 of the dosing interval This analysis

FIGURE 2 Epithelial lining fluid concentrations of clarithromycin and azithro-mycin with S pneumoniae MIC90


needs to be determined with each target pathogen to predict clinical differencesin the treatment of respiratory infections between these drugs

Finally as mentioned previously the macrolides and azalides have utilityin the treatment of respiratory tract infections caused by intercellular organismsExamination of Fig 3 allows one to understand the relationship between penetra-tion into mammalian tissue and the MIC of these target organisms Figure 3shows the penetration of these drugs into the alveolar macrophage which is usedas a marker for mammalian tissue uptake It can be seen that the AM concentra-tions for both drugs are well above the MIC for some typical intracellular patho-gens (T MIC for 100 of the dosing interval) One would predict that the twoagents would be equally effective in eradicating intracellular bacteria in a clinicalsetting

Pharmacodynamically the ketolide telithromycin demonstrates a concen-tration dependent kill rather than a time dependent kill In vitro studies also havesupported the theory that telithromycin exhibits primarily concentration depen-dent and inoculum dependent bacteriostatic activity [21] Additionally animalstudies have determined the AUCMIC ratio to be the best predictor of in vivoefficacy for telithromycin and therefore once daily dosing is an appropriate dos-ing regimen for this ketolide [22] A study evaluating telithromycinrsquos pharmaco-kinetics after an 800 mg oral dose demonstrated a Cmax of 190 mgl AUC of896 mghl and Tmax of 10 h [23] As a result of this favorable kinetic profileeffective AUCMIC ratios are observed for respiratory pathogens A summaryof MICs of telithromycin for these organisms is listed in Table 3 H influenzae

FIGURE 3 Alveolar macrophage concentrations of clarithromycin and azithro-mycin with S pneumoniae MIC90


TABLE 3 Ketolide Pharmaacodynamics

Telithromycin ABT773

MIC90 MIC90

Streptococcus pneumoniae 006 mgL 003mgLHaemophilus influenzae 2 mgL 4 mgLMoraxella cattarhalis 006 mgL 012 mgLMycoplasma pneumoniae 0004 mgL NRChlamydia pneumoniae 025 mgL NRLegionella spp 003 mgL 1 mgL

NR Not reported at this time

has an increased MIC for telithromycin but still demonstrates similar in vitroactivity to azithromycin and clarithromycin against this organism Treatmentsuccess relies heavily on factors other than the interaction of the antibiotic withthe bacteria These were previously described and include the patientrsquos immuno-competency ABT 773 demonstrates time dependent kill but unfortunately thereare few pharmacokinetic data published and therefore the TMIC cannot beaccurately determined at the present time However it is expected to demonstratean adequate pharmacodynamic profile against its target organisms ie thosecausing community-acquired respiratory infections

For good clinical cure of respiratory intracellular organisms the alveolarmacrophage drug concentration is an important consideration because this iswhere the organism is believed to reside A study evaluating steady state pharma-codynamics of the ketolide telithromycin in healthy volunteers demonstratedgood penetration into the bronchopulmonary tissues surpassing the plasma con-centration in these individuals The mean concentrations of this ketolide in thealveolar macrophages epithelial lining fluid and bronchial mucosa exceeds theMIC for most common respiratory pathogens [24] High intracellular concentra-tions of this drug were also observed in the white blood cells of healthy volun-teers again exceeding the MIC for the typical intracellular organisms for 100of the dosing interval [25]

It is unfortunate that one is exposed to the pseudo pharmacodynamic termpenetration ratio We call the term pseudo pharmacodynamic because the wayit is used implies that it has pharmacodynamic meaning but usually insufficientinformation is given to understand its meaning To illustrate this point we referyou to Table 4 which came from the same datasets Examination of this tablereveals that the clarithromycin AMplasma ratio goes from approximately 340to 1300 whereas that of azithromycin goes from approximately 1300 to 15000Also note that these data were obtained after the last dose of the drug was givenGenerally the ratio increases as a function of time after the last dose This is


TABLE 4 ClarithromycinAzithromycin AlveolarMacrophagePlasma Ratio

AM cell ratioTime afterdosing (h) Clarithromycin Azithromycin

4 543 12928 465 15237

12 1041 1280724 1265 14386

intuitively problematic because one expects to see concentrations decrease afterdosing Upon reflection one must realize that the ratio has a numerator and de-nominator To make the ratio larger one can increase the numerator or decreasethe denominator or both Usually insufficient data are presented to determinewhich is occurring and the meaning of the ratio is therefore unknown It is indeedunfortunate that when one sees a high ratio one assumes that the numerator islarge Examination of this table and the plasma and AM concentration data (Figs1 and 3) reveals that the ratio is increasing because the denominator (plasmaconcentrations) is decreasing much faster than the numerator (AM concentra-tions) after dosing is stopped The high ratios in this case are not a function ofsuperior tissue concentration (Fig 3) but of differences between the drugsrsquo AMand plasma concentrations (Fig 3) ie the lower the plasma concentration thehigher the ratio As a result one must evaluate lsquolsquopenetrationrsquorsquo data carefully tofully understand that importance of delivering drug to the body site where thebacteria may reside

4 APPROPRIATE PHARMACODYNAMIC MODEL

To understand the practical applicability of the current pharmacodynamic modelto drugs such as the macrolides and azalides we must remember two importantcriteria First the pharmacodynamic models index microbiological activity toserum or plasma concentrations The question is whether this is appropriate fora drug class that has a large degree of tissue distribution The second factor iswhere the pathogen reside in the body Figure 4 helps clarify this latter issue[1617] Bacteria that are not in intimate contact with mammalian cells eg ina mucus layer of the respiratory tract are not pathogens They may be detectedafter laboratory tests but they are colonizers For bacteria to be pathogens theyusually need to attach to a mammalian cell membrane (extracellular organisms)or penetrate into the mammalian cell (intracellular organisms) Let us considerextracellular organisms that act as pathogens and examine the existing pharmaco-dynamic model to see how it applies to this class of agents


FIGURE 4 Proposed stages of infection of the bronchial mucosa IgA immu-noglobulin A

Figure 5 schematically shows the typical time dependent pharmacodynamicmodel as it applies to β-lactam antibiotics These drugs do not penetrate intomammalian cells (the square boxes) The drug concentration shown by the Arsquosin the schematic represent the average interstitial fluid concentration which weassume is represented by the average serum or plasma concentration (which iswhat we usually measure) If this average concentration is high enough (2ndash4

FIGURE 5 Time-dependent pharmacodynamic model


FIGURE 6 Macrolide pharmacodynamic model in mammalian cells

times the MIC) and remains above the MIC for a long enough period of time (T MIC for 50 of the dosing interval) then the drug is expected to be clinicallyeffective in eradicating the pathogens A similar model is schematically shownin Fig 6 In this figure the macrolides and azalides penetrate into the mammaliancells If the average drug concentration is high enough (2ndash4 times the MIC) andis exposed to the bacteria for a long enough period of time (T MIC for 50of the dosing interval) then the bacteria will be exposed to lethal concentrationsof drug This occurs when the MIC of the bacteria is very low ie the bacteriais sensitive to the drug In essence the model collapses to the β-lactam modelIf the MIC of the bacteria were high relative to the average serum concentrationsthen this model would predict that the bacterial would not be eradicated This isillustrated in Table 5 which shows the pharmacodynamic prediction when the

TABLE 5 Macrolide PharmacodynamicAnalysismdashH influenzae

Macrolide T MIC CpkMIC AUCMIC

Erythromycin 250 mg 0 013 mdashClarithromycin 500 mg 3 025 12Azithromycin 250 mg 0 048 5


macrolides or azalides are targeted against an organism with a higher MIC suchas H influenzae Whether one prefers the pharmacodynamic parameter of AUCMIC T MIC or CpMIC the result is the same The pharmacodynamic parame-ters are too low to predict clinical success of these drugs against this pathogenClinical studies indicate that macrolidesazalides are effective in respiratory tractinfections when H influenzae is involved Obviously there is a mismatch betweenwhat nature is telling us and what the existing model is predicting It is our beliefthat this model does not fully apply when the pathogen has a relatively high MICSimply put if the model indexes microbiological data to average serum or plasmaconcentrations and the organism resides elsewhere (attached to the outer mem-brane of the bacteria) indexing microbiological activity to serum concentrationmay not be correct if the MIC is high To explain this further one must recognizethat although these drugs tend to easily move into tissues they do not remainthere forever They slowly (azithromycin) or not so slowly (clarithromycin) comeout of these tissue sites A concentration gradient probably exists with the highestconcentrations just outside the mammalian cell membrane the place where patho-gens are believed to reside These pathogens could be exposed to high concentra-tions of active antibiotic and be killed as a result This could explain the apparentparadox between clinical results and pharmacodynamic predictions for bacteriawith higher MICs As time goes by and resistance continues to develop the MICswill continue to increase At some point the concentrations will be too high to

FIGURE 7 Macrolide pharmacodynamic model in the presence of white bloodcells


even evoke this expanded model and we will clinically observe patient failuresupon therapy Of course the entire situation is further complicated by the hostrsquosimmune system (Fig 7) White blood cells are attracted to the site of infectionbecause the bacteria secrete chemotactic factors They are also capable of en-gulfing bacteria and killing them and it is believed that they actually bring activedrug to the lsquolsquobattlefieldrsquorsquo None of this has been adequately studied and representshypothesis to some extent It is necessary however to evoke an explanationlike the one presented above to explain the discrepancy between the existingpharmacodynamic models and human clinical empirical data The point is thatthe macrolide and azalide pharmacodynamic situation is rather complex and asimple pharmacodynamic model that indexes microbiological activity to serumconcentrations cannot fully explain such a complex situation

5 FUTURE CONSIDERATIONS

The macrolides azalides and ketolides are a useful class of drugs for the treat-ment of respiratory tract infections For these agents however owing to theirability to easily penetrate into mammalian tissue serum concentrations appearto be predictive of clinical outcome for only very sensitive organisms For organ-isms with higher MICs the drug concentrations at the site in the body where thebacteria reside may be of more clinical relevance however these data are difficultor even impossible to obtain in humans It is indeed unfortunate that the reportingof resistant organisms is not strongly base on the pharmacodynamic characteris-tics of these drugs They are determined by the setting of so-called breakpointswhich are an interpretation of mostly in vitro and some clinical and pharmacody-namic data These breakpoints can be somewhat arbitrarily set in spite of thegood intentions and hard work of the officials setting them It is possible as inthe case of macrolides to report many isolates as resistant but the reportingsystem does not provide any information about the magnitude of the resistanceKetolides which in a sense are macrolides with activity against macrolide resis-tant agents are an exception to this phenomenon at the present time Whetherthe drug (macrolide or azalide) can eradicate the organism depends on the serumand tissue concentrations achieved in relationship to the MIC If drug concentra-tions are high enough for a long enough time after the usual clinical doses then theorganism will be adversely affected (die) regardless of whether one classifies it asresistant or sensitive Clarithromycin has been developed in an extended releaseform in an attempt to increase drug concentrations over a longer period of timeThese tablets are taken once daily and have a steady state AUC equivalent to con-ventional immediate release clarithromycin which is taken twice daily [26]

Unfortunately the reporting of in vitro resistance data has a strong impacton the drug selection process because for the most part macrolide and azalidetherapy is empirical This issue is not easy to address because the existing phar-macodynamic data use serum or plasma as their index to microbiological efficacy


which may not be appropriate for the macrolides when they are targeted againstorganisms with higher MICs that reside in bodily fluids whose drug concentrationprofiles are not the same as that of serum As time passes and more organismsbecome resistant and therefore have higher MICs this situation will become moreconfused and one can predict that the macrolide class of antibiotics will probablybe replaced by the lsquolsquorespiratoryrsquorsquo quinolones for the empirical therapy of respira-tory tract infections This may be an unfortunate occurrence that may relegatethe macrolides to the role of adjunctive agent in the treatment or prophylaxisof infections believed to be caused by intracellular pathogens This would beunfortunate if it is based upon erroneous resistance data and as a result deprivespatients of an acceptable and somewhat unique (due to its high tissue penetrationand immunological properties) class of agents

REFERENCES

1 LA Mandell Community-acquired pneumonia Etiology epidemiology and treat-ment Chest 1995108(2)35Sndash41S

2 D Lieberman F Schlaeffer I Boldur D Lieberman S Horowitz MG Friedman MLeiononen O Horovitz E Manor A Porath Multiple pathogens in adult patientsadmitted with community-acquired pneumonia A one year prospective study of 346consecutive patients Thorax 199651179ndash184

3 HA Cassiere MS Niederman Community-acquired pneumonia Disease of theMonth 1998(Nov)622ndash675

4 SC Piscitelli LH Danziger KA Roldvold Clarithromycin and azithromycin Newmacrolide antibiotics Clin Pharm 199211137ndash152

5 MS Whitman AR Tunket Azithromycin and clarithromycin overview and compari-osn with erythromycin Infection Control Hosp Epidemiol 199213357ndash368

6 A Bauernfeind R Jungwirth E Eberlein Comparative pharmacodynamics of clar-ithromycin and azithromycin against respiratory pathogens Infection 199523(5)316ndash321

7 TM Ludden Pharmacokinetic interactions of the macrolide antibiotics Clin Pharma-cokinet 19851063ndash79

8 P Periti T Mazzei E Mini A Novelli Pharmacokinetic drug interactions of macro-lides Clin Pharmacokinet 199223106ndash131

9 C Agouridas A Bonnefoy JF Chanton In vitro antibacterial activity of HMR 3647a novel ketolide highly active against respiratory pathogens [abstract F-112] InProgram and Abstracts of the 37th Interscience Conference on AntimicrobialAgents and Chemotherapy (Toronto Ontario) Washington DC Am Soc Microbiol1997

10 C Marie-Bigdot D Decre C Muller E Salgado E Bergogne-Berezin In vitro activ-ity of a novel ketolide antimicrobial agent HMR 3647 compared to erythromycinroxithromycin azithromycin and clarithromycin [abstract F-117] In Program andAbstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemo-therapy (Toronto Ontario) Washington DC Am Soc Microbiol 1997


11 RP Smith AL Baltch W Ritz M Franke P Michelson Antibacterial effect ofketolides HMR 3004 and HMR 3647 against intracellular Legionella pneumophilia[abstract F-249] In Program and Abstracts of the 37th Interscience Conference onAntimicrobial Agents and Chemotherapy (Toronto Ontario) Washington DC AmSoc Microbiol 1997

12 RC Owens DP Nicolau R Quintiliani CH Nightingale Bactericidal activity of clar-ithromycin azithromycinand cefuroxime against penicillin-susceptible -intermedi-ate and -resistant pneumococci 20th Int Congress of Chemotherapy (Sydney Aus-tralia) June 29ndashJuly 3 1997

13 JG Den Hollander JD Knudsen JW Mouton F Fuursted N Frimodt-Moller HAVerbrugh F Espersen Comparison of pharmacodynamics of azithromycin anderythromycin in vitro and in vivo Antimicrob Agents Chemother 199842377ndash382

14 WA Craig Interrelationship between pharmacokinetics and pharmacodynamics indetermining dosage regimens for broad-spectrum cephalosporins Diagn Microb In-fect Dis 19952289ndash96

15 B Vogelman S Gudmundson J Leggett J Turnidge S Ebert WA Craig Correlationof antimicrobial pharmacokinetic parameters with therapeutic efficacy in animalmodel J Infect Dis 1998158831ndash847

16 F Kees M Wallenhofer H Grobecker Serum and cellular pharmacokinetics of clar-ithromycin 500mg qd and 250mg bid in volunteers Infection 199523168ndash172

17 G Foulds RM Shepard RB Johnson The pharmacokinetics of azithromycin in hu-man serum and tissues J Antimicrob Chemother 199025(suppl A)73ndash82

18 A Wildfeuer H Laufen T Zimmermann Uptake of azithromycin by various cellsand its intracellular activity under in vivo conditions Antimicrob Agents Chemother19964075ndash79

19 GW Amsden AN Nafziger G Foulds Pharmacokinetics in serum and leukocyteexposure of oral azithromycin 1500 milligrams given over a 3- or 5-day periodin healthy subjects Antimicrob Agents Chemother 199943163ndash165

20 G Panteix B Guillaumond R Harf A Desbos V Sapin M Leclercq M Perrin-Fayolle In-vitro concentration of azithromycin in human phagocytic cells J Antimi-crob Chemother 199331(suppl E)1ndash4

21 FJ Boswell JM Andrews R Wise Pharmacodynamic properties of HMR 3647 anovel ketolide demonstrated by studies of time-kill kinetics and post antibiotic effect[abstract F-254] In Program and Abstracts of the 37th Interscience Conference onAntimicrobial Agents and Chemotherapy (Toronto Ontario) Washington DC AmSoc Microbiol 1997

22 O Vesga C Bonnat WA Craig In vivo pharmacodynamic activity of HMR 3647a new ketolide [abstract F-255] In Program and Abstracts of the 37th InterscienceConference on Antimicrobial Agents and Chemotherapy (Toronto Ontario) Wash-ington DC Am Soc Microbiol 1997

23 F Namour DH Wessels MH Pascual D Reynolds E Sultan Dose proportionalityof the pharmacokinetics of HMR 3647 a new ketolide antimicrobial in healthy adultmales [abstract 79] In Program and Abstracts of the 37th Annual Meeting of theInfectious Diseases Society of America (Philadelphia PA) Alexandria VA InfectDis Soc America 1999


24 CM Serieys C Cantalloube P Soler HP Gia F Brunner HMR 3647 achieves highand sustained concentrations in broncho-pulmonary tissues [abstract P78] In Ab-stracts of the 21st International Congress of Chemotherapy (Birmingham UK) Ox-ford UK Br Soc Antimicrob Chemother 1999

25 HP Gia V Roeder F Namour E Sultan B Lenfant HMR 3647 achieves high andsustained concentrations in white blood cells in man [abstract P79] In Abstractsof the 21st International Congress of Chemotherapy (Birmingham UK) OxfordUK Br Soc Antimicrob Chemother 1999

26 LE Gustavson GX Cao SJ Semla RN Palmer Pharmacokinetics of a new extended-release clarithromycin tablet at doses of 500 and 1000 mg daily [abstract A-1191]In Program and Abstracts of the 39th Interscience Conference on AntimicrobialAgents and Chemotherapy (San Francisco CA) Washington DC Am Soc Micro-biol 1999

10

Metronidazole Clindamycin andStreptogramin Pharmacodynamics

Kenneth Lamp

Bristol-Myers SquibbPlainsboro New Jersey

Melinda K Lacy

University of Kansas Medical CenterKansas City Kansas

Collin Freeman

Bayer Corporation West Haven Connecticut

1 METRONIDAZOLE

Metronidazole (Flagyl) is a nitroimidazole antibiotic that has been available foruse in clinical practice for over 25 years Its original indication was for treatmentof infections caused by Trichomonas vaginalis but over the years it has beendiscovered to be useful in treating a variety of infections caused by various organ-isms Although the nitroimidazole class of antibiotics includes tinidazole secni-dazole and ornidazole the wealth of information about the pharmacokineticsand pharmacodynamics of these agents comes from published data about metroni-dazole However even with the vast amount of information about metronidazolethere is very little information regarding its pharmacodynamic properties com-

221

222 Lamp et al

pared to other antimicrobial agents such as the β-lactams aminoglycosides andfluoroquinolones The only distinguishing features of tinidazole ornidazole orsecnidazole are prolonged T12 values compared to metronidazole

11 Chemistry and Mechanism of Action

Metronidazole is a nitroimidazole (chemical name 1-[β-hydroxyethyl]-2-methyl-5-nitromidazole) that belongs to the same chemical class as tinidazole and ornida-zole (structure shown in Fig 1) The compound was discovered in the late 1950swhen researchers at RhonendashPoulenc Research Laboratories in France were tryingto create a synthetic product from a Streptomyces sppndashderived compound calledazomycin that would have appreciable activity against Trichomonas vaginalis[1]

From the research of different investigators metronidazolersquos mechanismof action is believed to involve four phases (1) entry into the bacterium cell (2)reduction of the nitro group cytotoxic effect of the reduced product and (4)liberation of end products that are inactive [2] It is the redox intermediate intra-cellular metabolites that are believed to be the key components of microorganismkilling for metronidazole The intracellular targets for these intermediates couldbe organismsrsquo RNA DNA or cellular proteins

12 Antimicrobial Activity

Following their work in 1959 Cosar and Julou and other researchers over thenext several years found a variety of potential human uses for metronidazole thatwent beyond treatment for T vaginalis infection The following is a summaryof metronidazolersquos antimicrobial activity

FIGURE 1 Metronidazole molecule

Metronidazole Clindamycin and Streptogramin 223

121 Protozoa

In addition to T vaginalis metronidazole has exhibited activity against otherprotozoan organisms including Giardia lamblia and Entamoeba histolytica [3ndash15]

122 Bacteria

Gram-Negative Bacteria Shinn [8] and later Tally et al [9] discoveredthe potential of using metronidazole for anaerobic infections Metronidazole hassince been used and studied extensively for treatment of various anaerobic infec-tions [10ndash12] Even today metronidazole is considered the gold standard bywhich other antimicrobials with perceived antianaerobic activity are comparedThis is due primarily to metronidazolersquos rapid killing of Bacteroides species andthe very low rate of resistance acquired by these bacteria [13ndash15] Other Bacteroi-des species and Fusobacterium spp have been reported as being at least as sus-ceptible to metronidazole as Bacteroides as well as Helicobacter (formerlyCampylobacter) pylori Actinobacillus and Prevotella although resistance tometronidazole may be common in various regions of the world [9131416ndash20]Minimum bactericidal concentrations (MBCs) of metronidazole when reportedin these studies are the same or within one dilution of the reported MICs

Gram-Positive Bacteria As for gram-positive anaerobes the peptostrep-tococci have usually been reported to exhibit MICs in the range of 025ndash40mgL [132122]

Susceptibility of Clostridium perfringens to metronidazole is also in theMIC range of 10ndash40 mgL although not all clostridia are so susceptible[13212421] Metronidazole is also considered to be one of the few drugs withactivity against Clostridium difficile [212526]

123 Other Organisms

Metronidazole has also been reported to have in vitro activity against Campylo-bacter fetus Gardnerella vaginalis Leptotrichia buccalis and Treponema pal-lidum to varying degrees [27ndash32]

13 Pharmacokinetic and Pharmacodynamic Properties

The following is a brief summary of the many trials that have studied metronida-zolersquos kinetics

131 Absorption

The absorption of metronidazole has been studied for oral tablets vaginal andrectal suppositories and topical gel

224 Lamp et al

Oral Absorption The oral absorption of metronidazole is excellent withbioavailability often reported as 90 [37ndash39] The oral Cmax with a 500 mgdose is approximately 80ndash130 mgL with a corresponding Tmax of 025ndash40 h[33ndash36]

Rectal Absorption Rectal administration of metronidazole 500 mg hasproduced Cmax values of approximately 40ndash55 mgL at Tmaxrsquos of 05ndash10 fora retention enema to 40ndash12 h for suppositories [11333738] Metronidazoleabsorption from the rectum has been reported as being approximately 67ndash82based upon calculations with equivalent intravenous (IV) doses used as references[38]

Intravaginal Absorption Intravaginal absorption of metronidazole hasvaried considerably depending on the vehicle used The 075 metronidazoleintravaginal gel at a dose of 50 g has produced Cmax values of 02ndash03 mgLwith Tmax values of 83ndash85 h [39] For vaginal suppositories and inserts the 500mg dose Cmax values have been approximately 19 mgL with corresponding Tmax

values of 77ndash20 h [3640] The bioavailability of vaginal suppositories is 25of an oral 500 mg dose but the vaginal gelrsquos bioavailability was 56 that of a500 mg IV dose [3639]

Topical Absorption Systemic absorption of 10 g of 075 metronidazolegel is reportedly quite low After 10 g of the gel was applied to the face of adultswith rosacea the resulting serum concentrations ranged from undetectable to 66ngmL in the following 24 h [4142]

132 Distribution

Protein binding of metronidazole is 20 [3543] Metronidazole crosses cellmembranes well in general and distributes well into a variety of tissues and fluidsThe reported volumes of distribution (Vd) in studies for various age groups haveranged from 051 to 11 Lkg [4344]

Single-dose studies using metronidazole 500 mg IV or oral doses havedetermined AUCs to be approximately 100ndash159 mg(L sdot h) [36] Oral or IV dosesof 10 g have displayed AUCs of 214ndash257 mg(L sdot h) [3345] The penetrationof metronidazole into polymorphonuclear leukocytes has been described asequivalent to the concentration found in the extracellular fluid [46]

133 Metabolism

Metronidazole undergoes hepatic metabolism forming five metabolites The twomajor metabolites are an acid metabolite and a hydroxy metabolite The acidmetabolitersquos activity is considered to be clinically negligible having only 5 ofthe activity of metronidazole [47]


Hydroxymetronidazole The hydroxy metabolite of metronidazole is themost clinically important because it also has antimicrobial activity but to a lesserextent than the parent compound Hydroxymetronidazole has been reported ashaving 30ndash65 of the activity of the parent compound with respect to similarisolates [192447]

134 Excretion

Metronidazole is primarily excreted in the bile as the parent drug and in the urineas its various metabolites Only about 6ndash18 of metronidazole is excreted in theurine unchanged [35] Hydroxymetronidazole is entirely excreted in the urinebeing approximately 25 of the original dose [48]

The elimination half-life (T12) has been reported to range from 6 to 10 h formetronidazole in volunteers and patients with normal hepatic and renal functions[3744] The hydroxy metabolitersquos T12 reportedly ranges from 8 to 12 h [36]Patients may have altered clearance values such as T12 values that are 10 h[49] Metronidazole appears to exhibit dose-dependent clearance in doses rangingfrom 250 to 2000 mg [4449]

14 Pharmacodynamics

141 Bactericidal Effect

Metronidazole appears to have an extremely rapid rate of killing against suscepti-ble anaerobes [1550]

Outcomes associated with the use of metronidazole in mixed aerobicndashan-aerobic infections such as intra-abdominal infections have yielded excellent re-sults in terms of bacterial eradication when an antianaerobic agent such as metro-nidazole was included as part of the therapeutic regimen [5152]

142 Concentration-Dependent Killing

Nix et al [53] discovered that metronidazole had a concentration-dependent kill-ing effect against T vaginalis under anaerobic conditions at concentrations thatranged from 01 to 80 mgL [53] Time-kill kinetic studies of ciprofloxacinalone (concentration 05 mgL MIC 025 mgL) compared to a combination ofciprofloxacin with metronidazole (100 and 400 mgL) displayed more rapidkilling with the combined antibiotics against C perfringens If this concentration-dependent effect truly exists for metronidazole it would suggest that the generaloptimal dosing strategy for metronidazole against organisms like T vaginalis andB fragilis would be to give higher doses less frequently rather then smaller dosesmore frequently similar to the use of aminoglycosides and fluoroquinolonesagainst susceptible gram-negative aerobic bacteria

226 Lamp et al


Like aminoglycosides and fluoroquinolones metronidazole appears to exhibit apostantibiotic effect (PAE) extending over 3 h [54]

144 Serum Bactericidal Titers

Serum bactericidal titers (SBTs) have been used in studies as an ex vivo methodof assessing metronidazolersquos effect on anaerobes primarily B fragalis Resultsfrom an SBT study reported a median metronidazole SBT of 12 against variousBacteroides species at 05 10 and 60 h after administration of a single doseof metronidazole 500 mg IV [55] Results with metronidazole against two strainsof B fragilis at the 6 h time point were 12 but titers related to FusobacteriumPeptostreptococcus and Eubacterium lentum were all 18 at all time points

Fluoroquinolones have often been studied for use in combination with met-ronidazole for antibacterial activity against gram-negative aerobes and anaerobesBoeckh et al [56] studied the effect of fluoroquinolones (ofloxacin ciprofloxacinenoxacin fleroxacin) in combination with antianaerobic antibiotics includingmetronidazole via SBTs SBTs were performed from serum samples obtainedat 1 2 6 and 8 h depending upon the antibiotic combination against variousgram-positive and gram-negative aerobic pathogens as well as strains of B frag-ilis and B thetaiotaomicron None of the combinations appeared to interferewith the individual antibioticsrsquo antibacterial activities and mean SBTs for theciprofloxacin-metronidazole ofloxacin-metronidazole enoxacin-metronidazoleand fleroxacin-metronidazole combinations were all in the 12ndash14 dilutionrange for the various strains and species of Bacteroides Similarly Pefanis etal [57] established greater effectiveness at eradicating mixed aerobicndashanaerobicgram-negative pathogens from experimentally induced abscesses in a rat modelbased upon reduced CFU counts

Serum bactericidal titer studies involving the combination of metronidazole10 g with extended spectrum cephalosporins have also yielded results in the14ndash18 range for B fragilis at the end of 12 or 24 h for ceftizoxime or ceftriax-one respectively [58ndash60]

15 SummarymdashMetronidazole

Although data are scarce metronidazolersquos concentration-dependent bactericidalactivity prolonged T12 and sustained bactericidal activity in plasma support theclinical evaluation of higher doses and longer dosing intervals In vitro and exvivo human studies have corroborated metronidazolersquos effectiveness as an anti-protozoal and antianaerobic agent

The rapid concentration-dependent killing of anaerobes by metronidazoleits active metabolite which helps to extend its effective duration of action thepenetrability of metronidazole into various tissues the near lack of resistance of


anaerobes like B fragilis to metronidazole and its low cost make metronidazolethe preferred agent for antianaerobic therapy in many cases

The other nitroimidazoles doubtless have identical pharmacodynamic prop-erties although their respective pharmacokinetics may be somewhat differentfrom those of metronidazole As newer nitroimidazoles are brought to market andare used with greater frequency metronidazolersquos use may continue to graduallydecline However the value of metronidazole is still readily apparent and it stillhas an important role in todayrsquos antimicrobial armamentarium

2 CLINDAMYCIN

Clindamycin (Cleocin Pharmacia amp Upjohn Co) a lincosamide antibiotic hasbeen used in clinical practice for over 30 years Compared to its parent compoundlincomycin clindamycin has improved oral absorption and a wider antibacterialspectrum of activity making it the most popular agent in this classification ofantibiotics It is commercially available as an intravenous formulation oral cap-sules and suspension as well as in several topical preparations (solution gellotion individual pledgets and vaginal cream) Clinically clindamycin is usedin the treatment of gram-positive and anaerobic bacterial infections of the abdo-men bone lung skin and soft tissues and pelvis and is often an alternative agentin those who are allergic to β-lactam antibiotics Additionally it is used in thetreatment of opportunistic protozoal infections of the lung and central nervoussystem in patients infected with the human immunodeficiency virus

Usual oral doses in adults range from 150 to 450 mg every 6 h whereasparenteral doses are generally 600ndash900 mg every 8 h [61]

21 Chemistry and Mechanism of Action of Clindamycin

Clindamycin (7-chloro-7-deoxylincomycin) is a semisynthetic derivative of lin-comycin and is therefore classified as a lincosamide antibiotic Similar to macro-lides streptogramins and chloramphenicol clindamycin exerts its activityagainst susceptible pathogens by binding to the 50S ribosomal subunit whichinhibits microbial protein synthesis through its effects on peptide chain initiation


Clindamycin is active against a variety of gram-positive and anaerobic bacterialpathogens [62] Most streptococci including Streptococcus pneumoniae andStreptococcus pyogenes are susceptible as are most methicillin-susceptiblestrains of Staphylococcus aureus However it is not active against enterococcior aerobic gram-negative organisms Clindamycin is active against a broad rangeof anaerobic gram-negative bacilli including Bacteroides species Fusobacteriumspecies anaerobic gram-positive bacilli such as Propionibacterium Eubacte-

228 Lamp et al

rium and Actinomyces and anaerobic gram-positive cocci such as PeptococcusPeptostreptococcus microaerophilic streptococci and most strains of Clostrid-ium perfringens It is also active against pathogens that cause bacterial vaginosiswhich include Bacteroides Gardnerella vaginalis Mobiluncus and Mycoplasmahominus

Additionally many protozoa including Toxoplasma gondii Plasmodiumfalciparum Babesia and Pneumocystis carinii are susceptible to clindamycin[6263]

Two recent surveillance studies in the United States demonstrated thatgreater than 95 of all pneumococcal strains were susceptible to clindamycinwith reported MIC90 values of 012 mgL [6465] In one of the studies 76 of969 S aureus isolates were susceptible with reported MIC50 and MIC90 valuesof 025 and 8 mgL [65] The susceptibility breakpoint is 05 mgL for Staphy-lococcus spp and 025 mgL for Streptococcus spp and S pneumoniae [66]

23 Pharmacokinetic Properties of Clindamycin

Clindamycin oral preparations are rapidly absorbed and reported oral bioavail-ability is around 90 Other notable pharmacokinetic properties include 90protein binding wide distribution in body fluids and tissues except for cerebralspinal fluid extensive hepatic metabolism to active metabolites and a half-lifeof around 20ndash24 h [616267ndash69] For severe liver dysfunction clindamycindoses should be reduced

For 600 mg intravenous doses peak serum concentrations are around 109gmL while trough concentrations are 20 and 11 mgL at 6 and 8 h respectively[61] Peak concentrations after intramuscular doses of 600 mg are 90 mgLwhich is slightly lower than those reported after intravenous dosing For 900 mgintravenous doses reported peak and trough concentrations at 8 h are 141 and17 mgmL respectively [61]

24 Pharmacodynamics of Clindamycin

Until recently little was known about the pharmacodynamic characteristics ofclindamycin Recent studies have demonstrated that clindamycin displays time-dependent pharmacodynamics against S pneumoniae methicillin-susceptible Saureus and B fragilis [6870] Furthermore clindamycin has an extended post-antibiotic effect against Staphylococcus [5471]

241 In Vitro Studies

Two time-kill studies have evaluated clindamycinrsquos activity against B fragilis[7172] Clindamycin was shown to display concentration-independent activityin a study conducted by Klepser et al [70] In this study clindamycin concentra-tions were evaluated over a range of 1ndash64 times the MIC Five strains of B


fragilis with clindamycin MICs of 003ndash80 mgL were studied Bactericidal ac-tivity (3 log decrease in the starting inoculum) was independent of clindamycinconcentration for three of the five strains A bacteristatic effect was noted for theisolate with an MIC of 80 mgL for all tested concentrations Against anotherstrain with an MIC of 10 mgL regrowth was observed at the MIC concentrationand bactericidal activity was observed for all other concentrations

Aldridge and Stratton [72] described concentration-dependent activity forclindamycin ceftizoxime and cefotetan against B fragilis However these inves-tigators studied only antibiotic concentrations over a range of 05ndash4 times theMIC In this study metronidazole chloramphenicol and cefoxitin were also eval-uated and reported clindamycin MIC values against the study isolates were 05and 10 mgL As described in the above study clindamycin clearly displaysconcentration-independent pharmacodynamic activity at concentrations that ex-ceed 4 times the MIC [70] Furthermore several in vitro and in vivo studiesdemonstrate that cephalosporins have time-dependent and not concentration-dependent pharmacodynamics which differs from the results reported by Al-dridge [54]

242 Serum Inhibitory and Bactericidal Titers

Klepser et al [68] evaluated the duration of serum bactericidal activity for 300mg oral and intravenous doses of clindamycin dosed every 8 h and every 12 hagainst two isolates each of S aureus S pneumoniae and B fragilis at steadystate in healthy volunteers Median MICs for the study isolates were 0125 and025 mgL for S aureus 0125 mgL for both S pneumoniae isolates and 05mgL for the B fragilis isolates For the every 8 h regimens measurable activitywas observed for 875ndash100 of the dosing interval against all study isolates Forthe every 12 h regimen bactericidal activity was observed for 50ndash77 of thedosing interval against S aureus 100 for S pneumoniae and 80ndash88 for Bfragilis These investigators concluded that dosing intervals of every 8 or every12 h for 300 mg oral or intravenous doses of clindamycin provided adequatecoverage against S aureus S pneumoniae and B fragilis except that an every8 h interval may be necessary for treatment of S aureus infections

Serum inhibitory titers were measured by Flaherty et al [67] in six volun-teers participating in a multiple dose intravenous clindamycin study against areference strain of B fragilis with an MIC of 025ndash05 mgL Clindamycin regi-mens assessed in this study were as follows 600 mg every 6 h 900 mg every8 h and 1200 mg every 12 h When considering the doses used in this study itis not surprising that activity was observed throughout the entire dosing intervalfor all regimens and that reciprocal inhibitory titers at the trough ranged from 5to 12

The synergistic effects of clindamycin with fluoroquinolones have alsobeen studied [7374] Increased serum bactericidal activity was observed when

230 Lamp et al

clindamycin was combined with ciprofloxacin ofloxacin and fleroxacin againstS aureus and S pneumoniae [73] This single-dose study evaluated 600 mg intra-venous and 300 mg oral doses of clindamycin given in combination with thequinolone test agents against both gram-positive organisms and anaerobes En-hanced activity was observed when clindamycin (600 mg IV) was given in combi-nation with ciprofloxacin (200 mg IV) and ofloxacin (200 mg IV) for S aureusand S pneumoniae Against S aureus reported reciprocal titers at 6 h were 54for ciprofloxacin-clindamycin compared to 23 for ciprofloxacin alone Similarlyofloxacin-clindamycin reciprocal titers of 41 were observed compared to 2 forofloxacin alone at 6 h Against S pneumoniae reciprocal titers at 6 h were 2and 136 for ciprofloxacin alone and ciprofloxacin-clindamycin respectively Forofloxacin and ofloxacin-clindamycin reciprocal titers of 2 and 135 were notedat 6 h Less activity was observed when oral clindamycin (300 mg) was givenin combination with oral enoxacin (400 mg) and fleroxacin (400 mg) than eitherquinolone alone at 8 h against S aureus

The serum bactericidal activity of an oral combination of ciprofloxacin (750mg every 12 h 3 doses) and clindamycin (300 mg every 6 h 5 doses) wasstudied by Weinstein et al [74] in healthy elderly volunteers Unlike the resultsreported by Boeckh et al [73] the bactericidal activity of ciprofloxacin againstS aureus was antagonized by clindamycin when clindamycin-susceptible strainswere tested However the reported MBCs for clindamycin against the three testS aureus strains were either 40 or 80 mgL This antagonistic effect was notobserved for the combination against S pyogenes and S pneumoniae in thisstudy


Several investigators have studied the postantibiotic effect (PAE) for clindamycinagainst S aureus [7576] Xue et al [76] reported that the duration of the PAEfor clindamycin is dependent on both concentration and duration of exposure toclindamycin with longer PAEs observed at higher concentrations In this studywhich used 21 clinical osteomyelitis isolates of S aureus observed PAEs rangedfrom 04 to 39 h Reported PAE values were not influenced by multiple dosingWhen considering clinically achievable concentrations and estimated PAE valuesof 24 h these investigators concluded that oral clindamycin could be adminis-tered at 300 mg every 8 h for the treatment of S aureus whereas recommendedintravenous dosing is 300 mg every 6 h

25 SummarymdashClindamycin

Similar to that observed for β-lactams macrolides vancomycin and others onlyrecently have the time-dependent pharmacodynamics of clindamycin been dis-covered Studies demonstrating serum bactericidal activity against susceptible


bacteria for either the majority or the entire dosing interval has been demonstratedwith lower doses than are currently recommended The application of the pharma-codynamics of clindamycin needs to be more fully evaluated in actual clinicalsituations

3 STREPTOGRAMIN

The streptogramin class of antibiotics are new introductions into the UnitedStates however they have been available in Europe for many years for mild tomoderate staphylococcal infections Recent modifications to the structure allowfor formulation of water-soluble forms of the compounds and subsequent usein intravenous solutions [77] These efforts have led to the combination drugquinupristin-dalfopristin (RP 59500 Synercid) and its evaluation for treatmentof severe antibiotic-resistant infections

31 Chemistry and Mechanism of Action of Streptogramin

Streptomyces pristinaespiralis produces two clinically unrelated antibiotics(streptogramin A and B) which in combination exert their antibacterial actionThe streptogramin A antibiotics are polyunsaturated cyclic macrolactones andinclude pristinamycin IIA virginiamycin M and dalfopristin Streptogramin Bantibiotics are cyclic depsipeptides and include pristinamycin IA quinupristinand virginiamycin S [78] Quinupristin was produced by modifying pristinamycinIA to quinuclindinylthiomethyl pristinamycin IA Dalfopristin is diethylamino-ethyl-sulfonyl-pristinamycin IIA [79] A mixture of quinupristin a streptograminB and dalfopristin a streptogramin A in a 3070 ratio is capable of synergisti-cally inhibiting protein synthesis Streptogramin A binds to the 50S and 70Ssubunits of the ribosome when they are not actively synthesizing protein It hasbeen suggested that they block the elongation phase of bacterial protein synthesisConversely streptogramin B can bind to active ribosomes at the 50S subunitThe synergistic activity of the streptogramins is achieved through a conforma-tional change in the 50S subunit after binding a type A streptogramin whichleads to a strengthening of the binding of the type B streptogramin Ultimatelythe streptogramin combination inhibits early- and late-stage protein synthesis[80]

32 Antimicrobial Activity of Streptogramin

321 Gram-Positive Bacteria

The quinupristin-dalfopristin combination inhibits growth of most gram-positivebacteria It has useful activity against Staphylococcus aureus Staphylococcusepidermidis Streptococcus pneumoniae Streptococcus agalactiae Streptococcus

232 Lamp et al

pyogenes viridans streptococci and Enterococcus faecium Enterococcus fae-calis is resistant to the streptogramins [81]

321 Gram-Negative Bacteria

Quinupristin-dalfopristin has acceptable activity for many respiratory pathogensMost Moraxella spp Legionella spp Mycoplasma spp and Neisseria spp havean MIC90 of 40 mgL Against Haemophilus influenzae it has less activitywith MIC90 values of 40ndash80 mgL [7981] Quinupristin-dalfopristin has noactivity against Enterobacteriaceae or Pseudomonas aeruginosa [81]

322 Other Bacteria

Quinupristin-dalfopristin maintains useful activity against both Bacteroides frag-ilis (MIC90 40 mgL) and other Bacteroides species (MIC90 4ndash8 mgL) [8283]

33 Pharmacokinetic Properties of Streptogramin

The pharmacokinetics of quinupristin-dalfopristin are reported in a very smallnumber of studies Further data is needed for complete dosing recommendationsespecially in patients

331 Absorption

Quinupristin-dalfopristin is available only as an injectable product although oralderivatives are being developed

Etienne et al [84] studied the pharmacokinetics in doses ranging from 14to 294 mgkg administered as a 1 h intravenous infusion Maximum concentra-tions at the end of the infusion rose linearly from 095 to 242 mgL for thedoses studied As would be expected the area under the timendashconcentration curve(AUC) also increased linearly from 159 to 377 mgsdothL for the 126 and 294mgkg doses Antibacterial activity was present for up to 6 h after the dosesmicrogsdothml or mgsdothL is the most frequently used units for AUC despite plasmaconcentrations below the typical MIC for staphylococci and streptococci prob-ably indicating the importance of active metabolites [84]

A more extensive study by Bergeron and Montay [85] used single dosesof 5 10 and 15 mgkg given intravenously over 1 h to 18 healthy volunteersAt the 3 doses quinupristin Cmax increased linearly (from 12 to 23 to 36 mgL) and dalfopristin Cmax values were 46 64 and 85 mgL The dalfopristinmetabolite RP 12536 appeared rapidly and increased disproportionally from 09to 25 to 38 mgL However a combined AUC0ndashinfin for both dalfopristin and itsmetabolite did increase linearly Quinupristin AUC values increased from 14 to30 to 47 mgsdothL [85]

332 Distribution

In the Etienne et al study the mean T12 for quinupristindalfopristin ranged from127 to 153 h The RP 12536 metabolite of dalfopristin had a T12 of 075ndash084 h


[84] Bergeron and Montay [85] reported that following biphasic plasma elimina-tion the elimination T12 values for quinupristin and dalfopristin were 093ndash096h and 039ndash091 h respectively Variability was higher for dalfopristin andT12 increased with increasing doses

Quinupristin and dalfopristin are highly protein bound in animal infectionmodels Quinupristin in plasma was bound approximately 80 in rats and 90in monkeys Dalfopristin binding appears to increase over time from 45 to90 or greater in rats and monkeys [85] Protein binding ranges from 23 to32 for quinupristin and from 50 to 56 for dalfopristin in humans [86]Volume of distribution (Vd) and Vd at steady state (Vdss) were 137ndash144 Lkgand 079ndash083 Llg respectively for quinupristin and were unaffected by doseDalfopristin Vd and Vdss rose with each dose For the 5 10 and 15 mgkg dosesVd was 054 088 and 18 Lkg while Vdss was 033 043 and 070 Lkg respec-tively [85]

Radiolabeled quinupristin and dalfopristin were injected into rats and mon-keys and radioactivity was assessed for distribution Either drug was rapidly andextensively distributed to tissues in both species High concentrations were seenin the gall bladder and in bile indicating biliary excretion Both componentsreached high concentrations in gastrointestinal tissues kidneys and liver Thebrain and spinal cord had very low concentrations of quinupristin or dalfopristin[85]

Bernard measured blister fluid penetration after a single 12 mgkg IV dosein six healthy male volunteers Although a microbiological assay method wasemployed that is incapable of differentiating between the two components ormetabolites the mean percent tissue penetration was 824 indicating good tis-sue penetration Six hours after the end of the infusion the combination wasdetectable at a concentration of 092 mgL which should be sufficient for bacte-rial activity [87]

333 Metabolism

Bergeron and Montay [85] reported that clearance rates were high and approxi-mated hepatic blood flow 11 L(hsdotkg) and 10 to 12 L(hsdotkg) for quinupristinanddalfopristin respectively Bernard et al [87] found similar values after a sin-gle 12 mgkg IV infusion reported as 74 Lh for quinupristin-dalfopristin

A single intravenous infusion of 430 mg for over 1 h was administered tosix healthy male volunteers The radioactivity of quinupristin and dalfopristinwas measured to determine their metabolic fate The two drugs are both metabo-lized to active metabolites in humans Quinupristinrsquos cysteine derivative (RP100391) accounts for 38 and 75 of total radioactivity in urine and fecesrespectively No unchanged dalfopristin was recovered in either urine or fecesIn urine dalfopristin was recovered as pristinamycin IIA (RP 12536) 70 andcysteine derivatives 30 [8588]

234 Lamp et al

334 Excretion

After IV administration both quinupristin and dalfopristin are excreted primarilyin the feces Fecal excretion as measured by radioactivity of quinupristin anddalfopristin accounted for 747 and 775 respectively The remainder of quin-upristin and dalfopristin was recovered in urine 15 and 187 respectivelyThirty-five percent of quinupristin in urine was recovered unchanged with a cor-responding value of 15 in feces [88]

Renal Dysfunction The pharmacokinetics of quinupristin and dalfopristinare not altered by the presence of continuous ambulatory peritoneal dialysis Theconcentrations of quinupristin and dalfopristin in dialysis effluent were very lowand below the MIC of most pathogens [86] Patients with severe renal failuremay have impaired clearance of quinupristin metabolites and Cmax and AUCvalues for dalfopristin were elevated 30 over those in healthy volunteers [86]

34 Pharmacodynamic Properties of Streptogramin


The MBC for streptogramin can be up to 5 dilutions higher than the MIC for Efaecium and 3ndash6 dilutions higher against S aureus Against streptococci theMIC and MBC were within one dilution for each other [89]

Quinupristin-dalfopristin has been evaluated in several studies using time-kill studies It exerts a time-dependent bacteriostatic and what is sometimesreferred to as slow bactericidal activity against E faecium and S aureus [8990]However in several studies quinupristin-dalfopristin has a good bactericidal ef-fect on certain strains of both organisms [9192] Further studies with manystrains of E faecium have shown that quinupristin-dalfopristin at a concentrationof 60 mgL can have a concentration-dependent bactericidal effect for isolateswith MBCs of 40 mgL however most strains appear to possess higher MBCs[91] The quinupristin-dalfopristin concentrationMBC ratio and quinupristinMICs were significantly correlated with the bactericidal rate [91] Other investi-gators have found that bactericidal activity is more likely to be demonstrated forenterococci in the log growth phase and those with at least intermediate suscepti-bility to erythromycin [93] Against S aureus quinupristin-dalfopristin also dem-onstrates a variable bactericidal rate presumably as a result of differences inMBCs Against methicillin-susceptible S aureus (MSSA) and methicillin-resis-tant S aureus (MRSA) quionupristin-dalfopristin decreased the inoculum 1ndash2log10 over 24 h compared to 3ndash4 log10 for oxacillin vancomycin or gentamicin[89] In other experiments quinupristin-dalfopristin has shown the ability to re-duce the inoculum by 2ndash3 log10 in only 6ndash12 h [92] The presence of a constitu-tively erythromycin-resistant phenotype is predictive of bacteriostatic activity[94]


Quinupristin-dalfopristin in early studies showed a consistent rapid bacteri-cidal activity against penicillin-susceptible S pneumoniae The inoculum wasdecreased by 2ndash3 log10 in only 10 min This bactericidal rate was faster than thatof all comparators including penicillin G erythromycin ciprofloxacin sparflox-acin and vancomycin However three out of ten strains only a bacteriostaticeffect was seen [95] Later studies with larger numbers of S pneumoniae isolatesdemonstrated its rapid bactericidal effect against all S pneumoniae regardless ofpenicillin or erythromycin susceptibility at 2 times the MIC [9697] Againstviridans streptococci quinupristin-dalfopristin exerts a bactericidal effect over12ndash24 h The bactericidal rate against viridans streptococci is less when the iso-lates are erythromycin-resistant through loss of quinupristin activity [98]


The influence of postantibiotic effect (PAE) on antibiotic dosing is controversialWith quinupristindalfopristin possessing short half-lives long PAEs may influ-ence the dosing schedule

The PAE against E faecium in in vitro experiments has produced variableresults Against E faecium the PAE after a 1 h exposure to 025 times the MICwas 85 and 26 h for vancomycin-susceptible and -resistant strains respectively[99] However others have found the PAE 4 times the MIC to be as short as02ndash30 h for vancomycin-resistant strains and inversely related to the MBC andquinupristin MIC [91] The presence of multiple and unmeasured antibiotic resis-tance may affect the results of PAE determinations for enterococci

Staphylococci are also reported to possess a long concentration-dependentPAE after exposure to quinupristindalfopristin The in vitro PAE at 10 timesthe MIC was 46ndash70 h for MSSA and MRSA either erythromycin-susceptibleor inducibly resistant For MRSA containing the constitutively erythromycin-resistant phenotype the PAE was reduced to 24 h [29] Other investigators havealso found a longer PAE in MSSA compared to MRSA at 4 times the MIC 7h versus 5 h respectively [100]

Pneumococci show concentration-dependent PAEs to quinupristin-dalfo-pristin The rapid bactericidal rate complicates the determination of PAE how-ever at 05 times the MIC the PAE ranges from 17 to 41 h [99]

In the mouse thigh infection model the PAEs against S aureus and Spneumoniae were 10 and 91 h respectively [101]

343 Experimental Infection Models

Few studies have been published with quinupristin-dalfopristin and limited infor-mation is available from in vitro or in vivo models These models are also limitedthrough the use of only a few bacterial strains in primarily endocarditis and maynot be completely predictive of results in humans for other types of infections

236 Lamp et al

In Vitro Models Quinupristindalfopristin was evaluated in an in vitroinfection model of simulated endocardial vegetations The drug was dosed tosimulate a human dose of 75 mgkg every 8 h for 96 h low-dose continuousinfusion (Css 15 mgL) and high-dose continuous infusion (Css 92 mgL)Two vancomycin-resistant and constitutively erythromycin-resistant E faeciumstrains were used and one of the isolates possessed an elevated MBC Againstthe isolate with a low MBC all regimens were bacteriostatic with the exceptionof the high-dose continuous infusion regimen which achieved bactericidal activ-ity after 90 h The addition of doxycycline to the every 8 h regimen resulted inincreased killing although it did not reach a value indicating synergy The isolatewith an elevated MBC was unaffected by quinupristindalfopristin as an every8 h regimen or low-dose continuous infusion Doxycycline possessed the greatestactivity alone and no increased killing was observed when it was administeredwith quinupristin-dalfopristin The use of a high-dose continuous infusion regi-men or the addition of doxycycline reduced or eliminated the appearance of resis-tant isolates during the model [102]

Quinupristin-dalfopristin activity against S aureus in a fibrin clot modelwas compared to vancomycin over 72 h A 75 mgkg every 8 h dose was simu-lated Quinupristin-dalfopristin was more active than vancomycin however nei-ther reached bactericidal activity This was despite both the MSSA and MRSAisolate possessing MBCs close to the MIC The combination of quinupristin-dalfopristin and vancomycin led to greater decreases in log10 counts (not syner-gistic) and prevented the emergence of quinupristin-dalfopristin resistance [103]

This same model further evaluated quinupristin-dalfopristin in simulateddoses of 75 mgkg given every 6 8 and 12 h and by continuous infusion (60mgL) against S aureus One strain was an MRSA with constitutive erythromy-cin resistance and the other was an MSSA and erythromycin-susceptible Allregimens were bactericidal against the MSSA strain but no regimens reachedthis level of activity against the MRSA isolate The AUC0-24 was significantlycorrelated with activity against the MRSA strain [104]

In Vivo Models Two inducibly erythromycin-resistant and one sensitiveE faecium strain were studied in a rabbit endocarditis model for 4 days Quinu-pristin-dalfopristin was given in a dose of 30 mgkg IM evert 8 h and compared tothrice daily regimens of amoxicillin or gentamicin or combinations Quinupristin-dalfopristin when given alone had equivalent activity to amoxicillin against theerythromycin-susceptible strain For the erythromycin-resistant strains quinu-pristin-dalfopristin was not effective Additional experiments against one of theerythromycin-resistant strains showed that combinations of quinupristin-dalfo-pristin with gentamicin or amoxicillin were more active than either of the drugsalone No information was presented on pharmacodynamic parameters [105]

Quinupristin-dalfopristin has been studied in a rabbit infected fibrin clotmodel against two strains each of S aureus and S epidermidis A rapid bacteri-


cidal rate was observed after the single dose of 50 mgkg for S aureus howeveronly bacteriostatic activity was seen against S epidermidis The elevated MBCsfor S epidermidis at the inoculum tested may explain the disparate results Alsothe Cmax obtained with this dose is considerably higher than that observed inhumans and may not be predictive of human experience [106]

An early rabbit endocarditis model administered quinupristin-dalfopristinin a dose of 20 mgkg intramuscularly (IM) every 6 h versus vancomycin 25 mgkg IV every 12 h for 4 days Three susceptible S aureus strains with increasingquinupristin-dalfopristin MICs (05ndash20 mgL) and MBCs (40ndash80 mgL) wereused The peak serum concentration of quinupristin-dalfopristin measured by bio-assay was 19 mgL with a T12 of 13 h Vancomycin was effective against allstrains whereas quinupristin-dalfopristin was effective against the strains withMICs of 05 and 10 mgL although less effective at sterilizing vegetations at anMIC of 10 mgL It failed against the strain with an MIC of 20 mgL Higherdoses of 40 mgkg in this model resulted in animal toxicity and death [107]Given the pharmacokinetic data these regimens would be expected to producea time above the MIC during the dosing interval (T MIC) of approximately57 36 and 0 for S aureus with MICs of 05 10 and 20 mgL respec-tively Therefore it appeared that quinupristin-dalfopristin required a T MICof approximately 50 for equivalent bactericidal activity

Quinupristin-dalfopristin was compared to vancomycin in another Saureus rabbit endocarditis model The isolate had an MIC and MBC of 05 mgL to quinupristin-dalfopristin Vancomycin and quinupristin-dalfopristin weredosed at 30 mgkg IM every 12 h for 4 days Quinupristin-dalfopristin was aseffective as vancomycin and no resistant isolates were recovered from vegeta-tions The T MIC for quinupristin-dalfopristin was 33 Concentrations werealso measured in the vegetations and at 1 h after a single injection the ratio ofvegetation to serum concentration was 41 These data along with a prolongedPAE may explain the efficacy of quinupristin-dalfopristin [108] However thisS aureus strain may not be representative of most clinical isolates due to itsequivalent MIC and MBC

A S aureus rat endocarditis model demonstrated that quinupristin-dalfo-pristin in a regimen simulating a human dose of 7 mgkg every 12 h was aseffective as vancomycin only against S aureus strains that were erythromycin-susceptible For constitutively erythromycin-resistant strains that had quinupristinMICs of 64 mgL the shorter dalfopristin T12 in rats did not allow for the T MIC (20) to be sufficient for efficacy A regimen that prolonged the dalfo-pristin concentration did allow the regimen to be effective for these macrolide-resistant strains The authors recommended that efficacy would be improvedthrough use of an every 8 h regimen or continuous infusion [24] Similar findingswere found in a rabbit endocarditis model evaluating the efficacy of quinupristin-dalfopristin against S aureus with variable erythromycin susceptibilities Quinu-pristin-dalfopristin doses were 30 mgkg IM every 8 or 12 h and 10 mgkg IV

238 Lamp et al

every 12 h These regimens were as effective as vancomycin against erythromy-cin-susceptible or inducibly resistant strains regardless of oxacillin susceptibilityQuinupristin-dalfopristin was less active in all doses against the two strains withconstitutively erythromycin-resistant phenotypes The AUC for quinupristin-dal-fopristin in plasma divided by the quinupristin MIC was most predictive of activ-ity The determination of quinupristin MICs may be necessary prior to beginningtherapy with quinupristin-dalfopristin for endocarditis [77]

A mouse S aureus septicemia model compared quinupristin-dalfopristinto vancomycin Only one S aureus strain was used and no MBC or other drugresistant data were reported Quinupristin-dalfopristin regimens of a single doseof 120 mgkg 60 mgkg every 12 h 40 mgkg every 8 h and 30 mgkg every6 h were administered for 24 h Quinupristin-dalfopristin in all doses demon-strated a faster bactericidal rate than vancomycin with some dose-dependent bac-tericidal activity being noted No data were presented on which parameter wasmore closely associated with activity because all AUCs were similar [109]

In a mouse thigh infection model the influence of dosing interval on theactivity of quinupristin-dalfopristin was evaluated against S aureus and S pneu-moniae The dose ranged from 125 to 800 mgkg and administered in one twoor four doses over 24 h A sigmoidal dosendashresponse model indicated that theAUC was best correlated with efficacy [101]

344 Synergy

The importance of synergistic combinations is well documented for certain en-terococcal and staphylococcal infections Given quinupristin-dalfopristinrsquos slowbactericidal activity against many of these strains synergy testing has been evalu-ated

As already mentioned quinupristin and dalfopristin are synergistic whenused in combination The IV product contains a quinupristin-dalfopristin ratioof 3070 The more variable T12 of dalfopristin and information on decreasedpenetration of dalfopristin into cardiac vegetations underlies the importance ofinvestigating the effect of different ratios on activity [104] Studies of in vitroand in vivo activity show that the combination remains synergistic and effectivein a range from 1684 to 8416 which should accommodate most clinical situa-tions [110]

Quinupristin-dalfopristin has no effect on the MIC of ciprofloxacin genta-micin or cefotaxime against E coli K pneumoniae C freundii E cloacae Smarcescens or P aeruginosa Quinupristin-dalfopristin increased the MIC forciprofloxacin against B fragilis in nine out of 20 strains but had no effect onother Bacteroides species or S aureus [82] The quinupristin-dalfopristin MICfor vancomycin-resistant E faecium was decreased when the combination com-bined with ciprofloxacin teicoplanin and tetracycline [100]

Using time-kill methods quinupristin-dalfopristin was not found to have


synergy against E faecium when combined with clinafloxacin LY333328 oreperozolid These isolates had quinupristin-dalfopristin MBCs of 80 mgL[111] No synergy was seen defined by an FIC index of 05 when quinupristin-dalfopristin was combined with vancomycin however time-kill studies indicatedsynergy against S aureus at higher inocula (5 107 CFUmL) [103] An induci-bly erythromycin-reistant gentamicin-resistant amoxicillin-sensitive E faeciumstrain was exposed to quinupristin-dalfopristin with and without gentamicin oramoxicillin but the combinations did not demonstrate faster killing rates [105]

35 SummarymdashStreptogramins

Quinupristin-dalfopristin has been shown effective against E faecium S aureusand S pneumoniae The pharmacodynamic parameter most associated with effi-cacy in animal models is the AUC The presence of erythromycin resistance hasa dramatic effect on the activity of the combination drug quinupristin-dalfopristinThis phenotype will lead to elevated quinupristin MICs and may need to be deter-mined prior to beginning therapy Further data are needed from patients to deter-mine the most appropriate dosing requirements that maximize its bactericidaleffect

REFERENCES

1 C Cosar L Joulou Activite de lrsquo(hydroxy-2-ethyl)-1-methyl-2-nitro-5-imidazole(8823 RP) vis-a-vis des infections experimentales a trichomonas vaginalis AnnInst Pasteur 195996238ndash241

2 M Muller Mode of action of metronidazole on anaerobic bacteria and protozoaSurgery 198393165ndash171

3 B Korner HK Jensen Sensitivity of Trichomonas vaginalis to metronidazole tini-dazole and nifuratel in vitro Br J Vener Dis 197652404ndash408

4 SD Sears J OrsquoHare In vitro susceptibility of Trochomonas vaginalis to 50 antimi-crobial agents Antimocrob Agents Chemother 198832144ndash146

5 L Jokipii AMM Jokipii In vitro susceptibility of Giardia lamblia trophozoites tometronidazole and tinidazole J Infect Dis 1980141317ndash325

6 VK Vinayak RC Mahajan NL Chitkara In-vitro activity of metronidazole on thecysts of Entamoeba histolytica Ind J Pathol Bacteriol 19751861ndash64

7 RC Mahajan NL Chitkara VK Vinayak DV Dutta In vitro comparative evaluationof tinidazole and metronidazole on strains of Entamoeba histolytica Indian J PatholBacteriol 197417226ndash228

8 DLS Shinn Metronidazole in acute ulcerative gingivitis Lancet 1962111919 FP Tally VL Sutter SM Finegold Metronidazole versus anaerobes In vitro data

and initial clinical observations Calif Med 197211722ndash2610 JP Rissing WL Moore Jr C Newman JK Crockett TB Buxton HT Edmondson

Treatment of anaerobic infections with metronidazole Curr Ther Res 198027651ndash663

240 Lamp et al

11 Study Group An evaluation of metronidazole in the prophylaxis and treatment ofanaerobic infections in surgical patients J Antimicrob Chemother 19751393ndash401

12 SJ Eykyn I Phillips Metronidazole and anaerobic sepsis Br Med J 197621418ndash1421

13 B Olsson-Liljequist CE Nord In vitro susceptibility of anaerobic bacteria to nitro-imidazoles Scand J Infect Dis 198126(suppl)42ndash45

14 KE Aldridge M Gelfand LB Reller LW Ayers CL Pierson F Schoenknect FTilton J Wilkins A Henderberg DD Schiro M Johnson A Janney CV SandersA five-year multicenter study of the susceptibility of the Bacteroides fragilis groupisolates to cephalosporins cephamycins penicillins clindamycin and metronida-zole in the United States Diagn Microbiol Infect Dis 199418235ndash241

15 JB Selkon The need for and choice of chemotherapy for anaerobic infectionsScand J Infect Dis 198126(suppl)19ndash23

16 GA Pankuch MR Jacobs PC Appelbaum Susceptibilities of 428 gram-positiveand -negative anaerobic bacteria to Bay y3118 compared with their susceptibilitiesto ciprofloxacin clindamycin metronidazole piperacillin piperacillin-tazobactamand cefoxitin Antimicrob Agents Chemother 1993371649ndash1654

17 CAM McNulty J Dent R Wise Susceptibility of clinical isolates of Campylobacterpyloridis to 11 antimicrobial agents Antimicrob Agents Chemother 198528837ndash838

18 M Lopez-Brea E Martin C Lopez-Lavid JC Sanz Susceptibility of Helicobacterpylori to metronidazole Eur J Clin Microbiol Infect Dis 1991101082ndash1083

19 MJAMP Pavicic AJ van Winkelhoff J de Graaff Synergistic effects betweenamoxicillin metronidazole and the hydroxymetabolite of metronidazole againstActinobacillus actinomycetemcomitans Antimicrob Agents Chemother 199135961ndash966

20 MJAMP Pavicic AJ van Winkelhoff J de Graaff In vitro susceptibilities of Actino-bacillus actinomycetemcomitans to a number of antimicrobial combinations Anti-microb Agents Chemother 1992362634ndash2638

21 AW Chow V Patten LB Guze Susceptibility of anaerobic bacteria to metronida-zole Relative resistance of non-spore-forming gram-positive bacilli J Infect Dis1975131182ndash185

22 AMM Jokipii L Jokipii Comparative activity of metronidazole and tinidazoleagainst Clostridium difficle and Peptostreptococcus anaerobius Antimicrob AgentsChemother 198731183ndash186

23 K Dornbusch CE Nord B Olsson-Liljeqvist Antibiotic susceptibility of anaerobicbacteria with special reference to Bacteroides fragilis Scand J Infect Dis 197919(suppl 19)17ndash25

24 JP OrsquoKeefe KA Troc KA Thompson Activity of metronidazole and its hydroxyand acid metabolites against clinical isolates of anaerobic bacteria AntimicrobAgents Chemother 198222426ndash430

25 JL Whiting N Cheng AW Chow Interactions of ciprofloxacin with clindamycinmetronidazole cefoxitin cefotaxime and mezlocillin against gram-positive andgram-negative anaerobic bacteria Antimicrob Agents Chemother 1987311379ndash1382


26 HM Wexler SM Finegold In vitro activity of cefotetan compared with that ofother antimicrobial agents against anaerobic bacteria Antimicrob Agents Chemo-ther 198832601ndash604

27 H Hof V Sticht-Groh Antibacterial effects of niridazole Its effect on microaero-philic campylobacter Infection 19841236ndash39

28 AM Freydiere Y Gille S Tigaud P Vincent In vitro susceptibilities of 40 Campy-lobacter fetus subsp jejuni strains to niridazole and metronidazole AntimicrobAgents Chemother 198425145ndash146

29 BM Jones I Geary AB Alawattegama GR Kinghorn BI Duerden In-vitro andin-vivo activity of metronidazole against Gardnerella vaginalis Bacteroides sppand Mobiluncus spp in bacterial vaginosis J Antimicrob Chemother 198516189ndash197

30 ABM Kharsany AA Hoosen J van den Ende Antimicrobial susceptibilities ofGardnerella vaginalis Antimicrob Agents Chemother 1993372733ndash2735

31 M Weinberger T Wu M Rubin VJ Gill PA Pizzo Leptotrichia buccalis bacter-emia in patients with cancer Report of four cases and review Rev Infect Dis 199113201ndash206

32 AH Davies Metronidazole in human infections with syphilis Br J Vener Dis 196743197ndash200

33 T Bergan E Arnold Pharmacokinetics of metronidazole in healthy volunteers aftertablets and suppositories Chemotherapy 198026231ndash241

34 GW Houghton J Smith PS Thorne R Templeton The pharmacokinetics of oraland intravenous metronidazole in man J Antimicrob Chemother 19795621ndash623

35 ED Ralph JT Clarke RD Libke RP Luthy WMM Kirby Pharmacokinetics ofmetronidazole as determined by bioassay Antimicrob Agents Chemother 19746691ndash696

36 J Mattila PT Mannisto R Mantyla S Nykanen U Lamminsivu Comparative phar-macokinetics of metronidazole and tinidazole as influenced by administration routeAntimicrob Agents Chemother 198323721ndash725

37 T Bergan O Leineboslash T Blom-Hagen B Salvesen Pharmacokinetics and bioavail-ability of metronidazole after tablets suppositories and intravenous administrationScand J Gastroenterol 198419(suppl 91)45ndash60

38 GW Houghton PS Thorne J Smith R Templeton Plasma metronidazole concen-trations after suppository administration In Phillips and Collier eds Metronida-zole Proceedings of the Second International Symposium on Anaerobic InfectionsGeneva April 197941ndash44

39 FE Cunningham DM Kraus L Brubaker JH Fischer Pharmacokinetics of intra-vaginal metronidazole gel J Clin Pharmacol 1994341060ndash1065

40 MM Alper N Barwin WM McLean IJ McGilveray S Sved Systemic absorptionof metronidazole by the vaginal route Obstet Gynecol 198565781ndash784

41 IK Aronson JA Rumsfield DP West J Alexander JH Fischer FP Paloucek Evalu-ation of topical metronidazole gel in acne rosacea Drug Intell Clin Pharm 198721346ndash351

42 LK Schmadel GK McEvoy Topical metronidazole A new therapy for rosaceaClin Pharm 1990994ndash101

242 Lamp et al

43 DE Schwartz F Jeunet Comparative pharmacokinetic studies of ornidazole andmetronidazole in man Chemotherapy 19762219ndash29

44 AH Lau K Emmons R Seligsohn Pharmacokinetics of intravenous metronidazoleat different dosages in healthy subjects Int J Clin Pharmacol Ther Toxicol 199129386ndash390

45 I Amon K Amon H Huller Pharmacokinetics and therapeutic efficacy of metroni-dazole at different dosages Int J Clin Pharmacol Ther Toxicol 197816384ndash386

46 WL Hand N King-Thompson JW Holman Entry of roxithromycin (RU 965) imi-penem cefotaxime trimethoprim and metronidazole into human polymorphonu-clear leukocytes Antimicrob Agents Chemother 1987311553ndash1557

47 KE Andersson Pharmacokinetics of nitroimidazoles Spectrum of adverse reac-tions Scand J Infect Dis 198126(suppl 26)60ndash67

48 I Nilsson-Ehle B Ursing P Nilsson-Ehle Liquid chromatographic assay for metro-nidazole and tinidazole Pharmacokinetic and metabolic studies in human subjectsAntimicrob Agents Chemother 198119754ndash760

49 TY Ti HS Lee YM Khoo Disposition of intravenous metronidazole in Asian sur-gical patients Antimicrob Agents Chemother 1996402248ndash2251

50 SK Spangler MR Jacobs PC Appelbaum Time-kill study of the activity of tro-vafloxacin compared with ciprofloxacin sparfloxacin metronidazole cefoxitinpiperacillin and piperacillintazobactam against six anaerobes J Antimicrob Che-mother 199739(suppl B)23ndash27

51 H Mattie AC Dijkmans C van Gulpen The pharmacokinetics of metronidazoleand tinidazole in patients with mixed aerobic-anaerobic infections J AntimicrobChemother 198210(suppl A)59ndash64

52 JG Bartlett TJ Louie SL Gorbach AB Onderonk Therapeutic efficacy of 29 anti-microbial regimens in experimental intraabdominal sepsis Rev Infect Dis 19813535ndash542

53 DE Nix R Tyrrell M Muller Pharmacodynamics of metronidazole determined bya time-kill assay for Trichomonas vaginalis Antimicrob Agents Chemother 1995391848ndash1852

54 WA Craig SC Ebert Killing and regrowth of bacteria in vitro A review ScandJ Infect Dis 199174(suppl 74)63ndash70

55 P Van der Auwera Y Van Laethem N Defresne M Husson J Klastersky Compar-ative serum bactericidal activity against test anaerobes in volunteers receiving imi-penem clindamycin latamoxef and metronidazole J Antimicrob Chemother 198719205ndash210

56 M Boeckh H Lode KM Deppermann S Grineisen F Shokry R Held K WernickeP Koeppe J Wagner C Krasemann K Borner Pharmacokinetics and serum bacteri-cidal activities of quinolones in combination with clindamycin metronidazole andornidazole Antimicrob Agents Chemother 1990342407ndash2414

57 A Pefanis C Thauvin-Elipoulos J Holden GM Eliopoulos MJ Ferraro RC Moell-ering Jr Activity of fleroxacin alone and in combination with clindamycin or metro-nidazole in experimental intra-abdominal abscesses Antimicrob Agents Chemother199438252ndash255

58 SF Kowalsky RM Echols EM McCormick Comparative serum bactericidal activ-


ity of ceftizoximemetronidazole ceftizoxime clindamycin and imipenem againstobligate anaerobic bacteria J Antimicrob Chemother 199025767ndash775

59 CD Freeman CH Nightingale DP Nicolau PP Belliveau PR Tessier Q Fu DXuan R Quintiliani Bactericidal activity of low-dose ceftizoxime plus metronida-zole compared with cefoxitin and ampicillin-sulbactam Pharmacotherapy 199414185ndash190

60 CD Freeman CH Nightingale DP Nicolau PP Belliveau R Quintiliani Serumbactericidal activity of ceftriaxone plus metronidazole against common intra-ab-dominal pathogens Am J Hosp Pharm 1994511782ndash1787

61 Pharmacia amp Upjohn Cleocin Phosphate manufacturerrsquos package insert Phar-macia amp Upjohn 1998

62 ME Falagas SL Gorbach Clindamycin and metronidazole Med Clin N Am 199579845ndash867

63 PG Kremsner Clindamycin in malaria treatment J Antimicrob Chemother 1990259ndash14

64 GV Doern MA Pfaller K Kugler J Freeman RN Jones Prevalence of antimicro-bial resistance among respiratory tract isolates of Streptococcus pneumoniae inNorth America 1997 results from the SENTRY antimicrobial surveillance pro-gram Clin Infect Dis 199827764ndash770

65 MA Pfaller RN Jones GV Doern K Kugler and the SENTRY Participants GroupBacterial pathogens isolated from patients with bloodstream infection Frequenciesof occurrence and antimicrobial susceptibility patterns from the SENTRY antimi-crobial surveillance program (United States and Canada 1997) Antimicrob AgentsChemother 1998421762ndash1770

66 National Committee for Clinical and Laboratory Standards Performance standardsfor antimicrobial susceptibility testing Eighth informational supplement NCCLSdocument M100-S8 Wayne PA 1998

67 JF Flaherty LC Rodondi BJ Guglielmo JC Fleishaker RJ Townsend JG Gamber-toglio Comparative pharmacokinetics and serum inhibitory activity of clindamycindifferent dosing regimens Antimicrob Agents Chemother 1988321825ndash1829

68 ME Klepser DP Nicolau R Quintiliani CH Nightingale Bactericidal activity oflow-dose clindamycin administered at 8- and 12-hour intervals against Staphylo-coccus aureus Streptococcus pneumoniae and Bacteroides fragilis AntimicrobAgents Chemother 199741630ndash635

69 JD Smilack WR Wilson FR Cockerill III Tetracyclines chloramphenicol erythro-mycin clindamycin and metronidazole Mayo Clin Proc 1991661270ndash1280

70 ME Klepser MA Banevicius R Quintiliani CH Nightingale Characterization ofbactericidal activity of clindamycin against Bacteroides fragilis via kill curve meth-ods Antimicrob Agents Chemother 1996401941ndash1944

71 WA Craig S Gudmundsson Postantibiotic effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams and Wilkins Baltimore 1996296ndash329

72 KE Aldridge CW Stratton Bactericidal activity of ceftizoxime cefotetan and clin-damycin against cefoxitan-resistant strains of the Bacteroides fragilis group J Anti-microb Chemother 199128701ndash705

73 M Boeckh H Lode KM Deppermann S Grineisen F Shokry R Held K WernickeP Koeppe J Wagner C Krasemann et al Pharmacokinetics and serum bactericidal

244 Lamp et al

activities of quinolones in combination with clindamycin metronidazole and orni-dazole Antimicrob Agents Chemother 1990342407ndash2414

74 MP Weinstein RG Deeter KA Swanson JS Gross Crossover assessment of serumbactericidal activity and pharmacokinetics of ciprofloxacin alone and in combina-tion in healthy elderly volunteers Antimicrob Agents Chemother 1991352352ndash2358


76 IB Xue PG Davey G Phillips Variations in postantibiotic effect of clindamycinagainst clinical isolates of Staphylococcus aureus and implications for dosing ofpatients with osteomyelitis Antimicrob Agents Chemother 1996401403ndash1407

77 B Fantin R Lequercq Y Merle L Saint-Julien C Veyrat J Duval C CarbonCritical influence of resistance to streptogramin B-type antibiotics on activity ofRP 59500 (quinupristin-dalfopristin) in experimental endocarditis due to Staphylo-coccus aureus Antimicrob Agents Chemother 1995 39400ndash405

78 R Rende-Fournier R Leclercq M Galimand J Duval P Courvalin Identificationof the satA gene encoding a streptogramin A acetyltransferase in Enterococcusfaecium BM4145 Antimicrob Agents Chemother 1993372119ndash2125

79 JM Andrews R Wise The in-vitro activity of a new semi-synthetic streptogramincompound RP 59500 against staphylococci and respiratory patohgens J Antimi-crob Chemother 199433849ndash853

80 C Cocito M Di Giambattista E Nyssen P Vannuffel Inhibition of protein synthesisby streptogramins and related antibiotics J Antimicrob Chemother 199739(supplA)7ndash13

81 DH Bouanchaud In-vitro and in-vivo antibacterial activity of quinupristindalfo-pristin J Antimicrob Chemother 199739(suppl A)15ndash21

82 HC Neu NX Chin JW Gu The in-vitro activity of new streptogramins RP 59500RP 57669 and RP 54476 alone and in combination J Antimicrob Chemother 199230(suppl A)83ndash94

83 PC Appelbaum SK Spangler MR Jacobs Susceptibility of 539 gram-positive andgram-negative anaerobes to new agents including RP59500 biapenem trospecto-mycin and piperacillintazobactam J Antimicrob Chemother 199332223ndash231

84 SD Etienne G Montay A Le Liboux A Frydman JJ Garaud A phase I double-blind placebo-controlled study of the tolerance and pharmacokinetic behaviour ofRP 59500 J Antimicrob Chemother 199230(suppl A)123ndash131

85 M Bergeron G Montay The pharmacokinetics of quinupristindalfopristin in labo-ratory animals and in humans J Antimicrob Chemother 199739(suppl A)129ndash138

86 CA Johnson CA Taulor SW Zimmerman WE Bridson P Chevlaier O PasquierRI Baybutt Pharmacokinetics of quinupristin-dalfopristin in continuous ambula-tory peritoneal dialysis patients Antimicrob Agents Chemother 1999 43152ndash156

87 E Bernard M Bensoussan F Bensoussan S Etienne I Cazenave E Carsenti-EtesseY Le Roux G Montay P Dellamonica Pharmacokinetics and suction blister fluidpenetration of a semisynthetic injectable streptogramin RP 59500 (RP 57669RP54476) Eur J Clin Microbiol Infect Dis 199413768ndash771


88 C Gaillard J Van Cantfort G Montay D Piffard A Le Liboux S Etienne AScheen A Frydman Disposition of the radiolabelled streptogramin RP 59500 inhealthy male volunteers In Programs and Abstracts of the 32nd Interscience Con-ference on Antimicrobial Agents and Chemotherapy Anaheim 1992 Abstract1317 Am Soc Microbiol Washington DC

89 RJ Fass In vitro activity of RP 59500 a semisynthetic injectable pristinamycinagainst staphylococci streptococci and enterococci Antimicrob Agents Chemo-ther 199135553ndash559

90 RL Hill CT Smith M Seyed-Akhavani MW Casewell Bactericidal and inhibitoryactivity of quinupristindalfopristin against vancomycin- and gentamicin-resistantEnterococcus faecium J Antimicrob Chemother 199739(suppl A)23ndash28

91 JR Aeschlimann MJ Rybak Pharmacodynamic analysis of the activity of quinu-pristin-dalfopristin against voncomycin-resistant Enterococcus faecium with dif-fering MBCs via time-kill-curve and postantibiotic effect method AntimicrobAgents Chemother 1998422188ndash2192

92 DE Low HL Nadler A review of in-vitro antibacterial activity of quinupristindalfopristin against methicillin-susceptible and -resistant Staphylococcus aureus JAntimicrob Chemother 199739(suppl A)53ndash58

93 F Caron HS Gold CB Wennersten MG Farris RC Moellering Jr GM EliopoulosInfluence of erythromycin resistance inoculum growth phase and incubation timeon assessment of the bactericidal activity of RP 59500 (quinupristin-dalfopristin)against vancomycin-resistant Enterococcus faecium Antimicrob Agents Chemo-ther 1997412749ndash2753

94 JM Entenza H Drugeon MP Glauser P Moreillon Treatment of experimental en-docarditis due to erythromycin-susceptible or -resistant methicillin-resistant Staph-ylococcus aureus with RP 59500 Antimicrob Agents Chemother 1995391419ndash1424

95 GA Pankuch MR Jacobs PC Applebaum Study of comparative antipneumococcalactivities of penicillin G RP 59500 erythromycin sparfloxacin ciprofloxacin andvancomycin by using time-kill methodology Antimicrob Agents Chemother 1994382065ndash2072

96 GA Pankuch MR Jacobs PC Applebaum MIC and time-kill study of antipneumo-coccal activities of RPR 106972 (a new oral streptogramin) RP 59500 (quinupris-tin-dalfopristin) pyostacine (RP 7293) penicillin G cefotaxime erythromycin andclarithromycin against 10 penicillin-susceptible and -resistant pneumococci Anti-microb Agents Chemother 1996402071ndash2074

97 GA Pankuch C Lichtenberger MR Jacobs PC Appelbaum Antipneumococcal ac-tivities of RP 59500 (quinupristin-dalfopristin) penicillin G erythromycin andsparfloxacin determined by MIC and rapid time-kill methodologies AntimicrobAgents Chemother 1996401653ndash1656

98 F LrsquoHeriteau JM Entenza F Lacassin C Leport MP Glauser P Moreillon RP59500 prophylaxis of experimental endocarditis due to erythromycin-susceptibleand -resistant isogenic pairs of viridans group streptococci Antimicrob AgentsChemother 1995391425ndash1429

99 GA Pankuch MR Jacobs PC Appelbaum Postantibiotic effect and postantibioticsub-MIC effect of quinupristin-dalfopristin against gram-positive and -negative or-ganisms Antimicrob Agents Chemother 1998423028ndash3031

246 Lamp et al

100 A Nougayrede N Berthaud DH Bouanchaud Post-antibiotic effects of RP 59500with Staphylococcus aureus J Antimicrob Chemother 199230(suppl A)101ndash106

101 W Craig S Ebert Pharmacodynamic activities of RP 50500 in an animal infectionmodel In Programs and Abstracts of the 33rd Interscience Conference on Antimi-crobial Agents and Chemotherapy New Orleans 1993 Abstract 470 Am Soc Mi-crobiol Washington DC

102 JR Aeschlimann MJ Zervos MJ Rybak Treatment of vancomycin-resistant En-terococcus faecium with RP 59500 (quinupristin-dalfopristin) administered by in-termittent or continuous infusion alone or in combination with doxycycline in anin vitro pharmacodynamic infection model with simulated endocardial vegetationsAntimicrob Agents Chemother 1998422710ndash2717

103 SL Kang MJ Rybak Pharmacodynamics of RP 59500 alone and in combinationwith vancomycin against Staphylococcus aureus in an in vitro-infected fibrin clotmodel Antimicrob Agents Chemother 1995391505ndash1511

104 MJ Rybak Houlihan RC Mercier GW Kaatz Pharmacodynamics of RP 59500(quinupristin-dalfopristin) administered by intermittent versus continuous infusionagainst Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infectionmodel Antimicrob Agents Chemother 1997411359ndash1363

105 B Fantin R Leclercq L Garry C Carbon Influence of inducible cross-resistanceto macrolides lincosamides and streptogramin B-type antibiotics in Enterococcusfaecium on activity of quinupristin-dalfopristin in vitro and in rabbits with experi-mental endocarditis Antimicrob Agents Chemother 199741931- 935

106 A Turcotte MG Bergeron Pharmacodynamic interaction between RP 59500 andgram-positive bacteria infecting fibrin clots Antimicrob Agents Chemother 1992362211ndash2215

107 HF Chambers Studies of RP 59500 in vitro and in a rabbit model of aortic valveendocarditis caused by methicillin-resistant Staphylococcus aureus J AntimicrobChemother 199230(supp A)117ndash122

108 B Fantin R Leclercq M Ottaviani JM Vallois B Maziere J Duval JJ PocidaloC Carbon In vivo activities and penetration of the two components of the strepto-gramin RP 59500 in cardiac vegetations of experimental endocarditis AntimicrobAgents Chemother 199438432ndash437

109 N Berthaud G Montay BJ Conard JF Desnottes Bactericidal activity and kineticsof RP 59500 in a mouse model of Staphylococcus aureus septicaemia J AntimicrobChemother 199536365ndash373

110 DH Bouanchaud In-vitro and in-vivo synergic activity and fractional inhibitoryconcentration (FIC) of the components of a semisynthetic streptogramin RP 59500J Antimicrob Chemother 199230(suppl A)95ndash99

111 RC Mercier SR Penzak MJ Rybak In vitro activities of an investigational quino-lone glycylcycline glycopeptide streptogramin and oxazolidinone tested aloneand in combinations against vancomycin-resistant Enterococcus faecium Antimi-crob Agents Chemother 1997412573ndash2575

11

Tetracycline Pharmacodynamics

Burke A Cunha

Winthrop-University Hospital Mineola and theState University of New York School of Medicine Stony Brook New York

Holly M Mattoes


1 INTRODUCTION

The first tetracycline to be discovered chlortetracycline was isolated fromStreptomyces aureufaciens in 1944 Since 1944 several tetracycline analogs havebeen developed including oxytetracycline which was introduced in 1950 tetracy-cline hydrochloride (1953) and demethylchlortetracycline (demeclocycline) Inthe late 1950s it was discovered that the 6-hydroxyl group could be removedfrom the basic tetracycline group which resulted in 6-deoxytetracyclines withsignificantly different microbiological and pharmacokinetic properties In the1960s the long-acting tetracyclines were introduced Doxycycline was isolatedin 1962 and minocycline was introduced in 1967 Although all tetracyclinesinhibit bacterial protein synthesis there are significant differences in inherentantibacterial activity between short-acting tetracyclines eg tetracycline andlong-acting second generation tetracyclines eg doxycycline and minocycline[1ndash3] The lsquolsquosecond generationrsquorsquo tetracyclines have better activity excellent phar-

247

248 Cunha and Mattoes

macokinetics better intestinal absorption better tissue penetration and decreasedtoxicity [4]

Recently the glycylcyclines minocycline derivatives have demonstratedpotent activity against a wide range of pathogens including vancomycin-resistantenterococcus (VRE) [5] The glycylcyclines have enhanced activity against aero-bic and anaerobic bacteria that are typically resistant to tetracycline antimicrobi-als [6] This enhanced activity is due to the agentsrsquo ability to overcome both ofthe major resistant mechanisms ribosomal protection and active efflux whichresult in tetracycline resistance As a result the glycylcycline spectrum of activityincludes penicillin-resistant Streptococcus pneumoniae VRE and anaerobes likedoxycycline [1ndash35]

2 PHARMACOKINETICS OF TETRACYCLINES

21 Tetracycline

The tetracycline class of antibiotics are variably absorbed and their pharmacoki-netic properties are often due to the differing relative lipid solubilities and protein-binding capacities Tetracycline has a shorter serum half-life (6 h) than that ofthe second-generation antimicrobials doxycycline (22 h) and minocycline (11ndash33 h) As a result the short half-life of tetracycline means it needs to be given asfour divided doses daily In patients with renal failure the half-life of tetracyclinedramatically increases and in patients with severe renal failure the half-life canbe prolonged to 57ndash120 h Doxycycline does not accumulate in renal failure andthe dose of doxycycline remains unchanged even in dialysis patients with renalfailure Among the class of tetracyclines protein binding is the lowest with tetra-cycline demonstrated to be 20ndash65 depending on the method of analysis [3]Unlike doxcycline the absorption of tetracycline is significantly affected by foodand milk decreasing by up to 50 Additionally tetracycline is chelated by ca-tions and therefore should not be used concurrently with antacids

Pharmacokinetic studies of tetracycline found that serum concentrationsafter a single 500 mg oral dose peaked at 3ndash43 microgmL after 2ndash4 h whereas inpatients with normal renal function at steady state the same dose achieved a peakconcentration of 2ndash5 microgmL A 250 mg oral dose had an average peak serumconcentration of 24 microg at 3 h and this dropped to 1 microgmL at 12 h An in vitromodel determined that at therapeutic doses the tissue distribution in the lungachieved concentrations of 02ndash2 microgmL Tissue penetration into the lungs ap-pears to be the same whether the lungs are healthy or diseased [7] Tetracyclinehas the ability to penetrate into reticuloendothelial cells which allows for its useagainst infections by intracellular pathogens such as Rickettsia Chlamydia andLegionella [8] Gram-positive resistance to tetracycline has dramatically limited

Tetracycline Pharmacodynamics 249

its use to treat common infections however doxycycline and minocycline haveno resistance after decades of extensive use [210]

22 Minocycline

Minocycline and doxycycline are considered second generation tetracyclinesowing to their pharmacokinetic advantages over tetracycline which result in en-hanced tissue and fluid penetration and more convenient once or twice daily dos-ing regimens Fewer data have been published regarding the pharmacokineticsof minocycline than that of doxycycline Minocycline may be given PO or IVand serum concentrations following a typical dosing regimen of an initial 200mg dose followed by 100 mg every 12 h results in steady state of 23ndash35 microgmL The half-life is approximately 17 h after a single dose and 21 h after multipledosing and unlike tetracycline the half-life is not prolonged or substantiallyincreased in renal failure [8] The absorption of minocycline is decreased byapproximately 20 following ingestion of food andor milk but is not clinicallysignificant Chelation occurs with minocycline therefore concomittant antaciduse should be avoided Similar to tetracycline doxycycline minocycline demon-strate high concentrations intracellularly which is useful in treating Chlamydiaand Legionella infections

23 Doxycycline

Doxycycline is highly protein-bound (82) and has the advantage of being 5times as lipid-soluble as tetracycline It is rapidly and almost completely (93)absorbed from the upper portion of the gastrointestinal tract following oral admin-istration tetracycline is less efficiently (25ndash80) absorbed Food does not havean important effect on the absorption of orally administered doxycycline andserum levels are not decreased in the presence of antacids gluten or metalliccations The absorption half-life of doxycycline is approximately 50 min anddetectable serum levels are achieved within 30 min following oral administrationPeak levels occur 2 h after dosing and the serum t12 is 22 h Oral antacidshowever have been found to increase the total clearance of IV doxycycline re-sulting in a decreased half-life [28]

Peak serum levels of doxycycline following a single 200 mg oral dose are27 microgmL (range 21ndash44 microgmL) A single 200 mg dose of doxycycline re-sults in serum levels of 16 microgmL (range of 15ndash17 microgmL) After a 400 mgoral dose doxycycline serum concentrations are approximately 40 microgmL Se-rum levels of doxycycline are the same with either oral or intravenous administra-tion after a steady state is achieved Intravenous administration of doxycyclineresults in higher initial serum concentrations 100 mg (IV) q12h 4 microgmL100 mg (IV) q8h 7 microgmL 200 mg (IV) q12h 15 microgmL Doxycycline is


widely distributed throughout body tissues Lung levels after 200 mg of doxycy-cline (IV) achieve concentrations of 68 microgg Average levels of doxycycline are59 microgg Doxycycline achieves therapeutic levels in the gallbladder bile ductwall and bile and readily penetrates respiratory secretions In patients with acutemaxillary sinusitis doxycycline reaches concentrations of 21 microgmL in sinussecretion Mean prostatic concentration of doxycycline is 163 microgg and doxy-cycline accumulates in prostatic tissue In patients given 200 mg doxycyclineorally followed by 100 mgday average ovarian levels ranged from 304 to 324microgg Levels of doxycycline in fallopian tubes were 321 microgg Mean doxycyclinelevels in breast milk at 24 h after drug administration were about 40 of serumlevels Moderate doxycycline concentrations are also found in umbilical bloodand amniotic fluid [1ndash3910]

Based on doxycyclines pharmacokinetics therapy should be initiated witha 72 h loading regimen because of its high lipid solubility for serious infectionseg Legionnairersquos disease A dose of 200 mg IV q12h provides rapid tissuesaturation of doxycycline and is effective in achieving maximum serum concen-trations rapidly because 5 serum half-lives are required before steady-state ki-netics are achieved Doxycycline 100 mg (IV) q12h basis requires 4ndash5 days oftherapy before a therapeutic effect can be achieved Since the serum t12 of doxy-cycline is 22 h an initial 72 h loading regimen is required if a rapid therapeuticeffect is desired [927] Doxycycline does not significantly accumulate in patientswith renal insufficiency Blood levels of doxycycline in patients with severelyimpaired renal function are the same as in patients with normal kidneys Thusdoxycycline should be administered in the usual dosage to patients with renalimpairment without risk of accumulation Hemodialysis has a negligible effecton the serum half-life of doxycycline and a dosage adjustment is not neededwhen doxycycline is administered in patients undergoing hemodialysis [1ndash3910]

24 Glycylcyclines

The glycylcyclines are a derivative of minocycline and have good activity againstgram-positivegram-negative pathogens GAR-936 a member of the glycylcy-cline class has good penetration into tissues with a high tissueplasma ratio anda large volume of distribution 101 Lkg in rats [11] A study evaluating healthysubjects found that the maximal serum concentration (Cmax) and area under theserum concentration versus time curve (AUC) values of this antimicrobial agentwas of proportional following a 1 h IV infusion of 125 mg resulting in a Cmax

of 011 microgmL and an AUC of 09 microgsdothmL Following a 300 mg dose theCmax and AUC increased to 28 and 179 microgsdothmL respectively When GAR-936 was dosed at 50 mgkg subcutaneously in a murine model of Pseudomonas


aeruginosa AUC values reached 633 microgsdothmL achieving better concentra-tions than a similarly dosed gentamicin which demonstrated an AUC of 511microgsdothmL [12] Additionally the half-life of GAR-936 was found to be long at36 h [13] Similarly to tetracycline and minocycline GAR-936 chelates cal-cium [11]

3 PHARMACODYNAMICS OF TETRACYCLINES

The tetracyclines are believed to be bacteriostatic when used in vitro howeverin high concentrations are bacteriocidal [3] Tetracyclines to have time-dependentkill meaning that the parameter of the time above the minimum inhibitory con-centration of the pathogen (T MIC) best describes the killing by these drugsFor time-dependent killing the killing is dependent on the length of time thebacteria are exposed to the antibiotic As the area under the concentration versustime curve for plasma (AUC) approaches 2ndash4 times the MIC value of the organ-ism the effect is maximized Here the rate of killing plateau and additional serumdrug concentration have a negligible effect and it is the time above the MIC isthe important parameter related to bacterial kill and antibacterial activity Gener-ally for these agents the T MIC should be at least 50 of the dosing intervalfor immunocompetent patients whereas immunocompromised patients may re-quire the T MIC 100 of the dosing interval

Tetracycline was once useful for a variety of gram-positive and gram-negative pathogens However as a result of increased resistance it is now typi-cally reserved for uncommon infections Additionally plasma-mediated resis-tance is occurring as a result of overuse in animal feeds containing tetracyclineand it appears that most acquired resistance of gram-positive and -negativepathogens is a result of this plasma-mediated problem [3] This makes it diffi-cult to treat infections by common gram-positive organisms which currentlydemonstrate a high level of resistance to tetracycline Tetracycline resistance isusually a result of active drug efflux due to an exogenous metalndashtetracyclineH antiporter Similarly doxycycline and minocycline do not share this resistanceproblem these second generation tetracyclines have excellent antimicrobial activ-ity [14]

Doxycycline is useful against a broad range of pathogens However it wasintroduced prior to the appreciation of current pharmacodynamic concepts andas a result it is only recently that the optimal dosing regimen for doxycyclinewas determined [15ndash20] Time-kill kinetic studies demonstrated that at low con-centrations ie at 2ndash4 times the MIC 100 mg (IVPO) q12h of doxycyclinekills the organisms tested in a time-dependent manner At higher serum concen-trations 200 mg (IVPO) q12h or 400 mg (IVPO) q24h ie 8ndash16 times the MIC


of the organisms doxycycline exhibits concentration-dependent killling [17ndash20](Fig 1) Additionally the postantibiotic effect (PAE) of doxycycline influencesthe pharmacodynamics of this agent PAE T C where T is the time requiredfor the counts of CFUmL in the test culture to increase by 1 log10 above thecount observed immediately after antibiotic removal and c is the time requiredfor the count of CFUmL in an untreated control culture to increase 1 log10 abovethe count observed immediately after completion of the same procedure used onthe test culture for antibiotic removal Doxycycline has a PAE for gram-positiveand gram-negative aerobic organisms that was found to be concentration-depen-dent Doxycycline has a PAE of 21ndash42 h with gram-positive and gram-negativeorganisms [20] (Fig 2) This is similar to the PAE of tetracycline which variesbetween 2 and 3 h

Glycylcyclines demonstrate time-dependent (concentration-independent)kinetics This was demonstrated in a thigh infection model which found thatT MIC was the best correlated pharmacodynamic model [21] The studycompared two glycylcyclines GAR 936 and WAY 152288 against a variety ofcommon gram-positive and gram-negative pathogens Whereas the glycylcy-cline GAR 936 only required the concentration of free drug to remain above theMIC for 50 of the dosing interval for effective eradication of Streptococcuspneumoniae Escherichia coli and Klebsiella pneumoniae similar killing withWAY 152288 required the MIC to be exceeded for at least 75 of the dosinginterval using the same isolates Because of the long half-lives and PAEs of theseagents the AUC also may play a predictive role in the killing of organisms[21]

It has been determined that for GAR 936 a single dose of 300 mg IV wouldprovide a concentration of 2 microgmL for 75 of the time [22] The glycylcyclineswould also have excellent activity against Staphylococcus aureus (MSSA) whichfor GAR 936 demonstrates MICs ranging from 006 to 1 microgmL and the MIC90

values for MSSA is 05 microgmL [23] An endocarditis model [24] found that whenserum levels fell below the MIC before 50 of the dosing interval GAR 936was as effective in eradicating VanA-resistant Enterococcus faecium as whenserum levels were constantly above the MIC throughout the dosing interval Theauthors concluded that this result may be due to the low clearance of the agentGAR 936 from the endocarditis vegetations as well as the long PAE againstenterococci [24]

Glycylcyclines has good activity against anaerobic bacteria covering mostcommon anerobic pathogens Unlike doxycycline and minocycline glycylcyineshave litle activity against Bacteroides fragilis and possibly Clostridium per-fringens [25] For Mycoplasma pneumoniae the susceptibility of GAR 936 mea-sured by the MIC50 was 02 microgmL twice that of doxycyclineminocycline andtwofold less than that of tetracycline [26]


FIGURE 1 Doxycycline kill curves for (a) Staphylococcus aureus (b) Strepto-coccus pneumoniae (c) Pasteurella multocida (d) Escherichia coli (With per-mission from Ref 20)


FIGURE 2 Doxycycline postantibiotic effects (PAEs) for (a) Staphylococcusaureus (b) Streptococcus pneumoniae (c) Pasteurella maltophilia (d) Esche-richia coli (With permission from Ref 20)


DISCUSSION

Although tetracyclines have been useful in treating a wide variety of pathogenssince the early 1900s increased resistance has made tetracycline second-line ther-apy until recently with the advent of doxycycline and minocycline Glycylcy-clines appear to be effective in treating a wide range of gram-positive gram-negative and anaerobic bacteria The second generation tetracyclines doxycy-cline and minocycline have increased pharmacokinetic and pharmacodynamicadvantages over tetracycline and other advantages in overall dosing allowingfor their potential once daily dosing as a result of their long PAEs and half-livesand increased activity against common pathogens which maintain their positionin the antibiotic armamentarism as first line agents [27ndash30]

REFERENCES

1 BA Cunha Clinical uses of tetracyclines In RK Blackwood JJ Hlavka JH Bootheds The Tetracyclines Springer-Verlag Berlin 1985393

2 BA Cunha JB Comer M Jonas The tetracyclines Med Clin North Am 198266293

3 NC Klein BA Cunha Tetracyclines Med Clin North Am 199579789ndash8014 N Joshi DQ Miller Doxycycline revisited Arch Intern Med 19971571421ndash

14285 PJ Petersen NV Jacobus WJ Weiss PE Sum RT Testa In vitro and in vivo antibac-

terial activities of a novel glycylcycline the 9-t-butylglycylamido derivative of mi-nocycline (GAR-936) Antimicrob Agents Chemother 199943738ndash744

6 RT Testa PJ Petersen NV Jacobus P Sum VJ Lee FP Tally In vitro and in vivoantibacterial activities of the glycylcyclines a new class of semisynthetic tetracy-clines Antimicrob Agents Chemother 1993372270ndash2277

7 MP Fournet R Zini L DeForges F Lange J Lange JP Tillement Tetracycline anderythromycin distribution in pathological lungs of humans and rat J Pharm Sci 1989781015ndash1019

8 GK McEvoy ed Tetracyclines In American Hospital Formularly Service DrugInformation American Society of Health System Pharmacists Bethesda MD 1997368ndash386

9 BA Cunha The pharmacokinetics of doxycycline Postgrad Med Commun 1979143ndash50

10 BA Cunha Doxycycline Antibiotics Clin 1999321ndash2711 NL Tombs Tissue distribution of GAR-936 a broad spectrum antibiotic in male

rats 39th Interscience Conference on Antimicrobial Agents and Chemotherapy1999 abstr P-413

12 SM Mikels AS Brown L Breden S Compton S Mitelman PJ Petersen WJ WeissIn vivo activities of GAR-936 (GAR) gentamicin (GEN) piperacillin (PIP) alone andin combination in a murine model of Pseudomonas aeruginosa pneumonia 39th Intersci-ence Conference on Antimicrobial Agents and Chemotherapy 1999 abstr P-414


13 G Muralidaharan J Getsy P Mayer I Paty M Micalizzi J Speth B Webster PMojaverian Pharmacokinetics (PK) safety and tolerability of GAR-936 a novelglycylcycline antibiotic in healthy subjects 39th Interscience Conference on Anti-microbial Agents and Chemotherapy 1999 abstr P-416

14 Y Someya A Yamaguchi T Sawai A novel glycylcycline 9-(nn-dimethylglycyl-amido)-6-demethyl-6-deoxytetracycline is neither transported nor recognized by thetransposon TN10-endoced metal-tetracyclineH antiporter Antimicrob AgentsChemother 199539247ndash249

15 WA Craig Pharmacokineticpharmacodynamic parameters Rationale for antibacte-rial dosing of mice and men Clin Infect Dis 1998261ndash12

16 K Totsuka K Shimizu In vitro and in vivo post-antibiotic effects (PAE) of CL331928 (DMG-DMDOT) a new glycylcycline against Staphylococcus aureus 34thInterscience Conference on Antimicrobial Agents and Chemotherapy 1994179abstr F114

17 WA Craig SC Ebert Killing and regrowth of bacteria in vitro A review Scand JInfect Dis Suppl 19917463ndash70

18 O Cars I Odenholt-Tornquist The post-antibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 199331159ndash166

19 WA Craig S Gudmundsson Post-antibiotic effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams and Wilkins Baltimore 1996296ndash329

20 BA Cunha P Domenico CB Cunha Pharmacodynamics of doxycycline Clin Mi-crob Infect Dis 20006270ndash273

21 ML van Ogtrop D Andes TJ Stamstad B Conklin WJ Weiss WA Craig O VesgaIn vivo pharmacodynamic activities of two glycylcyclines (GAR-936 and WAY152288) against various gram-positive and gram-negative bacteria AntimicrobialAgents Chemother 200044943ndash949

22 HW Boucher CB Wennersten RC Moellering GM Eliopoulos In vitro activity ofGAR-936 against gram-positive bacteria 39th Interscience Conference on Antimi-crobial Agents and Chemotherapy 1999 abstr P-406

23 E Mahalingam L Trepeski S Pongporter J De Azavedo DE Lo BN KreiswirthIn vitro activity of new glycylcycline Gar 936 against methicillin-resistant and-susceptible Staphylococcus aureus isolated in North America 39th InterscienceConference on Antimicrobial Agents and Chemotherapy 1999 abstr P-408

24 A Lefort L Massias A Saleh-Mghir M LaFaurie L Garry C Carbon B FantinPharmacodynamics of GAR-936 (GAR) in experimental endocarditis due to VANA-type Enterococcus faecium 40th Interscience Conference on AntimicrobialAgents and Chemotherapy 2000 abstr P-2256

25 M Hedberg CE Nord In vitro activity of anaerobic bacteria to GAR-936 a newglycylcycline 39th Interscience Conference on Antimicrobial Agents and Chemo-therapy 1999 abstr P-411

26 GE Kenny FD Cartwright The susceptibility of human mycoplasmas to a new gly-cylcycline GAR 936 39th Interscience Conference on Antimicrobial Agents andChemotherapy 1999 abstr P-412

27 VX Nguyen DE Nix S Gillikin JJ Schentag Effect of oral antacid administrationon the pharmacokinetics of intravenous doxycycline Antimicrob Agents Chemother198933434ndash436


28 HW Boucher CB Wennersten GM Eliopoulis In vitro activities of the glycyclineGAR-936 against gram positive bacteria Antimicrob Agents Chemother 2000442225ndash2229

29 KW Shea Y Ueno F Abumustafa SMH Qadri BA Cunha Doxycycline activityagainst streptococcus pneumoniae Chest 19951071775ndash1776

30 BA Cunha Doxycycline revisited Intern Med 19971065ndash69

12

Pharmacodynamics of Antivirals

George L Drusano Sandra L Preston

and Peter J Piliero

Albany Medical College Albany New York

1 INTRODUCTION

Although pharmacodynamic relationships for antibacterial agents have beensought and identified for a long period of time it has been a general feeling thatthe treatment of viral and fungal infections was different in kind A corollary tothis is that delineation of pharmacodynamic relationships would be much moredifficult

In reality the exact principles that govern the delineation of pharmaco-kineticpharmacodynamic (PKPD) relationships for antibacterials also governantifungals and as will be shown here antivirals

The first issue as always in the development of dynamics relationships isto decide upon an endpoint Most of the data for this chapter have been derivedfrom studies of the human immunodeficiency virus (HIV) Type 1 Consequentlythis chapter addresses the in vitro and in vivo data for this virus Other virusesshould behave in a similar fashion (as is seen with cytomegalovirus) An excep-tion to the rule is hepatitis C virus (among others) because of our inability toobtain more than one round of viral replication in vitro

With HIV-1 there are a number of endpoints that may be chosen Some are

259

260 Drusano et al

1 Survivorship2 Change in CD4 count consequent to therapy3 Change in viral load consequent to therapy4 Change in the hazard of emergence of resistance consequent to therapy5 Durability of maintaining the viral load below detectable levels

Survivorship is as always the ultimate test of an intervention With the adventof potent HIV chemotherapy this disease process has been converted from arelatively rapid killer to one that will likely take its toll over a number of decadesConsequently survivorship may not be the best endpoint to examine for clinicalstudies because of the long lead times In this chapter we concentrate on end-points 2ndash5 for clinical studies

In anti-HIV chemotherapy as in antibacterial chemotherapy one can gener-ate pharmacodynamic relationships from in vitro approaches as well as fromclinical studies One difference between these areas is the ease of employinganimal systems for the generation of dynamic relationships For antibacterialsthere are many examples of the generation of dynamic relationships for differentdrug classes [1ndash3] For HIV the cost and ethical implications of using simianmodels has prevented much work in this area The McCune model and its variantsare attractive but again because of the cost of maintaining the system little hasbeen done to develop dynamic relationships for HIV in animal systems Otherretroviruses have been employed [45] but it is difficult to draw inferences forhuman chemotherapy of HIV from a different pathogen with different pathoge-netic properties

Consequently in this chapter in vitro but not animal model systems arealso reviewed to add insight gleaned from these systems

2 IN VITRO SYSTEMS

Although one might consider determination of viral EC50 or EC90 to be a pharma-codynamic system measurement this chapter takes the view that this is simplya static measure of drug potency It will play (see below) an important role indetermining the final shape of the pharmacodynamic relationship when added toother measures Alone it is merely a measure of compound potency

For in vitro systems to be true pharmacodynamic systems there must bethe possibility of changing drug concentrations over time to examine the effectfor a pathogen of known susceptibility to the drug employed The only systemwith peer-reviewed publication for HIV-1 is shown in Fig 1 In addition it isimportant to factor in the effect of protein binding as only the free drug is virolog-ically active

Pharmacodynamics of Antivirals 261

FIGURE 1 Schematic diagram of the in vitro pharmacodynamic model sys-tem One of the two systems enclosed within a single incubator housed in abiological safety cabinet is shown Cells are grown and samples are removedfrom the extracapillary compartment of HF bioreactors Constant infusionoral or intravenous bolus doses are introduced through the dosing ports inthe diluent reservoir absorption compartment or central reservoir respec-tively Exposure of cells to fluctuating concentrations of D4T is affected byprogrammed dilution of drug within the central reservoir while the volumeof the central compartment is maintained constant by elimination The meanpore diameter of the HF capillaries (10 kDa) would prevent HIV or HIV-infectedcells from exiting the bioreactor and from circulating through the tubing (Fur-ther information is available upon request)

262 Drusano et al

3 PROTEIN BINDING

The effect of protein binding has been examined in greatest detail for its effecton the anti-HIV potency of a drug by this laboratory Bilello et al [6] examinedan HIV-1 protease inhibitor (A-80987) and determined the effect of protein bind-ing on free-drug concentration on drug uptake into infected cells and finallyon the virological effect of the drug This set of experiments has a lsquolsquotransitivelogicrsquorsquo organization

In the first experiment (Fig 2) increasing concentrations of the bindingprotein α1 acid glycoprotein were introduced into the test system and the con-centration of unbound drug was determined As can be seen increasing bindingprotein concentration leads to monotonically decreasing free-drug concentrations

In Fig 3 the amount of drug that penetrates infected CEM cells is shownas a function of the free fraction of the drug Increased external free-drug concen-tration results in an increased amount of intracellular drug (stop oil experiments)in a linear function Finally (Fig 4) the amount of intracellular drug is relatedto the decrement of p24 output as indexed through an inhibitory sigmoid-Emaxmodel It is clear through this series of experiments that only the free-drug con-centration can induce a decrease in viral production

These data make it clear that it is important to interpret drug concentrationsin the hollow fiber system as representing the external free-drug concentrationsnecessary to induce the desired antiviral effect

FIGURE 2 Relationship between increasing Prime1 acid glycoprotein concentra-tion and A-80987 free drug concentration


FIGURE 3 Relationship between increasing A-80987 free fraction and intracel-lular A-80987

FIGURE 4 Relationship (inhibitory sigmoid Emax) between intracellular A-80987 and HIV viral output from infected PBMCs

264 Drusano et al

The most obvious place to initiate evaluation of anti-HIV agents is withthe nucleoside analogs and with the HIV-1 protease inhibitors The first publishedhollow fiber evaluation of an antiviral was the nucleoside analog stavudine (d4T)Bilello et al [7] examined issues of dose finding and schedule dependency forthis drug

It is important to examine the development of stavudine in order to placeits evaluation in the hollow fiber system in the proper perspective The initialclinical evaluation of stavudine was initiated at a total daily dose of 2 mgkg perday The initial schedule chosen was every 8 h Whereas the initial dose had aneffect dose escalation to 12 mgkg per day produced no change in effect butmuch higher rates of drug-related neuropathy [8]

After this the dose was de-escalated to 4 mgkg per day and the schedulewas lengthened to every 12 h Further dose de-escalation was taken from thispoint on a 12 h schedule and nine dosing cohorts were eventually examinedThis process took approximately 2 years

The hollow fiber evaluation took approximately 3 months The identifieddose was ultimately the dose chosen by the phase III trial and has stood the testof time and usage It is at least to our knowledge the first prospective identifica-tion of a drug dose and schedule from an in vitro test system with clinical valida-tion The outcome of the experiments is illustrated in Fig 5 This evaluationmakes clear that for nucleoside analogs the pharmacodynamically linked variableis AUCEC90 With matching AUCs (Fig 5) there was no difference in outcomebetween the exposure being given in a continuous infusion mode and half theexposure every 12 h (data not shown) The reason is likely the phosphorylationof the parent compound into the virologically active form of the molecule (thetriphosphate for stavudine or diphosphate for prephosphorylated compoundssuch as tenofovir) It is clear from Figs 6 and 7 that there is full effect at 05mgkg every 12 h irrespective of starting challenge At half the exposure (025mgkg q12h) there is a hint of loss of control late in the experiment Finally at0125 mgkg q12h there is no discernible antiretroviral effect

HIV-1 protease inhibitors have also been examined in this system The firstto be examined was the early Abbott inhibitor A-77003 This drug was a proof-of-principle agent and was administered intravenously as a continuous infusionConsequently the hollow fiber evaluation was performed as a continuous infu-sion When protein binding was taken into account the concentrations requiredfor effect were above those tolerable clinically Consequently the drug was pre-dicted to fail Indeed an extensive phase III evaluation came to this conclusion[910]

The next protease inhibitor evaluated was amprenavir (141W94) Here[11] time EC90 was demonstrated to be the pharmacodynamically linked vari-able Dosing intervals of q12h and q8h with matching AUCs were compared to


FIGURE 5 The concentrationndashtime profile for stavudine in the hollow fiberunit is displayed for a 1 mg(kg sdot day) dose administered either as a continu-ous infusion or as 05 mgkg administered every 12 h The AUCs developedare identical The ability to suppress viral replication was identical for theregimens

continuous infusion of the same AUC Continuous infusion provided the mostrobust viral control followed closely by q8h dosing Every 12 h dosing lost asignificant fraction of the control of viral turnover This outcome was to be ex-pected as HIV-1 protease inhibitors are reversible inhibitors freely cross cellmembranes without the requirement for energy or a transporter and do not re-quire activation (phosphorylation) for effect as the nucleoside analogs do

A third protease inhibitor was examined the potent once-daily PI BMS232632 [12] In addition to the hollow fiber unit evaluation the technique ofMonte Carlo simulation was also brought to bear on the outcome of these experi-ments

In Fig 8 it is clear that time EC90 is the pharmacodynamically linkedvariable In the one instance complete control of viral replication in vitro isachieved with a continuous infusion regimen at 4 EC50 (about EC90ndash95) Thissame daily AUC administered as a bolus allows breakthrough growth Four timesthis daily AUC administered as a bolus regains control of the viral replicationIndeed this latter part of the experiment demonstrates that free-drug concentra-

266 Drusano et al

FIGURE 6 Panel a displays the increase in p24 output over time in a hollowfiber tube that was an untreated control () in a treated (05 mgkg q12h)tube where 11000 cells were chronically HIV-infected at start () or where1100 cells were HIV-infected at start () Panel b shows numbers of HIV DNAcopies in treated and untreated hollow fiber units


FIGURE 7 Panel a displays the increase in p24 output over time in a hollowfiber tube that was an untreated control () in a treated (025 mgkg q12h)tube where 11000 cells were chronically HIV-infected at start () or where1100 cells were HIV-infected at start () Panel b shows p24 output in atreated hollow fiber unit (0125 mgkg q12h []) and untreated control ()

268 Drusano et al

FIGURE 8 Effect of BMS 232632 on HIV replication Three infected hollow fiberunits were treated with BMS 232632 One tube was treated with a concentra-tion of four times the EC50 as a continuous infusion This produced a 24 hAUC of 4 24 EC50 The second tube received the same 24 h AUC butwas given in a peak-and-valley mode once daily The third tube received anexposure calculated a priori to provide a time EC90 that would give essen-tially the same suppression as the continuous infusion of 4 EC50

tions need to exceed the EC90 for approximately 80ndash85 of a dosing interval inorder to maintain control of the HIV turnover This served as the therapeutictarget for further evaluation

The sponsor provided pharmacokinetic data for BMS 232632 administeredto normal volunteers at doses of 400 mg and 600 mg orally once daily at steadystate Population pharmacokinetic analysis was performed and the mean parame-ter vector and covariance matrix were employed to perform Monte Carlo simula-tion The ability to attain the therapeutic target was assessed accounting for thepopulation variability in the handling of the drug This is demonstrated in Fig9 The viral isolate susceptibilities to BMS 232632 are displayed as EC50 valuesHowever an internal calculation corrects for the difference between EC50 andEC90 as well as for the protein binding of the drug The fractional target attainmentis for free drug being greater than the nominal EC90 for 85 of the dosing interval


FIGURE 9 A Monte Carlo simulation of 1000 subjects performed three timeswas employed to estimate the fraction of these subjects whose concentra-tionndashtime curve would produce maximal viral suppression on the basis ofthe data presented in Fig 2 The evaluation was performed for doses of ()400 mg and () 600 mg of BMS 232632 administered once daily by mouth() Forty-three isolates from a clinical trial of BMS 232632 were tested by theVirologics Phenosense assay

(the therapeutic target determined in Fig 8) As can be seen as the viral isolatesbecome less and less susceptible to BMS 232632 the greater the difference be-tween the 400 mg and 600 mg doses The sponsors also determined the viralsusceptibility to 43 clinical isolates from a phase III clinical trial of the drug inpatients who were HIV-treatment naive As can be seen all had EC50 valuesbelow 2 nM Consequently this allows us to take an expectation over the distribu-tion of measured EC50 values to determine the fraction of patients who will attaina maximal response to the drug under the assumptions that the viral susceptibilitydistribution is correct for naive patients and that the kinetics in normal volunteersand its distribution are a fair representation of the kinetics of the drug in infectedbut treatment-naive patients When this calculation is performed approximately69 of subjects taking the 400 mg dose will obtain a complete response whereasslightly greater than 74 of patients taking 600 mg will obtain a complete re-sponse

270 Drusano et al

A clear lesson learned regarding the chemotherapy of HIV is that combina-tion chemotherapy is more effective than monotherapy However little has beendone to determine optimal combination dosing regimens An initial effort to de-termine the interaction of antivirals in a fully parametric system was publishedby Drusano et al [13] The Greco model was fit to the data from an in vitroexamination of 141W94 (amprenavir an HIV-1 protease inhibitor) plus 1592U89(abacavir a nucleoside analog) This interaction is displayed in Figs 10ndash12 Fig-ure 10 displays the full effect surface from these two agents The effects of proteinbinding are taken into account as the effects are developed in the presence of

FIGURE 10 1592U89 and 141W94 combination study with albumin (40 mgmL) and α1 acid glycoprotein (1 mgmL) A three-dimensional response sur-face representing the interaction from the in vitro matrix is displayed Percentinhibition data from a 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide assay with HIV-1IIIB and MT-2 cells is displayed


FIGURE 11 Synergy plot drawn from the data displayed in Figure 10 Plottedat the 95 confidence level

physiological amounts of the binding proteins human albumin and human α1acid glycoprotein The drug interaction can be seen by subtracting the theoreticaladditive surface and the synergy surface is seen in Fig 11 It is important tonote that there in synergy across all concentrations of the agents As a modelwas fit to the data the weighted residuals are displayed in Fig 12 to demonstratethat the actual regression process was unbiased The actual degree of interactionis given by estimation of the interaction parameter α which is 1144 The 95confidence bound about the interaction parameter is from 0534 to 1754 As thisboundary does not cross zero the synergy is significant at the 005 level

This fully parametric analysis was employed in combination with MonteCarlo simulation to examine whether the drugs at the doses used clinically would

272 Drusano et al

FIGURE 12 Weighted residual plot from the fully parametric analysis The re-siduals are scattered about the zero line without bias

be synergistic and whether the dosing interval affected the outcome [14] Theanswer to both these questions was found to be yes The data are displayed inChapter 14

4 CLINICAL STUDIES OF ANTIVIRAL

PHARMACODYNAMICS

As noted previously the first decision required for determining a pharmacody-namics relationship in the clinic is the choice of an endpoint In this section thechange in CD4 counts the change from baseline viral load prevention of resis-tance and durability of maintenance of viral loads below detectability are theendpoints examined Where possible they are examined for both nucleoside ana-logs and HIV-1 protease inhibitors both alone and in combination

Use of nucleoside analogs as monotherapy occurred mostly at a time whenviral copy number determinations were not freely available Consequently virtu-ally all the available studies employed change in CD4 cell count or p24 as thedynamic endpoint


One of the first studies in this regard examined dideoxyinosine (ddI) usein a naive patient population [15] No concentrationndasheffect response was foundfor CD4 cells but a clear relationship was discerned between the number of CD4

cells present at baseline and the number of cells that returned with the initiationof ddI therapy (Fig 13) In addition this study found a relationship between ddIexposure (as indexed to the area under the plasma concentrationndashtime curveAUC) and the fall in p24 both within patients and across the population (Fig14) The finding reported in the study just cited was also found for zidovudine[16]

More recently Fletcher et al [17] examined the relationship between ddIAUC and fall in viral load in children (Fig 15) This study is flawed by the factthat the relationships were developed in patients receiving combination chemo-therapy without any effort being made to account for the interaction betweendrugs

With regard to emergence of resistance there was one important studylargely ignored that demonstrated the importance of viral susceptibility for effectKozal et al [18] examined patients switching from zidovudine to didanosine(ddI) Over half of such patients developed a mutation at codon 74 by week 24of ddI therapy that is known to confer ddI resistance The effect on the number

FIGURE 13 Linear regression between the number of CD4-positive T-lympho-cytes during therapy with dideoxyinosine and the baseline CD4 count

274 Drusano et al

FIGURE 14 Relation between the suppression of p24 antigen and the steady-state area under the plasma concentrationndashtime curve of dideoxyinosine

FIGURE 15 Didanosine AUC versus baseline to week 24 changes in plasmaHIV RNA levels The solid line represents the line of best fit as determinedwith linear regression the equation for this line is y 1337 (347 AUC)r 2 051 p 003


FIGURE 16 Mean CD4 T-cell changes before the appearance of the HIV-1 re-

verse transcriptase mutation at codon 74 and CD4 T-cell changes after the

appearance of the mutation in 38 patients switched from zidovudine to dida-nosine

of CD4 cells is demonstrated in Fig 16 At the time of appearance of the mutationthe CD4 count dips below the baseline number of CD4 cells indicating that theincrease in EC90 attendant to the mutation drives a loss of virological effect

There is considerably more information relating exposure to effect for theHIV-1 protease inhibitors Because of the time at which they were studied virtu-ally all of the data link some measure of exposure to the change from baselinein the viral load Early publications examining the relationship between indinavirexposure and the CD4 count were published by Stein and Drusano [1920] Inthe first of these publications it was demonstrated that the return of CD4 countwas related to the baseline CD4 count as with nucleoside analogs In the secondit was demonstrated that the return of CD4 cells had another component attachedto it The model was expanded to include the decline in viral load The returnof CD4 cell count was better explained by the larger model of baseline CD4 countplus viral load change as the independent variables than either alone

The first published paper examining the relationship between drug exposureand viral load decline studied high dose saquinavir Schapiro et al [21] demon-strated a relationship between the saquinavir AUC and the change in viral load

276 Drusano et al

FIGURE 17 Drug levels at week 4 (AUC to 24 h) plotted against the decreasein plasma HIV RNA levels at week 4 for each patient in whom pharmacokinet-ics were studied The line represents best fit and was determined using theleast squares algorithm r 0801 expressed as the Pearson correlation coef-ficient () Patients receiving 3600 mg of saquinavir per day () patients re-ceiving 7200 mg of saquinavir per day

A problem with this analysis is that the form of the function is not specifiedConsequently interpretation is difficult

Shortly thereafter Stein et al published a small phase III study of indinavir[22] This was the first lsquolsquohigh dosersquorsquo indinavir study (2400 mgday) and was thefirst to demonstrate robust viral suppression with this drug In addition the au-thors examined the relationship between indinavir exposure and both CD4 cellreturn and viral load decline These relationships are shown in Fig 18 One should

FIGURE 18 Modeling of data using a sigmoid Emax relationship and inhomoge-neous differential equations (a) The relationship of baseline CD4 lymphocytecount to the average CD4 lymphocyte count obtained over 24 weeks of ther-apy (b) The relationship of the inhibition of HIV generation to total drug expo-sure (AUC) (c) The relationship of the inhibition of HIV generation to Cmin

serum concentration


278 Drusano et al

not draw the conclusion that HIV suppression is linked to AUC (as this has aslightly better r2) In this study the drug was administered on a fixed dose andschedule maximizing the co-linearity (ie one could not make the AUC risewithout also increasing the Cmin) The in vitro studies simulated different dosesand schedules minimizing the co-linearity and definitively showed that time EC90 is the dynamically linked variable This is correlated with Cmin The compari-son of the in vitro and in vivo results also raises the issue of the importance ofthe EC90

Drusano et al demonstrated for both indinavir [23] and amprenavir [24]that normalizing the measure of drug exposure to the EC50 (or EC90) of a particularpatientrsquos isolate decreased the variance and increased the r2 This makes senseas the amount of exposure needed to suppress a sensitive isolate will on firstprinciples be less than that needed to suppress a resistant isolate (see above forthe case of ddI and CD4 cells)

An issue that is not addressed by these studies is the duration of HIV sup-pression and the closely linked issue of suppression of emergence of resistanceThe first study to examine this issue was that of Kempf et al [25] who showedthat obtaining a viral load below the detectability limit of the assay was importantto the duration of control of the infection Shortly thereafter Drusano et al [26]examined this issue both for protease inhibitor monotherapy with indinavir andfor the first time for combination therapy that included indinavir The results formonotherapy are displayed in Fig 19 It is clear that patients who attain viralloads that are below the detectability of the assay have the lowest hazard of losingcontrol of the infection or emergence of resistance The time to loss of controlof infection is clearly related to the nadir viral load attained

Of greater interest is the situation with combination chemotherapy as this isthe clinical norm This study also examined the influence of different combinationregimens on the hazard of loss of control of the viral infection Combinationregimens of zidovudine-indinavir zidovudine-didanosine-indinavir and zidovud-ine-lamivudine-indinavir were examined and compared to their monotherapyarms in a stratified analysis The results are displayed in Table 1 Only the regi-men of zidovudine-lamivudine-indinavir remained significantly different frommonotherapy after adjustment for the fall in viral copy number

This result caused the in vitro investigation of this regimen [27] A fullyparametric analysis demonstrated that the interaction among all three drugs waskey to the effect obtained in the clinical studies with this regimen This is shownin Table 2 where the αrsquos represent the interaction parameters There are threetwo-drug interaction parameters and one for the interaction of all three com-pounds Indinavir plus zidovudine interact in an additive manner as the value isclose to zero and the 95 confidence interval overlaps zero The same is trueof indinavir plus lamivudine Zidovudine plus lamivudine has a value that ispositive and a 95 confidence interval that does not overlap zero indicating a


FIGURE 19 KaplanndashMeier estimate of probability that patientrsquos isolate is notresistant to therapy (lack of sustained increase of 075 log10 copiesmL ofHIV-1 RNA from patientrsquos minimum level) at a given study day stratified byminimum HIV-1 RNA achieved Plot shows that probability of remaining sus-ceptible is largest for patients who achieve undetectable level of HIV-1 RNAVertical slashes on plot indicate censoring events

TABLE 1 Effect of Combination Therapy Versus IndinavirMonotherapy Before and After Adjusting for the Effect of the MinimumLevel of HIV-1 RNA

Combination P before Hazard ratiob

therapy adjustment Coefficienta (95 CI) P

IDVAZT 264 035 052 0705 (02541957) 497IDVAZTddI 002 136 090 0258 (00441514) 105IDVAZT3TC 001 168 080 0186 (00390893) 016

a Estimate SE b Hazard ratio is vs IDV monotherapy group in the same study

280 Drusano et al

TABLE 2 In Vitro Assessment of Drug Interactionof AZT-3TC-Indinavir

95 ConfidenceParametera Estimate interval

Econ 9899 9780ndash1002IC50IND 14690 12830ndash16560m IND 1711 1393ndash2030IC50AZT 1184 10820ndash12860mAZT 1289 5576ndash2020IC503TC 10290 1018ndash1041m3TC 6875 369ndash1006

αINDAZT 00001301 06191ndash06194αIND3TC 06881 005189ndash1428αAZT3TC 09692 09417ndash09966αINDAZT3TC 894 3434ndash1445

a Econ effect seen in the absence of drug (percent) IC50 concen-tration of drug necessary to reduce HIV-1 turnover by half whenused alone (nM) m slope parameter corresponding to the rateof rise of effect with increasing drug concentration Prime interac-tion parameter

degree of synergistic interaction that is statistically significant Finally the magni-tude of the synergistic interaction for the three-drug term is very large (and sig-nificant) It may be this exceptionally strong synergistic interaction that explainsthe superb results seen with this particular three-drug combination

It is of interest that lamivudine not only plays a key role in the regimenbut is also its Achillesrsquo heel Holder et al [28] demonstrated that when the tripleregimen fails in about 70 of cases the failure is due to an M184V mutationthat produces high level resistance to lamivudine So that although lamivudineappears to be a key part of this therapeutic regimen and its synergy it also hasthe lowest genetic barrier to resistance If there is some nonadherence to theregimen enough rounds of viral replication may occur to allow the point mutant(M184V) to be amplified in the total population When this clone becomes domi-nant in the population lamivudine will lose most of its contribution to the regi-men Most of the synergy is also lost and the result is viral rebound As Holderand colleagues demonstrated this occurs most of the time with mutation solelyaffecting the low genetic barrier drug

It is obvious then that optimal chemotherapeutic regimens for HIV (andother viral pathogens) are likely to require explicit modeling of the interactionof the drugs in the regimens


Burger et al [29] also examined this combination In a multivariate logisticregression with attaining a viral load below the limit of assay detectability as theendpoint they demonstrated that baseline viral load indinavir trough concentra-tions and prior HIV-1 protease inhibitor use influenced the probability of at-taining this endpoint

All of the above has related to HIV Other viruses can also have a pharma-codynamic evaluation elucidated For CMV there is a clear-cut dynamic relation-ship [30] that has been set forth in Chapter 14

In summary viruses follow the same laws of physics as bacterial pathogens(and fungal pathogens) It is important to delineate relationships both in vitroand in vivo between different measures of drug exposure and the endpoint thatis deemed important The in vitro investigations allow delineation of the truedynamically linked variable in a more straightforward manner The in vivo inves-tigations are important for validation The future is in generating exposurendashresponse relationships for combinations of agents In this way optimal therapyregimens can be generated to provide the greatest benefit for patients infectedwith viral pathogens

REFERENCES

1 B Vogelman S Gudmundsson J Leggett J Turnidge S Ebert WA Craig Correla-tion of antimicrobial parameters with therapeutic efficacy in an animal modelJ Infect Dis 1988 158831ndash847

2 GL Drusano DE Johnson M Rosen HC Standiford Pharmacodynamics of a fluor-oquinolone antimicrobial in a neutropenic rat model of Pseudomonas sepsis Antimi-crob Agents Chemother 1993 37483ndash490

3 A Louie P Kaw W Liu N Jumbe MH Miller GL Drusano Pharmacodynamics ofdaptomycin in a murine thigh model of Staphylococcus aureus infection AntimicrobAgents Chemother 2001 45845ndash851

4 JA Bilello JL Eiseman HC Standiford GL Drusano Impact of dosing scheduleupon suppression of a retrovirus in a murine model of AIDS encephalopathy Anti-microb Agents Chemother 1994 38628ndash631

5 JA Bilello JJ Kort C MacAuley TN Fredrickson RA Yetter JL Eiseman ZDVdelays but does not prevent the transmission of MAIDS by LP-BM MuLV-infectedmacrophage-monocytes J Acquir Immune Defic Syndr 1992 5571ndash576

6 JA Bilello PA Bilello K Stellrecht J Leonard D Norrbeck DJ Kempf T RobbinsGL Drusano The uptake and anti-HIV activity of A 80987 an inhibitor of the HIV-1 protease is reduced by human serum 1-acid glycoprotein Antimicrob Agents Che-mother 1996 401491ndash1497

7 JA Bilello G Bauer MN Dudley GA Cole GL Drusano The effect of 2prime3prime-di-deoxy-2prime3prime-didehydrothymidine (D4T) in an in vitro hollow fiber pharmacodynamicmodel system correlates with results of dose ranging clinical studies AntimicrobAgents Chemother 1994 381386ndash1391

8 MJ Browne KH Mayer SB Chafee MN Dudley MR Posner SM Steinberg KK

282 Drusano et al

Graham SM Geletko SH Zinner SL Denman et al J Infect Dis 1993 16721ndash29

9 JA Bilello PA Bilello JJ Kort MN Dudley J Leonard GL Drusano Efficacy ofconstant infusion of A 77003 an inhibitor of the HIV protease in limiting acuteHIV-1 infection in vitro Antimicrob Agents Chemother 1995 392523ndash2527

10 M Reedijk CA Boucher T van Bommel DD Ho TB Tzeng D Sereni P VeyssierS Jurruaans R Grannemann A Hsu et al Safety pharmacokinetics and antiviralactivity of A77002 a C2 symmetry-based human immunodeficiency virus proteaseinhibitor Antimicrob Agents Chemother 1995 391559ndash1564

11 GL Drusano Antiviral therapy of HIV and cytomegalovirus Abstracts of the 37thInterscience Conference on Antimicrobial Agents and Chemotherapy Abstract S-121 Sept 28ndashOct 1 1997 Toronto ON Canada

12 GL Drusano JA Bilello SL Preston E OrsquoMara S Kaul S Schnittman R EcholsHollow fiber unit evaluation of a new human immunodeficiency virus (HIV)-1 prote-ase inhibitor BMS232632 for determination of the linked pharmacodynamic vari-able J Infect Dis 2000 1831126ndash1129

13 GL Drusano DZ DrsquoArgenio W Symonds PA Bilello J McDowell B Sadler ABye JA Bilello Nucleoside analog 1592U89 and human immunodeficiency virusprotease inhibitor 141W94 are synergistic in vitro Antimicrob Agents Chemother1998 422153ndash2159

14 GL Drusano DZ DrsquoArgenio SL Preston C Barone W Symonds S LaFon M Rog-ers W Prince A Bye JA Bilello Use of drug effect interaction modeling with MonteCarlo simulation to examine the impact of dosing interval on the projected antiviralactivity of the combination of abacavir and amprenavir Antimicrob Agents Chemo-ther 2000 441655ndash1659

15 GL Drusano GJ Yuen JS Lambert M Seidlin R Dolin FT Valentine Quantitativerelationships between dideoxyinosine exposure and surrogate markers of responsein a phase I trial Ann Intern Med 1992 116562ndash566

16 GL Drusano FM Balis SR Gitterman PA Pizzo Quantitative relationships betweenzidovudine exposure and efficacy and toxicity Antimicrob Agents Chemother 199481726ndash1731

17 CV Fletcher RC Brundage RP Remmel LM Page D Weller NR Calles C SimonMW Kline Pharmacologic characteristics of indinavir didanosine and stavudine inhuman immunodeficiency virus-infected children receiving combination chemother-apy Antimicrob Agents Chemother 2000 441029ndash1034

18 MJ Kozal K Kroodsma MA Winters RW Shafer B Efron DA Katzenstein TCMerigan Didanosin resistance in HIV-infected patients switched from zidovudineto didanosine monotherapy Ann Intern Med 1994 121263ndash268

19 DS Stein GL Drusano Modeling of the change in CD4 lymphocyte cell counts inpatients before and after administration of the human immunodeficiency virus prote-ase inhibitor indinavir Antimicrob Agents Chemother 1997 41449ndash453

20 GL Drusano DS Stein Mathematical modeling of the interrelationship of CD4 lym-phocyte count and viral load changes induced by the protease inhibitor indinavirAntimicrob Agents Chemother 1998 42358ndash361

21 JM Schapiro MA Winters F Stewart B Efron J Norris MJ Kozal TC Merigan


The effect of high-dose saquinavir on viral load and CD4 T-cell counts in HIV-infected patients Ann Intern Med 1996 1241039ndash1050

22 DS Stein DG Fish JA Bilello J Chodakewitz E Emini C Hildebrand SL PrestonGL Martineau GL Drusano A 24 week open label phase I evaluation of the HIVprotease inhibitor MK-639 AIDS 1996 10485ndash492

23 GL Drusano This volume Chapter 1424 GL Drusano BM Sadler J Millard WT Symonds M Tisdale C Rawls A Bye

and the 141W94 International Product Development Team Pharmacodynamics of141W94 as determined by short term change in HIV RNA Influence of viral isolatebaseline EC50 Abstract A-16 37th Interscience Conference on Antimicrobial Agentsand Chemotherapy Sept 28ndashOct 1 1997 Toronto ON Canada

25 DJ Kempf RA Rode Y Xu E Sun ME Heath-Chiozzi J Valdes AJ Japour SDanner C Boucher A Molla JM Leonard The duration of viral suppression duringprotease inhibitor therapy for HIV-1 infection is predicted by plasma HIV-1 RNAat the nadir AIDS 1998 12F9ndashF14

26 GL Drusano JA Bilello DS Stein M Nessly A Meibohm EA Emini P DeutschJ Condra J Chodakewitz DJ Holder Factors influencing the emergence of resistanceto indinavir Role of virologic immunologic and pharmacologic variables J InfectDis 1998 178360ndash367

27 S Snyder DZ DrsquoArgenio O Weislow JA Bilello GL Drusano The triple combina-tion indinavir-zidovundine-lamivudine is highly synergistic Antimicrob AgentsChemother 2000 441051ndash1058

28 DJ Holder JH Condra WA Schleif J Chodakewitz EA Emini Virologic failureduring combination therapy with crixivan and RT inhibitors is often associated withexpression of resistance-associated mutations in RT only Conf Retroviruses Oppor-tun Infect 1999 Jan 31ndashFeb 46160 (abstr 492)

29 DM Burger RMW Hoetelmans PWH Hugen et al Low plasma concentrations ofindinavir are related to virological treatment failure in HIV-1-infected patients onindinavir containing triple therapy Antiviral Ther 1998 3215ndash220

30 GL Drusano F Aweeka J Gambertoglio M Jacobson M Polis HC Lane C Eaton SMartin-Munley Relationship between foscarnet exposure baseline cytomegalovirusblood culture and the time to progression of cytomegalovirus retinitis in HIV-positive patients AIDS 1996 101113ndash1119

13

Antifungal Pharmacodynamics

Michael E Klepser

University of Iowa Iowa City Iowa

Russell E Lewis

University of Houston College of Pharmacy Houston Texas

1 INTRODUCTION

Over the last two decades advances in the characterization of antibacterial phar-macodynamics have greatly improved therapeutic strategies in the use of antimi-crobial therapy Progress in our understanding of antifungal pharmacodynamicshowever remains severely limited Prospective clinical trials for serious fungalinfections are difficult to complete and rarely evaluate multiple treatment strate-gies [1] Moreover the high mortality associated with systemic fungal diseaselessens the likelihood that dose-ranging studies for antifungal agents will be pur-sued Perhaps more surprising however is the profound lack of in vitro andanimal data describing concentrationndasheffect relationships for antifungals Thislack of even fundamental knowledge regarding the pharmacodynamics of antifun-gals can even be appreciated in the clinical literature where optimal dosing strate-gies for amphotericin B remain largely undefined despite over 40 years of clinicaluse [23]

With the emergence of the AIDS epidemic widespread use of broad-spec-trum antibacterial therapy and a growing population of immunocompromisedpatients the spectrum of nosocomial pathogens has changed dramatically [4]Fungi are now the fourth most commonly isolated nosocomial bloodstream patho-gens in US hospitals and have been identified as an independent risk factor for

285

286 Klepser and Lewis

in-hospital death [56] This staggering increase in the frequency of fungal infec-tions coupled with the introduction of new antifungal agents has made the identi-fication of optimal antifungal treatment strategies even more critical Althoughstill in its infancy the study of antifungal pharmacodynamics is already providingimportant new information of the activity dosing and use of antifungals fortreatment of candidiasis and cryptococcosis

2 PROBLEMS WITH DESCRIBING ANTIFUNGAL

PHARMACODYNAMICS

21 In Vitro Data

One of the primary factors that has hindered the study of antifungal pharmaco-dynamics over the last two decades was the lack of standardized methods forperforming in vitro susceptibility testing of fungi Prior to the 1980s fungi wererelatively infrequent pathogens and amphotericin B was the sole systemic antifun-gal therapy available thus susceptibility testing of fungi was impractical andremained largely undeveloped With the frequency of fungal infections increasingand the number of new antifungal agents growing the development of standard-ized antifungal susceptibility testing methods has become a priority Problemswith inter- and intralaboratory reproducibility however hindered early effortsto develop antifungal susceptibility testing into a clinically useful tool One studyevaluating the interlaboratory reproducibility of antifungal susceptibility testingnoted a 512-fold variation in MIC values among collaborating laboratories thatused the same published but unstandardized methodology [7]

In the late 1990s the National Committee for Clinical Laboratory Standards(NCCLS) approved standardized methods for in vitro antifungal susceptibilitytesting and susceptibility breakpoints for Candida species [89] This step notonly enabled the quantitative description of antifungal resistance but also pro-vided the groundwork for performing in vitro pharmacodynamic work with anti-fungals

22 Animal Models

Historically animal (mostly rodent) models have been the preferred method oftesting antifungal efficacy Much of this deference to the use of animal modelswas a result of not having standardized in vitro susceptibility testing methodsfor fungi Moreover many fungal pathogens exhibit different morphology andgrowth characteristics in vivo from those of growth in culture media Candidaalbicans for example generally does not form the hyphal structures in culturemedia that are seen with in vivo tissue invasion [10ndash12] Published data fromanimal models however also suffer the limitation of unstandardized methodol-ogy and poor reproducibility [13] Selection of the endpoint for evaluation of

Antifungal Pharmacodynamics 287

study results in vivo ie survival versus mycology can profoundly influenceinterpretation of data Although survival is often the desired endpoint in vivoanimal studies are generally not conducted over a sufficient length of time toprovide an adequate opportunity for relapse of infection once therapy is discon-tinued Additionally it is reasonable to theorize that if fungi are detectable inbody tissues or fluids at the end of a treatment cycle these viable organisms mayproliferate and cause relapse after antifungal therapy is withdrawn Thereforecounts of viable fungi often provide the best information regarding in vivo anti-fungal efficacy This can be problematic however for extrapolating pharmacody-namic parameters predictive of in vivo antifungal efficacy Many antifungals pos-sess fungistatic activity thus the actual differences in counts of viable fungi invivo may be too small to delineate pharmacodynamic relationships [1314]

23 Clinical Data

Using clinical data to determine dosendashresponse relationships for antifungals canbe especially difficult The multiplicity of non-drug factors that play a part inthe development of severe fungal infections (ie previous chemotherapy intra-vascular catheters etc) profoundly influence the overall effectiveness of antifun-gal therapy in the individual patient Fungal infections are also a diagnostic chal-lenge and serial blood cultures are generally insensitive markers for diagnosisand response to antifungal therapy [15] Moreover monitoring of serum drugconcentration for antifungals is rarely performed thus comparison of differentclinical studies is unfeasible

Distinct diagnostic criteria compared to other systemic fungal infectionsgenerally make cryptococcal meningitis andor oropharyngeal candidiasis bestsuited to the extrapolation of outcome data with respect to antifungal therapyCryptococcal meningitis however may be preferred for outcome measurementbecause the mortality associated with oropharyngeal candidiasis is comparativelylower than that of other systemic infections

3 PHARMACODYNAMICS

31 Amphotericin B

Despite the availability of better-tolerated agents the broad spectrum of activityand potency of amphotericin B have maintained its role as the drug of choicefor deep-seated mycoses Amphotericin B is thought to act primarily by bindingto ergosterol in the fungal cell membrane resulting in intercalation of the mem-brane and leakage of intracellular contents [3] Amphotericin B has also beenshown to induce oxidative damage to the cell membrane and to stimulate hostimmune responses [16] which may contribute to the overall elimination of fungalpathogens from the infected host


Even with nearly 40 years of clinical use relatively few pharmacodynamicdata exist for this agent This lack of data is surprising considering the numeroustoxicities associated with amphotericin B therapy The recent introduction of lipidformulations of amphotericin B which have considerably different pharmacoki-netic characteristics than those of the standard formulations have further compli-cated issues regarding the optimal dosing of this agent

311 In Vitro Data

In vitro data describing the pharmacodynamics of amphotericin B are relativelyscarce Although the NCCLS has proposed standardized methods for microbrothsusceptibility testing of amphotericin B these methods are still not well suitedfor detecting amphotericin B resistance and are still under active investigation[891718]

Standardized methods for performing time-kill studies with amphotericinB against Candida and Cryptococcus spp however have been published [19]These methods have been used to evaluate concentrationndasheffect relationships ofamphotericin B against Candida albicans [20] and Cryptococcus neoformans[21] A representative graph from these studies is presented in Fig 1 Against

FIGURE 1 Representative graph of amphotericin B time-kill activity againstCandida and Cryptococcus species (From Refs 20 and 21)


both fungal species amphotericin B at concentrations above the MIC producedrapid fungicidal activity As concentrations of amphotericin B in the growth me-dia increased both the rate and extent of antifungal activity improved This im-provement in activity continued up to the maximal concentrations tested 16ndash32times the MIC of the test isolates This concentration-dependent relationship ofkilling has also been reported by other investigators [22]

The postantifungal effect (PAE) of amphotericin B has also been evaluatedfor both Candida and Cryptococcus species [23] Amphotericin B produces aprolonged PAE ranging from 05 to 106 h against Candida species and from 28to 104 h against C neoformans Additionally the authors noted that the PAEexerted by amphotericin B was dependent on the duration of exposure and theconcentrations of drug tested

According to these data it appears that amphotericin B exhibits concentra-tion-dependent antifungal activity in vitro However it is important to gaugein vitro results against clinically achievable drug concentrations Examining thesigmoidal dosendashresponse curves constructed from the time-kill data representa-tive of fully susceptible Candida and Cryptococcus spp (Fig 2) one notes thatthe transitional portion of the dose response curve for amphotericin B againstthese isolates (in vitro) occurred over a concentration range of 025ndash2 times the

FIGURE 2 Representative sigmoidal dosendashresponse curves of amphotericinB and fluconazole against Candida and Cryptococcus spp (From Refs20 and 21)


MIC (02ndash20 microgmL) This transitional portion represents a relatively broadrange of serum concentrations that can be achieved with clinically used dosagesof amphotericin B of 025ndash15 mgkg q24h [3] Therefore these data predictover a range of clinically achieved concentrations that amphotericin B would beexpected to exhibit concentration-dependent pharmacodynamics

312 In Vivo Data

Currently no studies animal or human specifically designed to evaluate the phar-macodynamic characteristics of amphotericin B have been published Howeversome animal and clinical studies do exist that have evaluated multiple doses ofamphotericin B under identical or similar testing conditions and can therefore becarefully evaluated for dosendashresponse relationships George et al [24] used arabbit model of aspergillosis to evaluate the efficacy of amphotericin B adminis-tered at 05 and 15 mg(kg sdot day) These investigators employed both clinical(survival) and mycological (tissue burden) endpoints to evaluate the efficacy ofthe two dosing regimens They reported that although there was no differencebetween the two amphotericin B regimens with respect to survival (both regimensyielded 100 survival) amphotericin B at 15 mg(kg sdot day) resulted in fewerpositive cultures and reduced fungal tissue burdens compared with the lower doseregimen

Pharmacodynamic data from controlled human studies are lacking There-fore we are left to evaluate data from multiple studies in order to construct apicture of pharmacodynamic relationships As mentioned one of the best patientpopulations available for comparison among studies are patients with cryptococ-cal meningitis Some of the primary reasons that this patient population lendsitself to interstudy comparisons are the defined criteria used to diagnose and de-fine disease the ability to detect the pathogen relatively reliably and the relativelystandard endpoints used for clinical and microbiological outcomes The majorproblem with extrapolating pharmacodynamic relationships from studies ofcryptococcal meningitis however is the lack of data describing amphotericin Bconcentrations at the site of infection (cerebrospinal fluid) This could somewhatskew the dosendashresponse relationships observed because distribution of amphoter-icin B into the CSF is generally poor (10) [3] Several studies evaluatingamphotericin B for the treatment of cryptococcal meningitis are presented inTable 1 Examination of these studies reveals a trend of improved outcomes withincreasing doses of amphotericin B It is interesting to note that mortality associ-ated with cryptococcal meningitis decreased from 14 at a dose of 03 mgkgper day to 29 at a dose of 10 mgkg per day [25ndash27]

313 Summary

In vitro and in vivo data both reveal improved fungicidal activity with amphoteri-cin B as concentrations or doses of the drug are increased Therefore in light of


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the long half-life and PAE associated with the drug it appears that the optimaldosing strategy for this agent would be to administer amphotericin B at relativelyhigh doses (ie 08 mgkg per day) at intervals of no less than every 24 h

32 Azole Pharmacodynamics

Fluconazole has been the most widely studied of the azole antifungals A syn-thetic triazole antifungal fluconazole exhibits fungistatic activity against a varietyof Candida species and C neoformans

321 In Vitro Data

Similar to amphotericin B limited data are available regarding the pharmacody-namic characteristics of fluconazole Klepser et al [2021] examined the fungi-static activity produced by fluconazole against C albicans and C neoformansusing time-kill methods According to their data the fungistatic activity producedby fluconazole was maximized at concentrations of 2ndash4 times the MIC of thetest isolates (Figs 2 and 3) Similar findings have been reported by other investi-gators [22] Additionally using an in vitro dynamic model of infection these

FIGURE 3 Representative graph of fluconazole time-kill activity against Can-dida and Cryptococcus spp (From Refs 20 and 21)


investigators compared the activity of two fluconazole dosing regimens 200 mgand 400 mg administered every 24 h against C albicans [28] No differenceswith respect to the rate or extent of activity produced by the two fluconazoleregimens were noted It is important to note that the fluconazole MICs for thetest isolates were 025 and 05 microgmL Given the long half-life of fluconazoleapproximately 20ndash50 h drug concentrations in the model for both regimenswould be expected to remain above 10 times the MIC for each regimen andisolate tested

Voriconazole is an investigational triazole antifungal with an enhancedspectrum of activity against Candida Cryptococcus and Aspergillus species Us-ing time-kill methods Klepser et al [29] examined the in vitro activity of vorico-nazole against C albicans C glabrata C tropicalis and C neoformans overconcentrations ranging from 0125 to 16 times the MIC of test isolates AgainstC albicans and C neoformans voriconazole produced fungistatic activity thatwas maximized at concentrations between 1 and 4 times the MIC

In vitro fluconazole produces a short PAE against Candida species [30ndash33] Against C albicans fluconazole produces a PAE of 05 h The durationof the PAE is not increased with higher concentrations or by increasing the dura-tion of exposure Some investigators have noted that the PAE induced by fluco-nazole may increase if human serum is added to the in vitro system [30] Thisobservation led the investigators to suggest that the PAE induced by fluconazolemay be significantly longer in vivo than in vitro

322 In Vivo Data

Using a murine model of deep-seated C albicans infection Louie et al [34]evaluated the pharmacodynamic characteristics of fluconazole In this study theinvestigators determined the range of concentrations over which response in themodel was noted to change from minimal to maximal (transition portion ofthe dosendashresponse curve) Then dose-fractionation studies were performed toevaluate the relationship between pharmacodynamic parameters such as peakMIC and AUCMIC ratios and time above the MIC and outcome at concentra-tions near the transition portion of the dosendashresponse curve Upon analysis theauthors determined that a concentration-associated parameter the AUCMIC ra-tio was best correlated with positive results Although these in vivo data mayappear to contradict previous in vitro findings some limitations of this studywarrant cautious interpretation of this study First the doses of fluconazole usedin the study produced serum concentrations in the model that ranged from approx-imately 4 to 8 microgmL well below clinically observed concentrations even withlow-dose 200 mg daily fluconazole Second viable colony counts of fungi inkidney homogenate served as the endpoint for the study and the authors statethat fungal densities were similar for groups that received the same daily doseof fluconazole However according to their data there does not appear to be a


difference among fungal densities in any of the dosing groups suggesting poorsensitivity in the model Finally the authors specifically studied the transitionportion of the dosendashresponse curve By definition the measured response shouldincrease as exposure to increased amounts of drug increase This portion of thecurve was noted to occur over a narrow range of concentrations If one were toexamine these data in the context of clinically achievable concentrations (Fig2) it would be apparent that this transition portion of the dosendashresponse curvewould be easily surpassed Therefore one might conclude that even though theactivity noted in the transition portion of the curve may correlate with the AUCMIC ratio under clinical conditions activity would be dictated according to theconcentration relationships of the flat portion or non-concentration-dependentportion of the curve against fully susceptible fungi (Fig 2)

323 Clinical Studies

Clinical trials designed specifically to study the pharmacodynamic properties offluconazole have not been completed However Witt et al [35] examined theactivity of fluconazole at doses ranging from 400 to 2000 mg in patients withcryptococcal meningitis A total of 12 15 20 and 10 patients received intrave-nous fluconazole at doses of 400 1200 1600 and 2000 mg respectively Theauthors reported that no association was observed between the dose of flucona-zole administered and outcome

324 Summary

Although the fungistatic activity of fluconazole may improve as drug concentra-tions increase to 2ndash4 times the MIC of a given fungus fluconazole appears toexhibit non-concentration-dependent characteristics over the range of concentra-tions yielded by clinically used doses Therefore it is not likely that the activityproduced by fluconazole will be improved via the administration of larger thannormal doses ie 400 mg daily unless the fungal pathogens exhibit relativelyhigh MICs (32ndash64 microgmL) to fluconazole

33 Flucytosine Pharmacodynamics

331 In Vitro Data

Flucytosine is a fluorinated pyrimidine that possesses antifungal activity againsta variety of fungal species including Candida and Cryptococcus In order to exertits antifungal activity flucytosine must be converted to fluorouracil by cytosinedeaminase located inside the fungal cell The use of flucytosine is frequentlylimited secondary to its narrow therapeutic index four times daily dosing intervaland rapid emergence of resistance if single-drug therapy is attempted

As with the other antifungal agents discussed few data currently exist re-


garding the pharmacodynamic characteristics of flucytosine Lewis et al [36]examined the influence of drug concentrations on the antifungal activity ex-pressed by flucytosine using time-kill methods Several Candida species and Cneoformans were tested at multiples of their flucytosine MICs ranging from 025to 64 times the MIC Against isolates of C albicans C glabrata C kruseiand C tropicalis flucytosine exhibited fungistatic activity that improved as theconcentration of flucytosine increased up to 16 times the MIC In contrast againstC neoformans there did not appear to be an identifiable relationship betweenconcentration and increased antifungal activity once flucytosine concentrationsexceeded the MIC of the isolate It should be noted that even at 16 times theMIC of the test isolates these concentrations are well within the range of clini-cally observed concentrations In fact steady-state serum and cerebrospinal fluidconcentrations in humans average 50ndash100 microgmL [2637ndash39] The MICs of theisolates used by Lewis et al ranged from 006 to 4 microgmL therefore clinicallyachievable concentrations would represent levels approximately 50ndash1600 timesthe MICs of these isolates As a result even though flucytosine does exhibitconcentration-dependent activity in vitro it is likely that concentration-indepen-dent activity would be observed clinically

A measurable PAE has been reported for flucytosine [2336] In generalflucytosine produces an in vitro PAE ranging from 05 to approximately 4 h Theduration of the PAE is prolonged as the concentration of drug tested increases[36]

332 In Vivo Data

Using a murine model of disseminated candidiasis Andes and colleagues exam-ined the pharmacodynamic characteristics of flucytosine [40] In their model theinvestigators determined that minimal concentration-dependent activity wasnoted They determined that the time that concentrations remained above theMIC of the pathogen and the AUCMIC ratio held the best correlation with ob-served effect It is important to note however that the half-life of flucytosine intheir model was approximately 035ndash045 h significantly less than the 6 h half-life reported for humans

333 Summary

Flucytosine exhibits non-concentration-dependent fungistatic activity over arange of clinically achievable concentrations Therefore given the relatively longhalf-life of this agent its narrow therapeutic window and correlation betweentime above the MIC and activity it appears reasonable to advocate the use oflow dose 100 mg(kg sdot day) regimens administered at extended intervals every8ndash12 h This strategy would appear to provide optimal activity while minimizingpatient exposure to the drug


34 Antifungal Combinations

With increasing reports of antifungal resistance and a limited number of therapeu-tic agents the use of combination antifungal regimens to offset toxicity and im-prove efficacy has become common clinical strategy Amphotericin Bndashflucyto-sine combination therapy has been proven to be an efficacious combination inthe treatment of cryptococcal meningitis [2638] Some clinical data are availableto suggest that azole-flucytosine combinations may be useful in the treatment offungal peritonitis [41]

One of the most controversial topics in antifungal therapeutics howeverconcerns azole-amphotericin B combinations Considering the pharmacology ofthese two antifungals this combination should result in antagonism Theoreti-cally the inhibition of ergosterol synthesis by fluconazole should result in de-creased binding and antagonism of amphotericin B activity Results from manyin vitro studies have supported this theory of azole-amphotericin B antagonismyet the results of in vivo rodent and rabbit studies have been unequivocal [42]Unlike many of the other azole antifungals fluconazole exhibits reversible bind-ing to the target enzyme 14α-demethylase and is relatively hydrophilic [43]Based of these differences and data from in vivo models several investigatorshave predicted that antagonism between amphotericin B and fluconazole wouldnot occur in vivo [4244ndash48]

If fluconazole does antagonize the activity of amphotericin B against yeasttwo important pharmacodynamic questions need to be answered (1) What isthe time course (rate) for the development of antagonismmdashis pre-exposure tofluconazole necessary (2) If antagonism does develop to what extent will overallantifungal activity be lost This second question is critical because it may explainwhy many in vivo models have failed to show increased mortality with an ampho-tericin B-fluconazole combination

Recently studies have been published examining these questions [2849]De novo ergosterol biosynthesis and incorporation into the fungal membrane inCandida species have been reported to occur over a period of approximately 6h [50] Considering the pharmacodynamics of each antifungal (Figs 1ndash3) it ap-pears that some amount of pre-exposure to fluconazole would be necessary toantagonize the activity of amphotericin B Ernst and coworkers noted that thefungicidal activity of amphotericin B is dramatically decreased when isolates arepre-exposed to fluconazole for more than 8 h The activity of amphotericin Bagainst yeast exposed to fluconazole for at least 8 h was indistinguishable fromthat of fluconazole alone and was fungistatic (2 log10 decrease from startinginoculum) A limitation of these studies however was the static nature of drugsin the test conditions

To test the likelihood of fluconazole antagonizing the fungicidal activityof amphotericin B over a range of dynamic concentrations Lewis et al [28]


developed an in vitro pharmacodynamic infection model of candidemia In thismodel the investigators simulated the human pharmacokinetic profile of five anti-fungal regimens plus control (1) fluconazole 200 mg q24h (2) fluconazole 400mg q24h (3) amphotericin B 1 mgkg per day (4) amphotericin B plus flucon-azole (400 mg) simultaneously and (5) amphotericin B administered 8 h afterthe fluconazole bolus Similar to previous time-kill studies amphotericin B ad-ministered 8 h after a fluconazole bolus drastically reduced amphotericin B fungi-cidal activity (Fig 4)

Data from these pharmacodynamic studies have several implications Firstthe time course and order of administration for amphotericin B and fluconazoleare important for the development of antagonism Therefore traditional methodsfor screening antifungal antagonism that rely on static concentration of antifun-gals administered simultaneously (ie checkerboard testing) may not necessarilydetect antagonism Especially notable the fungistatic activity of fluconazoleseems to persist despite the loss of amphotericin B fungicidal activity (Fig 4)If this antifungal activity persists in vivo it is unlikely that studies using mortalityas the principal endpoint would be able to detect antagonism of amphotericin BThis is also true clinically as fluconazole has been shown to be as efficaciousas moderate doses of amphotericin B for the treatment of systemic candidiasisin neutropenic and non-neutropenic patients [5152]

FIGURE 4 Representative graph of amphotericin B-fluconazole antifungalcombination activity tested in an in vitro pharmacodynamic infection modelagainst Candida albicans (From Ref 28)


4 CONCLUSIONS

Clearly the study of antifungal pharmacodynamics is still in its infancy Never-theless early in vitro and in vivo work has provided some important new informa-tion on the activity dosing and use of antifungals for the treatment of candidiasisand cryptococcosis The next challenge will be to bring pharmacodynamic datafrom benchtop and animal studies to the bedside Other systemic mycoses criti-cally need pharmacodynamic study Aspergillosis for example has become amajor cause of mortality among oncology patients yet no clinical trials existdescribing the optimal therapy for these fungal infections Future progress in thefield of antifungal pharmacodynamics will hopefully optimize our ability to treatmycoses despite a limited armamentarium

REFERENCES

1 JE Edwards Jr GP Bodey RA Bowden T Buchner BE de Pauw SG Filler MAGhannoum M Glauser R Herbrecht CA Kauffman S Kohno P Martino F Meu-nier T Mori MA Pfaller JH Rex TR Rogers RH Rubin J Solomkin C ViscoliTJ Walsh M White International Conference for the Development of a Consensuson the Management and Prevention of Severe Candidal Infections Clin Infect Dis19972543ndash59

2 MP Nagata CA Gentry EM Hampton Is there a therapeutic or pharmacokineticrationale for amphotericin B dosing in systemic Candida infections Ann Pharma-cother 199630811ndash818

3 HA Gallis RH DrewWW Pickard Amphotericin B 30 years of clinical experienceRev Infect Dis 199012308ndash329

4 SN Banerjee TG Emori DH Culver RP Gaynes WR Jarvis T Horan JR EdwardsJ Tolson T Henderson WJ Martone Secular trends in nosocomial primary blood-stream infections in the United States 1980ndash1989 National Nosocomial InfectionsSurveillance System Am J Med 19919186Sndash89S

5 D Pittet N Li RF Woolson RP Wenzel Microbiological factors influencing theoutcome of nosocomial bloodstream infections A 6-year validated population-based model Clin Infect Dis 1997241068ndash1078

6 MA Pfaller RN Jones GV Doern HS Sader RJ Hollis SA Messer Internationalsurveillance of bloodstream infections due to Candida species Frequency of occur-rence and antifungal susceptibilities of isolates collected in 1997 in the United StatesCanada and South America for the SENTRY Program The SENTRY ParticipantGroup J Clin Microbiol 1998361886ndash1889

7 DL Calhoun GD Roberts JN Galgiani JE Bennett DS Feingold J Jorgensen GSKobayashi S Shadomy Results of a survey of antifungal susceptibility tests in theUnited States and interlaboratory comparison of broth dilution testing of flucytosineand amphotericin B J Clin Microbiol 198623298ndash301

8 JH Rex MA Pfaller JN Galgiani MS Bartlett A Espinel-Ingroff MA GhannoumM Lancaster FC Odds MG Rinaldi TJ Walsh AL Barry Development of interpre-tive breakpoints for antifungal susceptibility testing Conceptual framework and


analysis of in vitrondashin vivo correlation data for fluconazole itraconazole and Can-dida infections Subcommittee on Antifungal Susceptibility Testing of the NationalCommittee for Clinical Laboratory Standards Clin Infect Dis 199724235ndash247

9 National Committee for Clinical Laboratory Standards 1997 Reference method forbroth dilution antifungal susceptibility testing of yeasts approved standard Vil-lanova PA NCCLS

10 RB Ashman A Fulurija TA Robertson JM Papadimitriou Rapid destruction ofskeletal muscle fibers by mycelial growth forms of Candida albicans Exp Mol Pathol199562109ndash117

11 JC Parker Jr JJ McCloskey KA Knauer Pathobiologic features of human candidia-sis A common deep mycosis of the brain heart and kidney in the altered host AmJ Clin Pathol 197665991ndash1000

12 TJ WalshWG Merz Pathologic features in the human alimentary tract associatedwith invasiveness of Candida tropicalis Am J Clin Pathol 198685498ndash502

13 DM Dixon In vivo models evaluating antifungal agents Methods Find Exp ClinPharmacol 19879729ndash738

14 ME Klepser RE Lewis MA Pfaller Therapy of Candida infections Susceptibilitytesting resistance and therapeutic options Ann Pharmacother 1998321353ndash1361

15 LA Mermel DG Maki Detection of bacteremia in adults Consequences of culturingan inadequate volume of blood Ann Intern Med 1993119270ndash272

16 G Medoff Controversial areas in antifungal chemotherapy Short-course and combi-nation therapy with amphotericin B Rev Infect Dis 19879403ndash407

17 JH Rex MA Pfaller M Lancaster FC Odds A Bolmstrom MG Rinaldi Qualitycontrol guidelines for National Committee for Clinical Laboratory StandardsmdashRec-ommended broth macrodilution testing of ketoconazole and itraconazole J Clin Mi-crobiol 199634816ndash817

18 JH Rex MA Pfaller MG Rinaldi A Polak JN Galgiani Antifungal susceptibilitytesting Clin Microbiol Rev 19936367ndash381

19 ME Klepser EJ Ernst RE Lewis ME Ernst MA Pfaller Influence of test conditionson antifungal time-kill curve results Proposal for standardized methods AntimicrobAgents Chemother 1998421207ndash1212

20 ME Klepser EJ Wolfe RN Jones CH Nightingale MA Pfaller Antifungal pharma-codynamic characteristics of fluconazole and amphotericin B tested against Candidaalbicans Antimicrob Agents Chemother 1997411392ndash1395

21 ME Klepser EJ Wolfe MA Pfaller Antifungal pharmacodynamic characteristicsof fluconazole and amphotericin B against Cryptococcus neoformans J AntimicrobChemother 199841397ndash401

22 F Meunier C Lambert P Van der Auwera In-vitro activity of SCH39304 in compar-ison with amphotericin B and fluconazole J Antimicrob Chemother 199025227ndash236

23 JD Turnidge S Gudmundsson B Vogelman WA Craig The postantibiotic effectof antifungal agents against common pathogenic yeasts J Antimicrob Chemother19943483ndash92

24 D George D Kordick P Miniter TF Patterson VT Andriole Combination therapyin experimental invasive aspergillosis J Infect Dis 1993168692ndash698

25 F de Lalla G Pellizzer A Vaglia V Manfrin M Franzetti P Fabris C Stecca Am-photericin B as primary therapy for cryptococcosis in patients with AIDS Reliability


of relatively high doses administered over a relatively short period Clin Infect Dis199520263ndash266

26 CM van der Horst MS Saag GA Cloud RJ Hamill JR Graybill JD Sobel PCJohnson CU Tuazon T Kerkering BL Moskovitz WG Powderly WE DismukesTreatment of cryptococcal meningitis associated with the acquired immunodefi-ciency syndrome N Engl J Med 199733715ndash21

27 MS Saag GA Cloud JR Graybill JD Sobel CU Tuazon PC Johnson WJ FesselBL Moskovitz B Wiesinger D Cosmatos L Riser C Thomas R Hafner WE Dis-mukes A comparison of itraconazole versus fluconazole as maintenance therapy forAIDS-associated cryptococcal meningitis National Institute of Allergy and Infec-tious Diseases Mycoses Study Group Clin Infect Dis 199928291ndash296

28 RE Lewis BC Lund ME Klepser EJ Ernst MA Pfaller Assessment of antifungalactivities of fluconazole and amphotericin B administered alone and in combinationagainst Candida albicans by using a dynamic in vitro mycotic infection model Anti-microb Agents Chemother 1998421382ndash1386

29 ME Klepser D Malone RE Lewis EJ Ernst MA Pfaller Evaluation of voriconazolepharmacodynamic characteristics using time-kill methodology Antimicrob AgentsChemother 2000441917ndash1920

30 F Minguez JE Lima MT Garcia J Prieto Influence of human serum on the postanti-fungal effect of four antifungal agents on Candida albicans Chemotherapy 199642273ndash279

31 F Minguez MT Garcia JE Lima MT Llorente J Prieto Postantifungal effect andeffects of low concentrations of amphotericin B and fluconazole on previouslytreated Candida albicans Scand J Infect Dis 199628503ndash506

32 F Minguez ML Chiu JE Lima R Nique J Prieto Activity of fluconazole Postanti-fungal effect effects of low concentrations and of pretreatment on the susceptibilityof Candida albicans to leucocytes J Antimicrob Chemother 19943493ndash100

33 EJ Ernst ME Klepser MA Pfaller Postantifungal effect of echinocaudin azole andpolyene agents against Candida albicans and Cryptococcus neoformans AntimicrobAgents Chemother 2000441108ndash1111

34 A Louie GL Drusano P Banerjee QF Liu W Liu P Kaw M Shayegani H TaberMH Miller Pharmacodynamics of fluconazole in a murine model of systemic candi-diasis Antimicrob Agents Chemother 1998421105ndash1109

35 MD Witt RJ Lewis RA Larsen EN Milefchik MA Leal RH Haubrich JA RichieJE Edwards Jr MA Ghannoum Identification of patients with acute AIDS-associ-ated cryptococcal meningitis who can be effectively treated with fluconazole Therole of antifungal susceptibility testing Clin Infect Dis 199622322ndash328

36 RE Lewis ME Klepser MA Pfaller In vitro pharmacodynamic characteristics offlucytosine determined by time kill methods Dicy Microbiol Infect Dis 200036101ndash105

37 JE Bennett Flucytosine Ann Intern Med 197786319ndash32138 JE Bennett WE Dismukes RJ Duma G Medoff MA Sande H Gallis J Leonard

BT Fields M Bradshaw H Haywood ZA McGee TR Cate CG Cobbs JF WarnerDW Alling A comparison of amphotericin B alone and combined with flucytosinein the treatment of cryptoccal meningitis N Engl J Med 1979301126ndash131

39 P Francis TJ Walsh Evolving role of flucytosine in immunocompromised patients


New insights into safety pharmacokinetics and antifungal therapy Clin Infect Dis1992151003ndash1018

40 DA Andes M van Ogtrop In vivo characterization of the pharmacodynamics offlucytosine in a neutropenic murine disseminated candidiasis model AntimicrobAgents Chemother 200044938ndash942

41 G Barbaro G Barbarini G Di Lorenzo Fluconazole vs itraconazole-flucytosine as-sociation in the treatment of esophageal candidiasis in AIDS patients A double-blind multicenter placebo-controlled study The Candida Esophagitis MulticenterItalian Study (CEMIS) Group Chest 19961101507ndash1514

42 AM Sugar Use of amphotericin B with azole antifungal drugs What are we doingAntimicrob Agents Chemother 1995391907ndash1912

43 SM Grant SP Clissold Fluconazole A review of its pharmacodynamic and pharma-cokinetic properties and therapeutic potential in superficial and systemic mycosesDrugs 199039877ndash916

44 M Scheven F Schwegler Antagonistic interactions between azoles and amphoteri-cin B with yeasts depend on azole lipophilia for special test conditions in vitroAntimicrob Agents Chemother 1995391779ndash1783

45 M Scheven C Scheven Quantitative screening for fluconazole-amphotericin B an-tagonism in several Candida albicans strains by a comparative agar diffusion assayMycoses 199639111ndash114

46 M Scheven L Senf Quantitative determination of fluconazole-amphotericin B an-tagonism to Candida albicans by agar diffusion Mycoses 199437205ndash207

47 AM Sugar Interactions of amphotericin B and SCH 39304 in the treatment of experi-mental murine candidiasis Lack of antagonism of a polyene-azole combinationAntimicrob Agents Chemother 1991351669ndash1671

48 AM Sugar CA Hitchcock PF Troke M Picard Combination therapy of murineinvasive candidiasis with fluconazole and amphotericin B Antimicrob Agents Che-mother 199539598ndash601

49 EJ Ernst ME Klepser MA Pfuller In vitro interaction of fluconazole and amphoteri-cin B administered sequentially against candida albicans effect of concentration andexposure time Diag Microbiol Infect Dis 199832205ndash210

50 SL Kelly J Rowe PF Watson Molecular genetic studies on the mode of action ofazole antifungal agents Biochem Soc Trans 199119796ndash798

51 EJ Anaissie RO Darouiche D Abi-Said O Uzun J Mera LO Gentry T WilliamsDP Kontoyiannis CL Karl GP Bodey Management of invasive candidal infectionsResults of a prospective randomized multicenter study of fluconazole versus am-photericin B and review of the literature Clin Infect Dis 199623964ndash972

52 JH Rex JE Bennett AM Sugar PG Pappas CM van der Horst JE Edwards RGWashburn WM Scheld AW Karchmer AP Dine A randomized trial comparingfluconazole with amphotericin B for the treatment of candidemia in patients withoutneutropenia Candidemia Study Group and the National Institute N Engl J Med19943311325ndash1330

53 MS Saag WG Powderly GA Cloud P Robinson MH Grieco PK Sharkey SEThompson AM Sugar CU Tuazon JF Fisher N Hyslop JM Jacobson R HafnerWE Dismukes Comparison of amphotericin B with fluconazole in the treatment ofacute AIDS associated cryptococcal meningitis N Engl J Med 199232683ndash89

14

Human Pharmacodynamics ofAnti-Infectives Determination fromClinical Trial Data

George L Drusano


1 INTRODUCTION

Determination of the relationship between drug exposure and response or be-tween drug exposure and toxicity is key to achieving the ultimate aim of chemo-therapy obtaining the maximal probability of a good therapeutic response whileengendering the smallest possible probability of toxicity

In the area of anti-infective chemotherapy there is a single difference fromother areas of clinical pharmacological investigation In other areas we deal withreceptors for the drug that are human in origin There are true between-patientdifferences in receptor affinity for the drug that are currently not measurableThis unmeasured variance leads to difficulty in generating pharmacodynamic re-lationships

In anti-infectives however we are dealing with an external invaderWhether we are dealing with bacteria viruses or fungi we can with few excep-tions (eg hepatitis C virus) grow the offending pathogen and obtain a measureof the drugrsquos potency for that particular pathogen These measures have differentnames depending on the pathogen [eg minimal inhibitory concentration (MIC)

303

304 Drusano

effective concentration that reduces growth by half (EC50) minimal fungicidal(static) concentration (MFC)] These measures of pathogen sensitivity to the drugcan then be used to normalize the drug exposure in the patient relative to theinvading pathogen This markedly reduces the observed variability and improvesthe ability to define a relationship between exposure and response

2 DETERMINANTS OF A PHARMACODYNAMIC

RELATIONSHIP FOR ANTI-INFECTIVES

21 Endpoints

In order to determine a relationship between exposure and response or betweenexposure and toxicity the first step is to identify an endpoint Such endpointsdiffer according to what is being studied Endpoints may be continuous in nature[eg change in glomerular filtration rate (GFR) after drug exposure] dichoto-mous or polytomous (eg success vs failure survival vs death a four-pointpain scale) or time to an event (time to death time to relapse time to viralclearance) As will be discussed later the endpoint chosen will in many waysdetermine a good deal of the rest of the analysis

The first and most important step in defining a pharmacodynamic relation-ship is to obtain solid response endpoint data If all other steps are performedwell but the endpoint data are poorly defined then the relationship will be poorat best and possibly misleading

22 Pathogen Identification and Susceptibility

Determination

If one is attempting to construct an effect relationship (ie between drug expo-sure and response) it is imperative that the offending pathogen be isolated andidentified and that the susceptibility of that specific pathogen to the drug beingused for therapy be measured This is straightforwardly performed in many clini-cal antibacterial trials because pathogen identification and MIC determination areintegral parts of making a clinical study case both clinically and microbiologicallyevaluable for the Food and Drug Administration

Amazingly little has been done regarding the EC50 of viruses and theirinfluence on outcome in clinical trials of antiviral chemotherapy It has only beenrecently with the availability of commercial homologous recombination assaysor rapid sequencing assays for HIV that determination of drug susceptibility hasbecome a part of the clinical trial arena Nonetheless EC50 has an important roleto play as demonstrated by the data in Figs 1andash1f In this evaluation the HIVprotease inhibitor indinavir was administered as a single agent Plasma concentra-tions were measured by HPLC and EC50 values were determined for indinavirby the ACTGDOD consensus assay A sigmoidal Emax effect model was fit to

Human Pharmacodynamics of Anti-Infectives 305

FIGURE 1 Pharmacodynamics of indinavir Emax model of plasma copy num-ber change For part explanations see text

306 Drusano

FIGURE 1 Continued


FIGURE 1 Continued

308 Drusano

the data with area under the plasma concentrationndashtime curve (AUC) peak con-centration and trough concentration each as the independent variable (Figs 1andash1c) In Figs 1dndash1f the drug exposures were normalized to the EC50 of that pa-tientrsquos virus [1] It can be seen that the normalization improves the fit of themodel to the data It should be noted that the normalization transforms severalpoints from well off the best-fit curve to an area where the fit improves This isbecause of the added information gained from treating a very susceptible viralstrain

It should also be noted that because the drug was administered at essentiallythe same dose and schedule in all the patients there is significant co-linearityThat is one cannot make the peak rise without also raising the trough and withoutincreasing the AUC Therefore one should not draw the inference from thesedata that the AUCEC50 ratio is the pharmacodynamically linked variable forindinavir Indeed from other sources of data the troughEC50 ratio or (perhapspreferably) the time EC95 is the linked variable for protease inhibitors [2]Clearly normalization to a measure of potency for the viral isolate to indinavirimproves the relationship between exposure and response

Much the same is true for any type of pathogen Our ability to grow theorganism and identify its sensitivity to the therapeutic agent is key to our abilityto formulate an exposurendashresponse relationship

However the measure of sensitivity of the pathogen to the drug althoughimportant is not a sufficient condition for the development of a dynamics rela-tionship In order to have the highest probability of attaining a robust dynamicsrelationship obtaining a good estimate of drug exposure for the individual patientis also critical This was seen in a neutropenic rat model of fluoroquinolone phar-macodynamics [3] Two stable mutants of a parental strain of Pseudomonas aeru-ginosa (MICs to the test fluoroquinolone of 1 4 and 8 mgL) were derivedTherapy with the same dose of drug produced a clear difference in response byMIC (80 mgkg once daily as therapy with survivorships of 70 15 and 0for the groups challenged with MICs of 1 4 and 8 mgL respectively) Howeverwhen the dose was altered (20 mgkg once daily for the MIC of 10 mgL chal-lenge strain) so that the peakMIC ratio and AUCMIC ratio were the same asthose seen for the challenge group with an MIC of 40 treated with 80 mgkgonce daily the survivorship curves were identical Because isogenic mutants wereemployed this demonstrates that both pieces of information (drug exposure plusa measure of drug susceptibility) are necessary for the best pharmacodynamicrelationships to be developed

23 Drug Exposure in Clinical Trial Patients

Amazingly few pharmacodynamic relationships have been derived in the anti-infective arena Part of the reason for this is that patients being treated for infec-


tions are often quite ill and unwilling or unable to undergo the rigors of a tradi-tional pharmacokinetic evaluation Often dose has been employed as a surrogatefor actual exposure estimates This has proven to be a failed strategy Dose isa poor measure of exposure There are true between-patient differences in thepharmacokinetic parameter values such as clearance and volume of distributionSuch true differences (but unmeasured when dose is used as a measure of expo-sure) translate into large differences in peak concentration trough concentrationand AUC in a population of patients receiving the same dose It should not besurprising that dose is a particularly poor measure of drug exposure and a poorexposure variable to employ in developing pharmacodynamic relationships

Figure 2 demonstrates the inadequacy of examining just dose as a measureof drug exposure This is the marginal density plot for clearance for levofloxacinThis drug was studied in 272 patients enrolled in the first study to prospectivelydevelop a relationship between exposure and response [4] This was done in amulticenter study that included 22 centers in the United States In the study proto-col patients with serum creatinine values in excess of 20 mgdL were excludedNonetheless by inspection the range of clearance exceeded tenfold This alsoindicates that the range of AUC for a fixed dose would exceed tenfold Obviouslyany attempt to link exposure to outcome employing dose as the measure of expo-sure would be doomed to failure

Over the past decade a number of mathematical techniques have foundtheir way into the toolbox of the kineticist or clinician wishing to construct suchrelationships The first is optimal sampling theory This technique allows identi-

FIGURE 2 Approximate marginal density for clearance of levofloxacin

310 Drusano

fication of sample times that are laden with lsquolsquoinformationrsquorsquo The definition oflsquolsquoinformationrsquorsquo is dependent upon the measure that is defined by the user Forinstance the most commonly employed measure is the determinant of the inverseFisher information matrix This is referred to as D-optimality It has several prop-erties that are desirable The answers obtained are independent of how the systemis parameterized and are also independent of units This measure also has theremarkable property of replicativeness That is if one defines a four-parametersystem there will be exactly four optimal sampling times If the investigatorwishes to make the sampling scheme more robust to errors D-optimality willtell the investigator to repeat one of the optimal sampling times This is becauseD-optimality is deterministic and is based upon the (incorrect) assumption thatthere is only one true parameter vector without true between-patient variabilityMost other measures of information content (eg C-optimality A-optimality)also suffer from being deterministic Publications by DrsquoArgenio [5] and Tod andRocchisani [6] extended optimal sampling into the stochastic framework and al-lowed true between-subject variability in the parameter values This allows theinvestigator to increase the number of samples and to have increasing amountsof information in the sampling scheme for patients whose values are more re-moved from the mean values

Traditional (deterministic) optimal sampling has been well validated Fur-ther it is possible to employ traditional optimal sampling and still obtain sam-pling schedule designs robust for a large portion of the population

One problem with optimal sampling strategy is that it assumes that theanswer is already known that is that one knows the true mean parameter vectorfor the model system This obviously places limitations on the use of optimalsampling strategy in the early phases of drug development when little is knownregarding the lsquolsquotruersquorsquo model to be employed for a specific drug and less is knownregarding the true mean parameter vector Nonetheless with only a little informa-tion regarding these issues optimal sampling has been employed successfully

There was no validation of this technique in patients until a series of studieswere published by Drusano and coworkers [7ndash10] In what was to our knowl-edge the first clinical validation of optimal sampling theory the drug ceftazidimewas examined in young patients with cystic fibrosis receiving a single dose [7]With the use of a Bayesian estimator the optimal sampling subset of the fullsampling set produced precise and unbiased estimates of the important pharmaco-kinetic parameter values

This group then examined the drug piperacillin in a population of septicneutropenic cancer patients [8] Whereas the study with ceftazidime was per-formed with a single dose of drug the study with piperacillin examined twoissues (1) whether optimal sampling would provide precise and unbiased esti-mates of parameter values in the steady-state situation and (2) whether obtaining


duplicate samples obtained at the specified sample times would improve the preci-sion of parameter estimation

The results demonstrated that optimal sampling would as expected pro-vide reasonably precise and unbiased parameter estimates Further this studyalso showed that resampling at the designated optimal times did not improve theprecision of parameter estimation The latter result was a bit of a surprise andflew in the face of the then-accepted theory regarding optimal sampling Optimalsampling theory assumes that the mean parameter vector is known without errorFurther true between-patient variance is not incorporated into the optimal sam-pling time calculation Given these limitations it is not surprising that when que-ried regarding the next most optimal time to obtain a sample after the originaloptimal times have all been obtained the theory forces one of the optimal timesto be repeated (property of replication) This strategy may improve the precisionfor the mean patient but in the clinical situation where one is trying to constructa population model (part of the creation of a pharmacodynamic model) it isimportant to recognize that true between-patient variance exists for the parametervalues

If an investigator is to limit the number of plasma samples obtained to anoptimal sampling set it is important to know how robust optimal sampling iswith regard to errors in nominal parameter values This group also addressed thisissue [10] Theophylline has been demonstrated to have its clearance altered bysmoking cigarettes The degree of this alteration has been on the order of a 50increase in the mean clearance of the population It was felt that by studying apopulation of smokers as well as a population of nonsmokers and employingoptimal sampling strategies for both smokers and nonsmokers they could examinehow badly optimal sampling sets performed when systematic errors on the orderof 50 (either high or low) were introduced into the nominal value for clearanceThis study demonstrated that errors of this magnitude did not introduce significantbias or imprecision into the overall estimation of theophylline clearance Furtherbecause this study was performed in two stages after the first stage they embed-ded a sampling set that was calculated by employing the patientrsquos initial parame-ter values estimated from the full sample set obtained during the first stage Theydemonstrated that the patientrsquos own optimal samples provide excellent precisionand minimal bias for the second stage of the study (patient by patient) Such afinding is important in that it means that toxic drugs can be adequately controlledwith minimal sample acquisition if patients are to be dosed over a relatively longperiod of time (as is the case in antiretroviral chemotherapy) Likewise obtaininginformation about the patientrsquos parameter values for effect control with limitedsample acquisition also becomes possible in the routine clinical situation

Others have recognized the importance of optimal sampling theory in guid-ing the acquisition of plasma samples in the clinical trial setting for the develop-

312 Drusano

TABLE 1 Precision () of Kinetic Parameters of Theophylline asDetermined from Different Optimal Sampling Strategies Relative to ThoseDetermined from the Full Sampling Strategya

Vc Vss Varea SCl T12β

Correct7 220 126 130 297 299Wrong7 166 101 104 356 398Patientrsquos7 228 134 130 298 366Patientrsquos4 260 220 228 299 377

a Correct7 represents the 7 sample times derived from the lsquolsquocorrectrsquorsquo prior populationWrong7 represents the seven sample times derived from the lsquolsquowrongrsquorsquo prior populationPatientrsquos7 and Patientrsquos4 represent the seven and four sample times derived from thepatientrsquos own prior parameter values

ment of exposurendashresponse relationships Forrestrsquos group [11] developed an opti-mal sampling strategy for ciprofloxacin that is useful in the environment ofseriously ill hospitalized patients with lower respiratory tract infections Fletcherand colleagues [12] adapted optimal sampling strategy to the AIDS arena for thedevelopment of concentration-controlled trials

The second technique is population pharmacokinetic modeling Credit forthe initial development of this technique reflects to Sheiner Beal and colleagues[13ndash15] After the initial development of the NONMEM system other groupsdeveloped population modeling programsmdashMallet (NPML) [16] Schumitzky etal (NPEM) [17] the PPHARM system [18] Davidian and Gallant [19] Lind-strom and Bates [20] and Forrest et al [21] among others Population modelingallows the development of a mean parameter vector for the model without requir-ing that every patient have a robust sampling set It also provides an estimate ofthe covariance matrix allowing construction of parameter distributions and alsoallowing Monte Carlo simulation which has recently been shown to be useful inevaluation of doses and schedules Of course the important issue with populationmodeling is that the data must be well timed The looseness of execution oftenassociated with performing population PK modeling is not an excuse for poortiming of sample collection Such poor attention to detail can severely impactupon the estimates rendering them either biased or imprecise Nonetheless itshould be recognized that the ability to perform population modeling has resultedin nothing short of a revolution in our ability to obtain information about drugdisposition in ill target patients The data presented in Fig 2 are from an analysisemploying NPEM [4] Ill patients with community-acquired infections were stud-ied with each patient having an optimal sampling set of seven plasma determina-tions each guided by stochastic design theory


Once population modeling has been performed it is then useful to performmaximal a posteriori probability (MAP) Bayesian estimation This allows pointestimates of the model parameters to be obtained for all the patients in the popula-tion Measures of drug exposure [peak concentration trough concentration areaunder the plasma concentrationndashtime curve (AUC)] can be calculated and thennormalized to the potency parameter (eg peakMIC ratio AUCMIC ratiotime MIC) It is now possible to examine the relationship between exposureand response andor toxicity

In the study cited above a parameter vector was calculated for each patientby Bayesian estimation The plasma drug concentrations were then simulated forthe specific times they were obtained and a predicted versus observed plot wasproduced Figure 3 displays this analysis The best-fit line was

Observed 1001 predicted 00054 r2 0966 p 0001

Once robust estimates of parameter values are obtained for each patientit is straightforward to attempt to link measures of exposure (peak concentrationMIC or AUCMIC ratio time MIC etc) to outcomes For continuous outcomevariables (eg viral copy number CD4 counts) continuous functions such as atraditional sigmoidal Emax effect function would be a natural choice (see Figs1andash1f)

However clinical trials frequently have either dichotomous outcome vari-ables (eg successfailure eradicationpersistence) or have time-to-event end-

FIGURE 3 Scatterplot and least squares line for the entire population

314 Drusano

points (eg time to death time to opportunistic infection time to lesion changein CMV retinitis) For dichotomous outcome variables logistic regression analy-sis is a natural choice For the prospective study examining levofloxacin citedabove we had an analysis plan that tested 13 covariates univariately [422]Model building then ensued from the covariates that significantly altered theprobability of a good clinical or microbiological outcome (separate sets of analy-ses) The final models for clinical and microbiological outcomes are displayedgraphically in Figs 4 and 5 respectively

It should also be noted that small boxes on the probability curve denoteindependent variable lsquolsquobreakpointsrsquorsquo These are arrived at through classificationand regression tree (CART) analysis These merely indicate that patients whoseindependent variable (here peak concentrationMIC ratio) has a value equal toor greater than the breakpoint value have a significantly higher probability ofobtaining a good outcome CART is a useful adjunctive technique in pharmaco-dynamic analyses but should probably be seen as an exploratory tool and onefor rational setting of breakpoints Logistic regression should be seen as the pri-mary tool for analysis with dichotomous endpoints

In addition to modeling successfailure logistic regression can also be em-ployed to model the probability of occurrence of toxicity An example can beseen in the analysis of aminoglycoside-related nephrotoxicity published by Rybaket al [23] These authors performed a prospective randomized double-blind trialin which patients received their aminoglycoside either once daily or twice daily

FIGURE 4 Logistic regression relationship between levofloxacin peakMIC ra-tio and the probability of a good clinical outcome


FIGURE 5 Logistic regression relationship between levofloxacin peakMIC ra-tio and the probability of organism eradication

In the final model the schedule of administration the daily AUC of aminoglyco-side and the concurrent use of vancomycin all independently influenced the prob-ability of occurrence of aminoglycoside-related nephrotoxicity

Sometimes as with the therapy of cytomegalovirus retinitis the endpointexamined is the time to an event here the time to CMV lesion progression Inthis circumstance after having performed the Bayesian estimation the measuresof exposure may be employed as covariates in a Cox proportional hazards modelanalysis This semiparametric approach is a useful way to approach such analysesFor those instances where fuller knowledge of the shape of the hazard functionis available fully parametric analyses (eg Weibull analysis) can be performed

In an analysis of the use of foscarnet for the therapy of cytomegalovirusretinitis Drusano et al [24] performed a population pharmacokinetic analysisfollowed by Bayesian estimation The exposures then became part of the pharma-codynamic analysis Five covariates were examined (1) baseline CD4 count (2)peak CD4 count during therapy (3) whether or not the patient had a baselineblood culture positive for CMV (4) the peak concentration achieved and (5) theAUC achieved Trough concentrations were not considered because they wouldgenerally be below the level of assay detection In fact all five covariates signifi-cantly shifted the hazard function With model building only AUC and the base-line CMV blood culture status remained in the final model Figure 7 demonstratesthe exposure response from the final Cox model

Monte Carlo simulation has recently been demonstrated to be useful forthe evaluation of doses and schedules for anti-infective agents This technique

316 Drusano

FIGURE 6 Probability of aminoglycoside toxicity (a) Twice daily dosingmdashvancomycin use and daily AUC (b) Once daily dosingmdashvancomycin use anddaily AUC


FIGURE 7 Exposure response of final Cox model (a) Baseline cytomegalovi-rus blood culture negative (b) baseline cytomegalovirus blood culture posi-tive () Lowest AUC observed in the population ( ) 20th 50th and80th percentiles respectively of AUC in the population

was first applied for this purpose by Drusano at a meeting of the FDA Anti-Infective Drug Products Advisory Committee [25] Two applications are demon-strated here The first is for dose adequacy and for preclinical MIC breakpointdetermination The second is for the evaluation of the dosing schedule

To evaluate the adequacy of a 500 mg dose of the fluoroquinolone lev-ofloxacin Drusano and Craig collaborated for the following analysis The meanparameter vector and covariance matrix from the levofloxacin study cited earlierwere employed to create a 10000 subject Monte Carlo simulation The AUCdistribution for a 500 mg IV dose for these subjects was generated The data

318 Drusano

FIGURE 8 Levofloxacin 10000-subject Monte Carlo simulation Pneumococ-cal target attainment with a 500 mg qid dose () 1 Log drop target () stasistarget () MIC distribution

from the levofloxacin TRUST (Tracking Resistance in the United States Today)study was employed for the MIC distribution for Streptococcus pneumoniae Thekey for this analysis was to set a lsquolsquotarget goalrsquorsquo Craigrsquos mouse thigh modelallowed setting the AUCMIC target goal of 270 (total drug) associated withstasis and 345 (total drug) for a drop in the CFU of one log10 unit (associatedwith the shutoff of bacteremia) for levofloxacin [26] Each of the 10000 AUCsin the distribution were divided by the MIC range from 0125 to 40 (in a twofolddilution series) The resultant values were compared to the target goals and thefrequency with which the target was achieved was ascertained The outcome ofthis analysis is displayed in Fig 8

It is obvious that the goal attainment rate is 100 for both targets until anMIC of 05 mgL is reached At 10 mgL both target attainments decline but

TABLE 2 Levofloxacin 10000-Subject Monte Carlo Simulation TargetAttainment Over a 4296 Isolate Database of Streptococcus pneumoniae

Target 1 Log drop (345 AUCMIC ratio) Stasis (27 AUCMIC ratio)Attainment 947 02 978 01

Source PK parameters from Ref 33 isolate MICs from the 1998ndash1999 TRUST studytarget attainment data from Ref 26


FIGURE 9 One randomly chosen concentration time profile resulting fromMonte Carlo simulations of different dosage schedules for abacavir and am-prenavir with 500 subjects (andashd) Concentrationndashtime profiles (a c) () Am-prenavir 800 mg g8h () abacavir 300 mg q12h (b d) () Amprenavir 1200mg q12h () abacavir 300 mg q12h (e f) Effectndashtime profiles for a specificpatient

320 Drusano

FIGURE 10 Average percent of maximal antiretroviral effect for combinationtherapy with abacavir plus amprenavir Amprenavir schedule of administra-tion was 800 mg (q8h) or 1200 mg (q12h) Abacavir schedule was 300 mg(q12h) in both groups Results weere calculated from a Greco interactionmodel Drug concentrations were from Monte Carlo simulation

both are in excess of 90 Only after this do we see a large decline in targetattainment

It is possible to remove the variability in the MIC by performing an expecta-tion over the MIC distribution In essence we can multiply the target attainmentrate by the fraction of the strains of pneumococcus represented at each levofloxa-cin MIC value This gives us an estimate of the target attainment rate in a clinicaltrial subject to the assumptions that the MIC distribution is representative of thatseen in clinical trials and that the AUC distribution is likewise representative ofa clinical trial The target attainment rates are shown in Table 2

This example demonstrates that a 500 mg dose of levofloxacin would likelybe adequate for pneumococcal infections given the distribution of the AUCs forthe drug and the distribution of the MICs This has been demonstrated in clinicaltrials of levofloxacin in community-acquired pneumonia [2227]

It is also possible to examine schedule with this technique Drusano et al[28] examined the combination of abcavir plus amprenavir for HIV The interac-tion of the two agents was quantitated in the presence of human binding proteinsin vitro using the Greco interaction equation [29] Population pharmacokineticmodels were then derived from clinical trial data for both drugs Monte Carlosimulations were derived of the effectndashtime curves for 500 subjects In the simu-lations doses of 300 mg of abacavir every 12 h (q12h) plus 800 mg of amprenavir


every 8 h (q8h) were simulated as well as doses of abacavir 300 mg every 12h plus 1200 mg of amprenavir every 12 h In Figs 9a and 9b the mean concentra-tionndashtime profiles are shown for the various simulations for 500 subjects Figures9c and 9d show one subject selected from the population Figures 9e and 9f showthe effect vs time curves derived from that specific patient at steady state forthe different schedules of administration

The effectndashtime curves can be integrated over a 24 h steady-state interval

FIGURE 11 Frequency histograms for average percent of maximal effect forabacaviramprenavir combination therapy In (a) abacavir was 300 mg (q12h)and amprenavir dose was 800 mg (q8h) In (b) abacavir was 300 mg (q12h)and amprenavir was 1200 mg (q12h) Values were determined as in Fig-ure 10

322 Drusano

and divided by the interval length (24 h) An average percent of maximal effectresults from the calculation These are plotted in Fig 10 for the two schedulesof administration for all 500 simulated subjects

It is obvious from inspection that the schedule of administration that ismore fractionated for the protease inhibitor (amprenavir q8h) is providing greatereffects This can clearly be seen in the frequency histograms presented in Fig11 Irrespective of how one tests the differences between regimens (frequency90 maximal effect frequency 70 maximal effect difference betweenmean percent maximal effects) the more fractionated regimen is always statisti-cally significantly superior

Consequently Monte Carlo simulation can be used for both dose and regi-men evaluations and can also set preclinical and after the human pharmacody-namic trials have been performed clinical MIC breakpoints It is obvious thatthere are other uses for this flexible and powerful technique for the clinical arena(eg drug penetration to the site of infection [30])

Each of the foregoing examples leads to a simple paradigm It is straightfor-ward to attempt to define pharmacodynamic relationships in the clinical trial set-ting The approach is set forth in Table 3 Once such relationships are definedparticularly when relationships are available for both efficacy and toxicity as isthe case for aminoglycoside antibiotics [2331] the true goal of anti-infectivechemotherapy (maximal effect with minimal toxicity) can be sought by usingstochastic control techniques [32] As part of the approach it is clear that plasmaconcentration determination is a requirement It should be brought home force-

TABLE 3 Paradigm for the Development of ExposurendashResponseRelationships for Anti-Infective Agents

1 Decide on an endpoint2 Make potency measurements on pathogens from trials (MIC EC50 etc)3 Obtain exposure estimates for patients from those trials

a Stochastic design for sampling schemeb Population pharmacokinetic modelingc MAP-Bayesian estimation for individual-patient exposure esti-

mates4 Decide on an endpoint analysis (the following are examples only)

a Sigmoidal Emax analysis for a continuous variableb Logistic regression for dichotomouspolytomous outcomesc Cox proportional hazards modeling (or a variant) for time-to-event

datad Classification and regression tree analysis for breakpoint determi-

nation5 Stochastic control when effectndashtoxicity relationships are available


fully to pharmaceutical sponsors that the paradigm has shifted We can success-fully seek and generate such relationships In the lsquolsquoold daysrsquorsquo the necessity forconcentration determination was the death knell for the development of a com-pound Now it is clear that for the patientrsquos sake we can achieve maximal proba-bility of a good clinical and microbiological outcome coupled with the minimalprobability of toxicity and (perhaps) emergence of resistance by measuring drugconcentrations in plasma This should be the new (and positive) way of differenti-ating drugs We should prefer those that provide us the possibility of having arational basis for producing the best patient outcomes Again third party payorsalso need to understand that drugs with such relationships developed should havepriority on clinical pathways because they provide maximal probability of re-sponse with minimal probability of toxicity

REFERENCES

1 GL Drusano Antiviral therapy of HIV and cytomegalovirus In Symposium 130-A S-121 37th Interscience Conference on Antimicrobial Agents and ChemotherapyToronto Canada Sept 28ndashOct 1 1997 Am Soc Microbiol Washington DC

2 GL Drusano JA Bilello SL Preston E OrsquoMara S Kaul S Schittman R EcholsHollow fiber unit evaluation of BMS232632 a new HIV-1 protease inhibitor forthe linked pharmacodynamic variable In Session 171 Presentation 1662 40th In-terscience Conference on Antimicrobial Agents and Chemotherapy Toronto Can-ada Sept 17ndash20 2000 Am Soc Microbiol Washington DC

3 GL Drusano DE Johnson M Rosen HC Standiford Pharmacodynamics of a fluor-oquinolone antimicrobial in a neutropenic rat model of Pseudomonas sepsis Antimi-crob Agents Chemother 199337483ndash490

4 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornseif V ReichlJ Natarajan FA Wong M Corrado Levofloxacin population pharmacokinetics inhospitalized patients with serious community-acquired infection and creation of ademographics model for prediction of individual drug clearance Antimicrob AgentsChemother 199842631ndash639

5 DZ DrsquoArgenio Incorporating prior parameter uncertainty in the design of samplingschedules for pharmacokinetic parameter estimation experiments Math Biosci 199099105ndash118

6 M Tod J-M Rocchisani Implementation of OSPOP an algorithm for the estimationof optimal sampling times in pharmacokinetics by the ED EID and API criteriaComput Methods Programs Biomed 19965013ndash22

7 GL Drusano A Forrest MJ Snyder MD Reed JL Blumer An evaluation of optimalsampling strategy and adaptive study design Clin Pharmacol Ther 198844232ndash238

8 GL Drusano A Forrest KI Plaisance JC Wade A prospective evaluation of optimalsampling theory in the determination of the steady state pharmacokinetics of pipera-cillin in febrile neutropenic cancer patients Clin Pharmacol Ther 198945635ndash641

9 GJ Yuen GL Drusano A Forrest KI Plaisance ES Caplan Prospective use of opti-

324 Drusano

mal sampling theory Steady-state ciprofloxacin pharmacokinetics in critically illtrauma patients Clin Pharmacol Ther 198946(4)451ndash457

10 GL Drusano A Forrest JG Yuen KI Plaisance Optimal sampling theory Effectof error in a nominal parameter value on bias and precision of parameter estimationJ Clin Pharmacol 199434967ndash974

11 AD Kashuba CH Ballow A Forrest Development and evaluation of a Bayesianpharmacokinetic estimator and optimal sparse sampling strategies for ceftazidimeAntimicrob Agents Chemother 1996401860ndash1865

12 SE Noormohamed WK Henry FS Rhames HH Balfour Jr CV Fletcher Strategiesfor control of zidovudine concentrations in serum Antimicrob Agents Chemother1995392792ndash2797

13 TH Grasela LB Sheiner Population pharmacokinetics of procainamide from routineclinical data Clin Pharmacokinet 19849545ndash554

14 SL Beal LB Sheiner Estimating population kinetics CRC Crit Rev Bioeng 19828195ndash222

15 LB Sheiner B Rosenberg VV Marathe Estimation of population characteristics ofpharmacokinetic parameters from routine clinical data J Pharmacokinet Biopharm19775445ndash479

16 A Mallet A maximum likelihood estimation method for random coefficient regres-sion models Biometrika 198673645ndash656

17 A Schumitzky R Jelliffe M Van Guilder NPEM2 A program for pharmacokineticpopulation analysis Clin Pharmacol Ther 199455163

18 F Mentre R Gomeni A two step iterative algorithm for estimation in non-linearmixed effects models with an evaluation in population pharmacokinetics J Bio-pharm Stat 19955141ndash158

19 M Davidian AR Gallant Smooth nonparametric maximum likelihood estimationfor population pharmacokinetics with application to quinidine J Pharmacokinet Bi-opharm 199220529ndash556

20 M Lindstrom D Bates Nonlinear mixed effects models for repeated measures dataBiometrics 199046673ndash687

21 A Forrest CH Ballow DE Nix MC Birmingham JJ Schentag Development of apopulation pharmacokinetic model and optimal sampling strategies for intravenousciprofloxacin Antimicrob Agents Chemother 1993371065ndash1072

22 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornseif V Reichl JNatarajan M Corrado Prospective development of pharmacodynamic relationshipsbetween measures of levofloxacin exposure and measures of patient outcome Anew paradigm for early clinical trials J Am Med Assoc 1998279125ndash129

23 MJ Rybak BJ Abate SL Kang MJ Ruffing SA Lerner GL Drusano Prospectiveevaluation of the effect of an aminoglycoside dosing regimen on rates of observednephrotoxicity and ototoxicity Antimicrob Agents Chemother 1999431549ndash1555

24 GL Drusano F Aweeka J Gambertoglio M Jacobson M Polis HC Lane C Eaton SMartin-Munley Relationship between foscarnet exposure baseline cytomegalovirusblood culture and the time to progression of cytomegalovirus retinitis in HIV-posi-tive patients AIDS 1996101113ndash1119

25 GL Drusano SL Preston C Hardalo R Hare C Banfield D Andes WA Craig Useof pre-clinical data for the choice of a phase IIIII dose for SCH27899 with applica-


tion to identification of a pre-clinical MIC breakpoint Antimicrob Agents Chemo-ther 20014513ndash22

26 Craig WA Personal communication27 TM File Jr J Segreti L Dunbar R Player R Kohler RR Williams C Kojak A

Rubin A multicenter randomized study comparing the efficacy and safety of intra-venous andor oral levofloxacin versus ceftriaxone andor cefuroxim axetil in treat-ment of adults with community-acquired pneumonia Antimicrob Agents Chemother1997411965ndash1972

28 GL Drusano DZ DrsquoArgenio SL Preston C Barone W Symonds S LaFon M Rog-ers W Prince A Bye JA Bilello Use of drug effect interaction modeling with MonteCarlo simulation to examine the impact of dosing interval on the projected antiviralactivity of the combination of abacavir and amprenavir Antimicrob Agents Chemo-ther 2000441655ndash1659

29 GL Drusano DZ DrsquoArgenio W Symonds PA Bilello J McDowell B Sadler ABye JA Bilello Nucleoside analog 1592U89 and human immunodeficiency virusprotease inhibitor 141W94 are synergistic in vitro Antimicrob Agents Chemother1998422153ndash2159

30 GL Drusano SL Preston M Van Guilder D North M Gombert M Oefelein LBoccumini B Weisinger M Corrado J Kahn A population pharmacokinetic analy-sis of the prostate penetration of levofloxacin Antimicrob Agents Chemother 2000442046ndash2051

31 AD Kashuba AN Nafziger GL Drusano JS Bertino Jr Optimizing aminoglycosidetherapy for nosocomial pneumonia caused by gram-negative bacteria AntimicrobAgents Chemother 199943623ndash629

32 A Schumitzky Applications of stochastic control theory to optimal design of dosageregimens In DZ DrsquoArgenio ed Advanced Methods of Pharmacokinetic and Phar-macodynamic Systems Analysis Plenum New York 1991137ndash152

33 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornseif V ReichlJ Natarajan FA Wong M Corrado Levofloxacin population pharmacokinetics andcreation of a demographic model for prediction of individual drug clearance in pa-tients with serious community-acquired infection Antimicrob Agents Chemother1998421098ndash1104

15

Antibacterial Resistance

Philip D Lister

Creighton University School of Medicine Omaha Nebraska

1 INTRODUCTION

Bacterial resistance to antibiotics is an inescapable response to the overuse ofantimicrobial agents in the environment It can arise from the selection of resistantsubpopulations among susceptible strains or from the increased prevalence ofstrains expressing intrinsic or acquired resistance The impact of antibiotic resis-tance on patients and society is overwhelming as infections caused by resistantpathogens are associated with higher rates of morbidity and mortality than infec-tions caused by pathogens [12] Furthermore microbial drug resistance has beenprojected to add between $100 million and $30 billion annually to health carecosts [3] Recognition of the factors associated with increasing resistance is essen-tial for the development of new strategies to slow the evolution and progressionof antimicrobial resistance among bacteria

The heavy use and abuse of some antimicrobial agents have been directlylinked to some of the more serious resistance problems challenging clinicianstoday [45] Perhaps the best example is the association between excessive ceftaz-idime use and the development and spread of multidrug resistance among gram-negative bacteria [67] With a diminishment in the number of novel agents enter-ing the clinical arena the judicious use of currently available antibiotics becomesessential if we are to preserve their clinical efficacy Drug control strategies must

327

328 Lister

not only involve tighter regulation on the use of antibiotics but also must involvea more scientific approach to dosing strategies The field of antimicrobial pharma-codynamics strives to establish relationships between the pharmacokinetics of adrug and the effective treatment of infections However just as important are therelationships between pharmacokinetics and selection of resistance during ther-apy This chapter will review some of the most critical resistance problems facingclinicians today the problems encountered with accurate detection of resistancein the clinical laboratory and the role of pharmacodynamic strategies in slowingthe emergence of resistance among bacteria

2 EMERGING RESISTANCE PROBLEMS IN

INFECTIOUS DISEASES


211 Enterococcus Species

Among the numerous species of enterococci E faecalis and E faecium are thetwo most important clinically In addition to causing serious hospital-acquiredinfections such as bacteremia and endocarditis [8] E faecalis and E faeciumare two of the most intrinsically resistant bacteria challenging clinicians today[9] These two pathogens naturally exhibit low-level resistance to the β-lactamsaminoglycosides clindamycin trimethoprim-sulfamethoxazole and other classesof antimicrobial agents When bacterial killing is required for clinical cure thecombination of a cell-wall-active agent (penicillin or vancomycin) with an amino-glycoside provides one of the true examples of antimicrobial synergy

Unfortunately E faecalis and E faecium have not been satisfied with theirintrinsic resistance and have been acquiring high-level resistance to these antibi-otics as well [9] Once high-level resistance to either the β-lactams or aminogly-cosides is acquired synergistic killing is lost therapy of serious infections iscompromised and the search for more effective antimicrobial agents or alterna-tive combinations becomes essential

212 Staphylococcus aureus

In contrast to the impressive intrinsic resistance displayed by the enterococci Saureus represents a prototype bacterium for rapid acquisition and developmentof resistance in response to antibiotics in the environment Soon after penicillinbecame available for clinical use Spink and Ferris [10] reported their isolationof a penicillin-resistant S aureus that produced a penicillin-inactivating enzymePenicillinases in S aureus are usually encoded on a plasmid and can spread easilyamong the genus Currently 70ndash90 of staphylococci are resistant to penicillinand the aminopenicillins [11] To circumvent this problem penicillinase-resistantpenicillins such as methicillin and oxacillin were synthesized and introduced inthe early 1960s However the first methicillin-resistant S aureus (MRSA) was

Antibacterial Resistance 329

discovered as early as 1961 [12] Resistance to this class of penicillins is notmediated through enzymatic inactivation but rather is mediated through the ex-pression of a low-affinity penicillin binding protein PBP2prime [13] By the late1980s 25 of all staphylococcal isolates in the United States were resistant tomethicillin [14] with prevalence rates varying markedly between geographicalareas and between individual institutions

Strains of MRSAs are frequently resistant to multiple antibiotics includingthe penicillins cephalosporins cephamycins carbapenems β-lactamasendashinhibi-tor combinations aminoglycosides macrolides tetracyclines and sulfonamides[11] The glycopeptides vancomycin and teicoplanin remain the preferred therapyfor MRSA infections However strains of S aureus exhibiting intermediate resis-tance to both vancomycin and teicoplanin have been reported [1516]

213 Streptococcus pneumoniae

Like S aureus S pneumoniae has shown a remarkable ability to evolve anddevelop resistance to antibiotics in the environment In contrast to S aureushowever the rate of resistance development is much slower among pneumococciClinical isolates of S pneumoniae remained susceptible to penicillin until the1960s when the first intermediate-resistant isolates were reported in Boston in1965 [17] Thereafter pneumococcal susceptibility to penicillin gradually de-creased with reports of intermediate resistance increasing globally and the firstreports of high-level resistance and clinical failures with penicillin appearing inthe literature [18ndash20] In the United States the prevalence of penicillin-nonsus-ceptible pneumococci steadily increased through the 1980s and 1990s with 40of pneumococci across the country having now lost their susceptibility to penicil-lin [21]

The mechanism of penicillin resistance among S pneumoniae involves theacquisition of lsquolsquoresistantrsquorsquo PBP gene segments from other streptococci in theenvironment [2223] As susceptibility to penicillin decreases a concurrent de-crease in susceptibility to other penicillins cephalosporins cephamycins andcarbapenems is also observed [24] In addition penicillin-resistant pneumococciare increasing their resistance to other classes of antimicrobial agents includingthe macrolides tetracyclines chloramphenicol and trimethoprim-sulfamethoxa-zole [24] The increased prevalence of multidrug-resistant pneumococci presentsa therapeutic dilemma for the treatment to serious pneumococcal infections

A summary of emerging resistance problems among gram-positive bacteriais provided in Table 1


221 Haemophilus influenzae

It is now well established that ampicillin resistance among H influenzae strainscan result from mutations affecting the PBP targets decreased permeability

330 Lister

TABLE 1 Emerging Resistance Problems Among Gram-Positive Pathogens

Bacterial species Class of antibiotics Molecular mechanisms

Enterococcus faecium High-level β-lactam Low-affinity PBPsEnterococcus faecalis β-lactamase inactivation

Vancomycin Production of alteredcell-wall precursorsthat vancomycinbinds with lower af-finity

Aminoglycosides Inactivating enzymesStaphylococcus aureus Penicillin and Penicillinase

aminopenicillinsAll β-lactams Acquired low-affinity

PBP2primeVancomycin Increased targets in

outer cell wallStreptococcus pneu- β-Lactams Acquired lsquolsquoresistantrsquorsquo

moniae PBP gene segments

through the outer membrane andor acquisition of plasmid-mediated β-lacta-mases [25] Of these three resistance mechanisms production of β-lactamaseaccounts for 90 of ampicillin resistance among H influenzae globally withTEM-1 being the most common enzyme produced [25] On average 30ndash37 ofH influenzae strains from every region of the United States produce β-lactamase[21] Although β-lactamase-producing strains remain susceptible to the β-lacta-masendashinhibitor combinations and extended-spectrum cephalosporins permeabil-ity andor PBP changes can provide resistance to these agents as well [2126]In addition to β-lactam resistance H influenzae are developing impressive resis-tance to the macrolide antibiotics with 54 of over 3000 isolates exhibitingresistance in a 1998 national survey [21]

222 Escherichia coli and Klebsiella pneumoniae

Strains of E coli and K pneumoniae have shown a remarkable ability to evolveand adapt to the threat of antibiotics in the environment especially to the β-lactam class Resistance to β-lactams among these two species is mediated pri-marily through the production of β-lactamases Whereas both species encode forβ-lactamase on their chromosome plasmid-encoded β-lactamases are the pre-dominant mechanism of resistance with TEM-1 and SHV-1 being the most com-mon enzymes produced [6] TEM-1 and SHV-1 are considered broad-spectrumβ-lactamases and account for the majority of E coli and K pneumoniae resistanceto the penicillins and early narrow-spectrum cephalosporins Two approaches


used to circumvent β-lactamase-mediated resistance have been to develop lsquolsquoen-zyme-resistantrsquorsquo cephalosporins and to combine an inhibitor of β-lactamases withenzyme-labile penicillins E coli and K pneumoniae have evolved impressivelyin response to these two approaches and have rapidly developed resistance tothe extended-spectrum lsquolsquothird generationrsquorsquo cephalosporins and the β-lactamasendashinhibitor combinations

In response to the β-lactamasendashinhibitor combinations E coli and K pneu-moniae have mutated to either increase production of their plasmid-encoded β-lactamases [2728] or decrease the sensitivity of their β-lactamases to inhibitionby clavulanate tazobactam and sulbactam [2930] In response to the extended-spectrum cephalosporins (ceftazidime cefotaxime ceftriaxone) E coli and Kpneumoniae have evolved through two distinct pathways They have either mu-tated the active sites of their TEM-1 or SHV-1 enzymes to increase the hydrolysisof these drugs or they have acquired plasmid-encoded AmpC cephalosporinasesthat originated from the chromosomal AmpCs of other species (see Sections223 and 224) and are inherently capable of providing resistance to the ex-tended-spectrum cephalosporins The mutated variants of TEM-1 and SHV-1have been termed extended-spectrum β-lactamases (ESBLs) because they haveextended the resistance potential of E coli and K pneumoniae to include theextended-spectrum cephalosporins and aztreonam Unfortunately many strainsproducing these enzymes appear falsely susceptible to the newer cephalosporinsand aztreonam in routine susceptibility tests [3132] and the scope of this resis-tance problem remains largely unknown Although most ESBL-producing bacte-ria are also resistant to ampicillin-sulbactam amoxicillin-clavulanate and ticar-cillin-clavulanate many remain susceptible to piperacillin-tazobactam Similarlymany ESBL-producing E coli and K pneumoniae remain susceptible to thelsquolsquofourth generationrsquorsquo cephalosporin cefepime However the clinical relevance ofpiperacillin-tazobactam and cefepime susceptibility has not been established andthere are concerns of false susceptibility similar to that observed with the ex-tended-spectrum cephalosporins and aztreonam Further complicating therapy ofESBL-producing bacteria is that these enzymes are encoded on plasmids that alsocarry genes encoding resistance to other classes of antimicrobial agents [3334]Therefore it is not uncommon to find ESBL-producing bacteria that are alsoresistant to the aminoglycosides trimethoprim-sulfamethoxazole chlorampheni-col and the tetracyclines The most reliably active agents for treatment of ESBL-producing E coli and K pneumoniae are the carbapenems cephamycins andfluoroquinolones However strains of K pneumoniae resistant to all availableantimicrobial agents have been isolated [35] and may signal the beginning of theprophesied lsquolsquopost-antibiotic erarsquorsquo

223 Other Enterobacteriaceae

Although the predominant ESBL-producing bacteria are E coli and K pneumon-iae ESBLs have also been observed in species of Enterobacter Citrobacter

332 Lister

Proteus Salmonella Serratia and other species of Klebsiella [63637] How-ever it is an inducible chromosomal AmpC cephalosporinase that is most com-monly involved in the development of β-lactam resistance among some of thesespecies [38] Since the 1980s Enterobacteriaceae possessing inducible AmpCcephalosporinases have played an important role in the emergence of resistance

TABLE 2 Emerging Resistance Problems Among Gram-NegativePathogens

Bacterial species Antibiotic class Molecular mechanisms

H influenzae Penicillins TEM-1 β-lactamasePBP affinity decreaseOuter membrane per-

meabilityK pneumoniae E coli Penicillins and narrow- TEM-1 and SHV-1

spectrum cephalo- β-lactamasessporins

β-Lactamasendashinhibitor Inhibitor-resistantcombinations β-lactamases or

hyperproductionof TEM-1SHV-1

Penicillins cephalospo- Extended-spectrumrins inhibitorndashpenicil- β-lactamaseslin combinationsand monobactams

Cephamycins Plasmid-encodedAmpC β-lactamase

E cloacae E aerogen- Penicillins cephalospo- Chromosomal AmpCese S marcescens rins inhibitorndashpenicil- β-lactamaseC freundii lin combinations

cephamycins andmonobactams

Carbapenems CarbapenemasesAmpC outer mem-

brane porinsP aeruginosa Antipseudomonal peni- Plasmid-encoded

cillins β-lactamasesPenicillins cephalospo- Chromosomal AmpC

rins inhibitorndashpenicil- β-lactamaselin combinationsand monobactams

Multiple drug classes Outer membrane porinsAntibiotic efflux pumps


to the antipseudomonal penicillins extended-spectrum cephalosporins and az-treonam Studies have shown that the rapid development of resistance to virtuallyall β-lactams is associated with mutant subpopulations that constitutively producehigh levels of their AmpC cephalosporinase [39] With one mutant cell beingpresent among every 106ndash107 viable cells high-level β-lactam resistance has beenshown to emerge in anywhere from 19 to 80 of patients infected with isolatespossessing an inducible AmpC cephalosporinase [6] Although the emergence ofAmpC-mediated resistance has been observed during therapy with many of theavailable β-lactam antibiotics there appears to be a particular association withextended-spectrum cephalosporins most particularly ceftazidime [2] In contrastto the β-lactam class of drugs carbapenem resistance is very rare among membersof the Enterobacteriaceae When resistance is observed it is due to either theproduction of a β-lactamase capable of hydrolyzing carbapenem antibiotics (car-bapenemase) or a reduction in the accumulation of drug in the periplasmic space[384041]

224 Pseudomonas aeruginosa

Among the gram-negative bacterial species P aeruginosa is one of the mosttalented in terms of rapidly developing resistance to antibiotics Similar to theEnterobacteriaceae the mechanisms responsible for β-lactam resistance amongP aeruginosa often involve the production of β-lactamases both plasmid- andchromosomally encoded and the emergence of high-level β-lactam resistanceduring therapy is also primarily due to the selection of mutants that overproducetheir chromosomal AmpC cephalosporinase [42] In contrast to the Enterobacteri-aceae however rates of imipenem resistance among P aeruginosa can exceed50 [43] The β-lactams and carbapenem antibiotics are not the only classes thatare impacted by P aeruginosarsquos ability to rapidly develop resistance Due to itsability to rapidly alter outer membrane permeability P aeruginosa can developresistance during the course of therapy with virtually any antibiotic

A summary of emerging resistance problems among gram-negative bacteriais provided in Table 2

3 DETECTION OF RESISTANCE IN THE

CLINICAL LABORATORY

31 Clinical Relevance of Susceptibility Tests

The susceptibility of a bacterial pathogen to antibiotics is an important therapeuticguide for clinicians However antimicrobial susceptibility is just one piece of acomplex puzzle determining clinical outcome Just as important are the pharma-cokinetics of the antibiotic at the site of infection the physical and chemicalenvironment at the site of infection the pharmacodynamic interactions between

334 Lister

the antibiotic and target bacterium the immune status of the host the need forsurgical intervention and the emergence of resistance during therapy The inter-play between some or all of these factors can lead to a clinical failure even whenthe bacterium exhibits susceptibility to an antibiotic in vitro Conversely clinicalsuccess can sometimes be achieved in cases when the bacterial pathogen exhibitsresistance in vitro This is especially true when interpretive breakpoints for resis-tance are based upon serum pharmacokinetics yet the antimicrobial agents areknown to concentrate to much higher levels at the site of infection

There have been relatively few studies investigating the predictive valueof susceptibility tests and comparison of their data is difficult due to differencesin susceptibility tests used definitions of sensitivity or resistance microorgan-isms studied and criteria used for clinical success or failure [44] Despite theseproblems general conclusions can be reached The correlation between in vitrosusceptibility and favorable clinical outcome is moderately good at best mostlydue to the multitude of other factors that can significantly affect therapeutic suc-cess Although the correlation between in vitro resistance and clinical failure ismuch stronger the relationship is not 100 [44] This is not surprising howeverconsidering that in vitro susceptibility assays are performed in the absence ofhost defense mechanisms and that interpretive breakpoints based on serum phar-macokinetics are inappropriate for drugs that concentrate to higher levels in tis-sues or urine Despite these imperfections there is a clinical relevance to thedata obtained from in vitro susceptibility assays especially when antimicrobialresistance is accurately detected Unfortunately most efforts directed toward im-proving susceptibility testing to date have focused on achieving a reproducibledetection of susceptibility As the threat of antimicrobial resistance continues toincrease in our environment it becomes essential that scientists shift the focusof susceptibility assays from the reproducible detection of susceptibility to theaccurate detection of old and newly evolving resistance mechanisms There arethree general approaches that can be used to detect resistance in clinical isolates(1) detection of resistance genes (2) detection of gene products that mediateresistance and (3) detection of phenotypic resistance through routine susceptibil-ity assays These three approaches will be reviewed in this chapter

32 Methods for the Detection of Resistance Genes

One advantage of using genetic techniques to detect resistance is that they cangive the clinician therapeutic guidance faster than routine culture and susceptibil-ity methods In addition genetic techniques can sometimes be more accurate thansusceptibility tests in defining the resistance mechanisms of a bacterium Forexample oxacillin resistance among strains of S aureus can be mediated eitherthrough the production of PBP2prime or through the hyperproduction of a penicillinase[45] It is not always possible to differentiate between these two resistance mecha-


nisms based on routine susceptibility data yet the therapeutic implications areimportant [46] A genetically based assay that accurately detects mecA in staphy-lococci could provide important therapeutic information to the clinician and pre-vent the unnecessary use of vancomycin in some cases It is important to notehowever that these genetically based assays are useful only when they detect aresistance gene in a bacterial isolate A negative result is of little use to the clini-cian because other unrelated resistance mechanisms may be operative in thebacterium Therefore a negative result should never be used as a confirmationof susceptibility

Two approaches are currently used to detect resistance genes (1) directdetection of genes with DNA probes and (2) amplification of genes using poly-merase chain reaction (PCR) methodology Variations of these methodologiesare currently being developed to target resistance genes in numerous bacterialspecies However this section will only briefly review those genetic methods thattarget aminoglycoside and glycopeptide resistance in enterococci and β-lactamresistance mediated through mecA and β-lactamases (see Table 3)

321 Glycopeptide and Aminoglycoside Resistance

To date there have been four different genotypes of vancomycin resistance char-acterized vanA vanB vanC and vanD [5] Of these vanA and vanB are the mostimportant clinically providing high-level vancomycin resistance among strainsof E faecalis and E faecium Gene probes and PCR assays have been designedto detect the vanA vanB vanC genotypes [4748] and PCR assays are currentlybeing evaluated for the identification of vancomycin-resistant enterococci in fecalsamples [49]

Owing to the diversity of aminoglycoside-modifying enzymes amonggram-negative bacteria it is very difficult to find a consensus sequence that wouldallow for detection of multiple genes using one DNA probe or PCR primer setIn contrast the genes encoding aminoglycoside-modifying enzymes in gram-pos-itive bacteria are much more conserved To date most efforts have focused ondetecting the genes encoding ANT(6) and AAC(6prime)-APH(2Prime) which providehigh-level aminoglycoside resistance to enterococci [50]

322 β-Lactam Resistance

Among gram-positive bacteria most attention has focused on the genetic detec-tion of mecA-mediated resistance in isolated colonies of staphylococci or bloodculture [5152] These assays can require as few as 500 staphylococci to obtaina positive reaction using a PCR enzyme-linked colorimetric assay

The enzymes that are responsible for β-lactam resistance among gram-neg-ative bacteria are diverse not only in their function but also in their genetic codeDNA probes and PCR primer sets have already been prepared for several plas-mid-encoded β-lactamases [5354] and DNA probes have been used to detect

336 Lister

TABLE 3 Assays to Detect Antimicrobial Resistance Genes

Bacterial species Resistance mechanism Genes targeted

Enterococcus faecium Vancomycin resistance vanAEnterococcus fae- through VanA VanB vanBcalis Enterococcus or VanC vanCspp

High-level aminoglyco- ant(6prime)side through inactivat- aac(6prime)-aph(2Prime)ing enzymes ANT(6)or AAC(6prime)-APH(2Prime)

Staphylococcus aureus β-Lactam resistance mecAthrough production oflow-affinity PBP2prime

Escherichia coli Resistance to penicillins blaTEM

Klebsiella pneumoniae and early cephalospo- blaSHV

rins through TEM-1or SHV-1 β-lactamase

Haemophilus influenzae Resistance to penicillins blaTEM

through production blaROB

of TEM-1 or ROB-1β-lactamases

Enterobacteriaceae Resistance to penicil- Oligonucleotide typinglins cephalosporins to detect nucleotideinhibitorndashpenicillin changes at criticalcombinations and basepairs of blaTEM

monobactams and blaSHV

through productionof ESBLs

the blaTEM gene (TEM-1) in urethral exudates urine and spinal fluid [55ndash57]However because of the diversity of β-lactamase produced by gram-negativebacteria samples may need to be tested with multiple probes or PCR primer setsFor example to accurately detect β-lactamase-mediated resistance in E coli andK pneumoniae probes or primer sets for TEM-1 SHV-1 and a multitude ofother β-lactamases may need to be tested on a single isolate Similarly to accu-rately detect β-lactamase-mediated resistance among H influenzae probes orprimer sets for TEM-1 or ROB-1 should be evaluated even though TEM-1 isby far the most predominant enzyme Even with the development of probes toevery known β-lactamase gene this genetically based approach would not detectresistance mediated by porin or PBP changes and thus a negative result shouldbe used as a confirmation of susceptibility


The ever-evolving family of ESBLs presents an additional problem forgenetically-based assays Because these enzymes have evolved over time throughpoint mutations within the active sites of TEM-1 and SHV-1 [3337] probes andprimer sets designed for blaTEM and blaSHV will detect an ESBL gene Howeverthese probes cannot differentiate an ESBL-encoding gene from a gene encodingTEM-1 or SHV-1 Considering the therapeutic implications associated withESBLs it is critical that any genetically-based assays demonstrate this differentia-tion capability Mabilat and Courvalin [58] described an oligonucleotide-basedhybridization method that can differentiate β-lactamase genes based on nucleo-tide changes at critical basepairs However because of the logistics and labor-intensive nature of this technology oligonucleotide typing presents a greater ben-efit for epidemiological studies at this time As technologies continue to evolvethe direct detection of ESBLs may become a reality in the clinical laboratory

33 Methods For Detection of Gene Products That

Mediate Resistance

Similar to the genetically-based methods discussed above methods that directlydetect the active products of resistance genes can provide valuable therapeuticinformation to the clinician faster than routine culture and susceptibility methodsA positive result from these tests can guide the clinician by inferring resistanceto those drugs known to be affected by the expressed resistance mechanismHowever similar to genetically based methods a negative result does not ensurethe lack of resistance as other mechanisms of resistance may be operative Asummary of the methods used for direct detection of β-lactamases and chloram-phenicol acetyltransferase (CAT) is provided in Table 4

331 Direct Detection of β-Lactamases

There are three direct β-lactamase detection assays used clinically (1) iodometricassay (2) acidometric assay and (3) chromogenic assay The iodometric andacidometric assays are based upon the colorimetric detection of penicillinoic acidproduced from β-lactamase hydrolysis of penicillin [5960] In the acidometricassay hydrolysis of citrate-buffered penicillin causes a decrease in pH and acolor change of phenol red to yellow In the iodometric assay penicillinoic acidreduces iron and prevents the interaction of iron with starch Without the hydroly-sis of penicillin to penicillinoic acid iron and starch interact and the mediumturns bluish purple In the chromogenic assay the chromogenic cephalosporinnitrocefin can either be incorporated into liquid suspension or impregnated intosterile paper disks Hydrolysis of nitrocefin results in an electron shift and a redproduct [61] The disk chromogenic assay commercially known as the cefinasedisk is the most common method used for β-lactamase detection in clinical labo-ratories

338 Lister

TA

BLE

4A

ssay

sto

Det

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edia

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mic

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nce

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esis

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anis

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Hae

mo

ph

ilus

infl

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zae

Mo

ra-

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smid

-en

cod

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lact

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enic

illin

oic

acid

xella

cata

rrh

alis

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nte

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spp

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eiss

eria

go

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eae

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pH

chan

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ph

eno

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tap

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sau

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nndashs

tarc

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hro

mo

gen

icn

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cefi

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spen

sio

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pre

gn

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pap

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del

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shif

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The direct detection of β-lactamase in species of staphylococci entero-cocci H influenzae Neisseria gonorrhea and Moraxella catarrhalis indicatesresistance to all penicillin antibiotics but does not indicate resistance to all thecephalosporins Furthermore a negative reaction does not rule out penicillin re-sistance because some strains are resistant to penicillins through altered PBPsandor decreased permeability [62] Another consideration is the inducibility ofpenicillinase production in staphylococci Unlike the constitutive productionof β-lactamase by H influenzae most staphylococci produce detectable levelsof their penicillinase only after induction with a β-lactam drug [63] Thereforeany negative results from uninduced cultures of staphylococci must be repeatedwith cultures grown in the presence of subinhibitory concentrations of a β-lactamantibiotic

332 Detection of CAT

The majority of chloramphenicol resistance is mediated through the enzymechloramphenicol acetyltransferase (CAT) [64] A rapid method for detectingCAT in cultures of H influenzae has been described [65] and is available com-mercially as a kit This assay has also been used to detect the production of CATin S pneumoniae [66] and Salmonella spp [67] In this assay acetyl coenzymeA is converted to hydrogen sulfide coenzyme A during the reaction of CAT withchloramphenicol The free sulfhydryl of coenzyme A then reacts with 55prime-dithi-obis(2-nitrobenzoic acid) and a resultant color change from pale yellow to deepyellow is observed Similar to β-lactamase detection assays a negative resultdoes not ensure that the bacterium is susceptible to chloramphenicol

34 Susceptibility Methods for Phenotypic Detection

of Resistance

Assays that evaluate the growth of bacteria on or in antibiotic-containing mediahave been the standard approach to susceptibility testing for decades Theseassays can be either qualitative quantitative or semiquantitative Qualitativeassays provide susceptible intermediate and resistance phenotypes but do notprovide information on how susceptible or resistant a bacterium is to antibioticsIn contrast quantitative assays provide a minimum inhibitory concentration(MIC) and thus provide information on the degree of susceptibility or resistanceSemiquantitative assays fall between the qualitative and quantitative methodsmdashthey evaluate susceptibility using a short range of antibiotic concentrationsaround an important lsquolsquobreakpointrsquorsquo concentration or a single concentration thatcan differentiate between mechanisms of resistance

341 In Vivo Correlation of Susceptibility Assays

The primary criticism of any in vitro susceptibility test is that the in vivo environ-ment is not simulated and that the assays are artificially evaluating antimicrobial

340 Lister

susceptibility The four most important criticisms focus on the growth of bacteriain nutrient-rich media the size of starting inocula the absence of serum proteinsthat bind antibiotics and the lack of pharmacodynamic correlations

Growth In or On Artificial Media When the interactions of an antibioticwith a bacterium are evaluated in vitro the bacteria are usually growing logarith-mically in or on nutrient-rich media This is in contrast to the slow growth orstasis governed by the nutrient-deprived environment of most infections There-fore cells that are growing rapidly in vitro may appear more susceptible togrowth-phase-dependent antibiotics such as β-lactam antibiotics than theywould actually be in the infected patient In addition other factors such as pHosmolarity and levels of gas exchange which can play an important role in theinteractions of antibiotics with bacteria may also be misrepresented in broth cul-tures and in the growing bacterial colony [68]

Inoculum Size Because the load of bacteria can vary substantially frompatient to patient and between different sites of infection it is difficult to definea clinically relevant inoculum Therefore scientists have chosen to focus theirattention on standardizing inocula so that the reproducibility of data is maximized[6970] Unfortunately accurate reproducibility is best achieved through reducingthe inoculum size to provide a more uniform bacterial population By reducingthe inoculum size one also increases the apparent activity of some antibiotics[6871] and limits the ability to detect resistant subpopulations that may causetherapeutic failure [72 73]

Regardless of the inoculum size selected however the methods used toachieve the standard inoculum are also important Standard inocula can be ob-tained either by growing the bacteria in broth to a desired turbidity or throughthe suspension of colonies from agar cultures to achieve a desired turbidity Ithas been suggested that inocula be prepared from at least four or five coloniesto ensure that all phenotypes are present [74] However others have suggestedthat five colonies is less than adequate to represent the clinical situation [75]

Protein Binding The degree of protein binding can vary widely betweenand within the different classes of antibiotics For example just among the β-lactam antibiotics percent protein binding can range from 5 to 90 [76]Although the degree of protein binding has been shown to influence the move-ment of antibiotics from the bloodstream to extravascular spaces [77] the impactof protein binding on antibacterial activity remains unsettled The interaction be-tween antibiotics and serum proteins is a dynamic reversible process with drugmolecules constantly binding and unbinding at a rapid rate [78] In studies withciprofloxacin and ofloxacin which exhibit 20ndash40 binding to serum proteinsthe addition of 40ndash50 serum to MIC assays did not affect the potency of thesefluoroquinolones [79ndash82] In studies with trovafloxacin which exhibits 70 pro-


tein binding intra- and interspecies variations have been observed ranging fromno effect at all to significant reductions in potency in the presence of serumproteins [83] Just as conflicting are data from studies with cefonicid which ishighly protein-bound and exhibits a significant decrease in in vitro potency inthe presence of serum proteins [84] Although protein binding has been used toexplain clinical failures with cefonicid when the pathogens are susceptible invitro [85] the efficacy of cefonicid against K pneumoniae was actually enhancedby the administration of excess serum albumin to mice [86] The best explanationfor this observation is that the binding of cefonicid to the excess serum albuminextended its elimination half-life and increased the time for which concentrationsremained above the MIC

Pharmacodynamic Correlations One of the major drawbacks of mostsusceptibility assays is that they evaluate only the inhibition of visible growthWith the exception of broth dilution methodologies susceptibility assays cannotdifferentiate bacteriostasis from bactericidal killing and this differentiation canbe critical for successful treatment of some serious infections Another drawbackof in vitro susceptibility assays is that they evaluate antibioticndashbacterium interac-tions using static concentrations over limited periods of time In contrast duringthe treatment of an infection bacteria are exposed to constantly changing concen-trations of antibiotic over several days Therefore important pharmacodynamicinteractions may be missed during routine susceptibility assays especially theoutgrowth of slow-growing mutant subpopulations that could result in therapeuticfailure in the patient

Whereas it is obvious that in vitro susceptibility assays are far from perfectand fall far short of mimicking the in vivo environment the data obtained fromthem can be useful if their limitations are recognized and more attention is di-rected toward their accurate detection of antibacterial resistance The salient as-pects of currently used quantitative semi quantitative and qualitative assays willbe reviewed

342 Quantitative Assays

The four quantitative assays used in research and clinical laboratories are themacrobroth dilution assay microbroth dilution assay agar dilution assay and E-test These assays provide MIC data that can be directly compared to achievabledrug concentrations in the patient especially at the site of infection Thereforequantitative assays provide clinicians with the type of information that allowsthem to optimize therapeutic strategies to not only help the patient but also slowthe emergence of resistance

Macrobroth Dilution The macrobroth dilution assay is the most conser-vative of the quantitative assays and is considered the gold standard against whichall susceptibility tests are evaluated One advantage of macrobroth dilution assays

342 Lister

is that they allow evaluation of bacterial killing as well as evaluation of the MICHowever because of the laboriousness of this method its use in clinical labora-tories is not practical To perform a macrobroth dilution assay twofold serialdilutions of antibiotic are prepared in 1ndash3 mL of broth media and each tube isinoculated with 5 105 colony-forming units (CFU) per milliliter of the testbacterial culture [87] Therefore the total number of bacteria inoculated into eachtube ranges from 5 105 to 1 106 CFU Although the macrobroth dilutionassay is the most reliable for detecting resistance among clinical isolates thestarting inoculum is often insufficient to evaluate the presence of mutant subpopu-lations that could cause therapeutic failures

Microbroth Dilution Because of the laboriousness of macrobroth dilutionassays a microbroth dilution version was developed This assay is performed insmall plastic trays with individual wells containing 50ndash100 microL of media andtwofold serial dilutions of antibiotic Although microdilution assays also use start-ing inocula of approximately 105 CFUmL the total number of bacteria inocu-lated into each well is only 104 CFU Considering the established relationshipbetween inoculum size and susceptibility it is not surprising that bacteria appearto be more susceptible in microdilution assays than in macrodilution assays Fur-thermore with an even lower starting inoculum microdilution assays are evenless likely to detect resistant subpopulations that could cause therapeutic failure

Agar Dilution The basic principle of the agar dilution assay is similarto that of broth dilution methods with the exception that drug is incorporatedinto an agar medium and bacteria are inoculated on top of the agar One majorlimitation of agar dilution assays is that they can only evaluate the inhibition ofvisible growth and cannot distinguish bacteriostasis from bacterial killing Likemicrobroth dilution assays agar dilution assays are hindered by a starting inocu-lum of only 104 CFU which make bacteria appear more susceptible than withmacrobroth methods [88ndash90] Furthermore the detection of resistant subpopula-tions is even less likely with agar dilution assays than with either of the brothdilution methods The reason for this is that one resistant cell will grow into onlya single colony on an agar plate and this single mutant colony would likely beignored In contrast one mutant cell in a broth culture would eventually growto turbidity and would not likely be ignored

E-test The E-test is a quantitative assay that represents a marriage be-tween agar dilution and the disk diffusion assay discussed in Section 345 Inthis test a standard inoculum of bacteria (05 McFarland turbidity) is used tocreate a confluent lawn of growth on an agar plate Onto this lawn of bacteriaare placed plastic-coated strips impregnated with a continuous and exponentialgradient of antibiotic concentrations As antibiotic diffuses from the E-test stripinto the agar medium it interacts with the lawn culture of bacteria In either the


absence of drug or presence of subinhibitory drug concentrations confluentgrowth is observed However near the E-test strips an elliptical zone of inhibitionis observed reflecting the increasing concentrations of antibiotic along the stripThe MIC is obtained from the point of intersection between the ellipse of growthinhibition and the antibiotic gradient on the E-test strip Unlike in the other dilu-tion-based methods the E-test inoculum is not measured by the number of viablebacteria per milliliter or spot However the density of the inoculum still playsa critical role in the data obtained If the inoculum is too light an unwantedadvantage is provided to the drug diffusing from the E-test strip thus resultingin larger zones of inhibition and lower MICs Even with the strictest of standardprocedures bacteria appear more susceptible with E-test methodology than withbroth dilution assays

Problems Associated with Interpretive Breakpoints Once an MIC is ob-tained from a quantitative assay the next step is to determine the susceptibleintermediate or resistant phenotype of the bacterium These phenotypes are deter-mined by comparing MICs to established interpretive breakpoints When drugpharmacokinetics are considered during the establishment of interpretive criteriabreakpoints are based on the serum pharmacokinetics of the drug There are twocritical flaws with this approach The first problem is that some are known toconcentrate to much higher levels in tissues and urine than their peak concentra-tions in serum Therefore establishing susceptible breakpoints based on the se-rum pharmacokinetics of a drug that concentrates outside the bloodstream mayincorrectly classify bacteria as nonsusceptible when in fact therapeutic levelswould be achieved at the site of infection The second problem is just the converseof the first in that drug access to the central nervous system and other restrictedareas is often limited and only a fraction of serum antibiotic is able to penetrateTherefore establishing breakpoints based on serum pharmacokinetics may actu-ally overestimate the activity of some drugs for treatment of infections in inacces-sible sites This is best illustrated by the problems encountered with establishinginterpretive breakpoints for cephalosporins in the treatment of pneumococcalmeningitis As susceptibility to cephalosporins began to decrease with increasingproblems of penicillin resistance clinical failures with cephalosporins in the treat-ment of meningitis were observed despite in vitro susceptibility based on theestablished interpretive breakpoints Therefore it was apparent that the originalinterpretive breakpoints for cephalosporins did not relate to pneumococcal infec-tions within the central nervous system and re-evaluation was necessary

Even when interpretive breakpoints do correlate with the pharmacokineticsof a drug problems with detection of resistance can occur The best example toillustrate this problem is the dilemma of detecting ESBL-mediated resistanceamong E coli and K pneumoniae As previously discussed the MICs of ceftazi-dime and cefotaxime against ESBL-producing E coli and K pneumoniae are

344 Lister

often in the susceptible or intermediate range [91] Therefore current interpretivecriteria do not reliably identify ESBL-producing bacteria as resistant and theresults can be detrimental to the infected patient To address this problem newassays and screening criteria are being developed to increase the detection ofESBL-mediated resistance in the clinical laboratory However similar problemswill likely occur with other antibiotics and bacterial species as mechanisms ofantimicrobial resistance continue to evolve and spread in the environment

343 Automated Susceptibility Systems

There are a number of semiautomated or fully automated systems available forsusceptibility testing in clinical laboratories Most of these involve the automatedmonitoring of bacterial growth in drug-free and drug-containing wells Data areanalyzed using algorithms that are then converted into quantitative results Toevaluate multiple drugs and drug classes on a single susceptibility card thesesystems are limited in the number of drug concentrations that can be testedTherefore susceptibility is measured with just a narrow range of drug concentra-tions around a critical lsquolsquobreakpointrsquorsquo concentration and semiquantitative data areobtained The major drawback of these systems is that they rely on the assumptionthat the breakpoint concentrations selected have clinical relevance Thereforeany errors in selecting the appropriate lsquolsquobreakpointrsquorsquo concentrations can providepotentially harmful susceptibility data for patients Another disadvantage ofbreakpoint assays is they have no way of detecting declining susceptibility in theenvironment Only when susceptibility crosses the critical breakpoint is resistancedetected This creates a problem for the detection of those resistance mechanismsthat fail to increase MICs above the susceptible breakpoint ie ESBLs Onefinal problem associated with automated susceptibility systems is that they aredesigned to provide rapid results within hours rather than the usual overnightincubations associated with routine dilution assays This rapid approach has beenshown to be unreliable in detecting resistance to some antibiotics and researchcontinues to solve these problems

344 Resistance Screening Assays

Similar to the automated susceptibility systems resistance screening assays aresemiquantitative assays that evaluate growth or lack of growth at a single drugconcentration In contrast to automated systems however the concentrations cho-sen for these assays are not lsquolsquobreakpointrsquorsquo concentrations but rather concentra-tions that allow for differentiation between resistance mechanisms intrinsic andhigh-level resistance and truly susceptible and falsely susceptible populationsA summary of the screening assays for mecA-mediated methicillin resistanceamong staphylococci vancomycin and high-level aminoglycoside resistanceamong enterococci and ESBL-mediated resistance among E coli K pneumon-iae and K oxytoca is provided in Table 5


TA

BLE

5S

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Det

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ate

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Res

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esis

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ech

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mM

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Sta

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β-

Lact

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Ino

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te10

4C

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ace

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ple

-af

fin

ity

PB

P2prime

fro

mm

ecA

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ted

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h4

sod

ium

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rid

ean

d6

microgm

Lo

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llin

A

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wth

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r24

ho

fin

cub

atio

nat

37o C

isin

dic

ativ

eo

fm

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dre

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toal

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ams

En

tero

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V

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Ino

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ith

6microg

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A

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In

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106

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ith

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Pre

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cula

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Lin

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roth

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tain

ing

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leb

siel

lap

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nia

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ases

microgm

Lo

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fpo

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idim

ece

fota

xim

eK

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lao

xyto

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ftri

axo

ne

or

aztr

eon

am

Pre

sen

ceo

fvi

sib

leg

row

thaf

ter

24h

of

incu

bat

ion

issu

gg

esti

veo

fE

SB

Lp

rod

uct

ion

C

on

firm

ato

ryas

say

req

uir

ed

346 Lister

MRSA Screen The therapeutic importance of differentiating between dif-ferent mechanisms of methicillin resistance in staphylococci has been discussedBecause hyperproduction of β-lactamase or alterations in PBPs unrelated to mecAresult in borderline resistance strains expressing these mechanisms of resistanceare typically inhibited by concentrations of oxacillin that do not inhibit the growthof MRSA expressing PBP2prime An agar-based oxacillin screening assay has beendescribed that differentiates mecA-mediated resistance from other mechanismsof oxacillinmethicillin resistance [87] In this assay agar supplemented with 4sodium chloride and 6 microgmL of oxacillin is inoculated with 104 CFU of bacteriaas in agar dilution methodology Growth on this agar screen indicates that theisolate is expressing mecA Although this screening assay is not 100 accurateit does allow for the differentiation of the majority of MRSA and can be usedto guide appropriate therapy

High-Level Aminoglycoside Resistance Screen Enterococci are intrinsi-cally resistant to aminoglycosides with MICs in the range of 8ndash256 microgmL [9]Although intrinsic resistance prevents aminoglycosides from being used as singleagents for serious enterococcal infections it does not interfere with their syner-gistic interactions with cell-wall-active agents In contrast the acquisition ofhigh-level resistance is characterized by much higher MICs and does interferewith synergistic killing Broth- and agar-based screening assays have been devel-oped to detect high-level aminoglycoside resistance among enterococcal isolates[87] Due to cross-resistance the only two aminoglycosides that need to be testedare gentamicin and streptomycin In the agar screening methodology 500 microgmL of gentamicin or 2000 microgmL of streptomycin is incorporated into agar me-dium and 106 CFUspot of enterococcal isolates is inoculated onto the agar sur-face much as in agar dilution methodology After 24ndash48 h of incubation thepresence of more than one colony or hazy growth indicates high-level resistanceto aminoglycosides For broth screening purposes a microdilution theme is usedand high-level aminoglycoside resistance is detected by the visible growth of a5 105 CFUmL inoculum in the presence of 500 microgmL of gentamicin or 1000microgmL of streptomycin

Vancomycin Resistance Screen Because vancomycin resistance can below level with strains expressing the VanB and VanC phenotypes routine suscep-tibility methods may fail to detect these resistance mechanisms [9293] There-fore an agar screening method is used that evaluates the growth of enterococcalisolates on agar containing 6 microgmL of vancomycin [8793] After 24 h of incuba-tion the presence of more than one colony indicates resistance to vancomycin

ESBL Screen Although MICs of extended-spectrum cephalosporins andaztreonam against ESBL-producing bacteria do not always exceed susceptiblebreakpoints they are elevated compared to those of strains lacking ESBLs TheNCCLS recommends a broth-based screening assay for E coli K pneumoniae


and K oxytoca that evaluates the growth of these three species in the presenceof 1 microgmL of cefpodoxime ceftazidime cefotaxime ceftriaxone or aztreonam[94] Visible growth after 16ndash20 h of incubation is suggestive of ESBL produc-tion and this phenotype must then be confirmed Confirmation of ESBL produc-tion involves the measurement ceftazidime-clavulanate and cefotaxime-clavula-nate MICs and comparison with the MICs obtained with each cephalosporinalone A threefold or greater difference confirms the production of an ESBLSimilar assays are being developed for some automated systems and the E-testassay

345 Qualitative Assays

Qualitative assays provide simple lsquolsquoyesrsquorsquo or lsquolsquonorsquorsquo susceptibility informationthrough establishment of susceptible intermediate or resistant phenotypes How-ever quantitative information on the level of susceptibility is not provided

Disk Diffusion Assay The disk diffusion assay is well standardized re-producible and inexpensive and is relatively easy to perform Because of thesequalities the disk diffusion is the most common susceptibility assay used in clini-cal laboratories Although similar in methodology to the E-test disk diffusionassays use paper disks impregnated with a single drug concentration rather thanthe range of antibiotic concentrations associated with E-test strips This differenceresults in circular zones of growth inhibition the size of which ultimately deter-mines the susceptibility phenotype of the bacterium Although the susceptibilityof a bacterium to an antibiotic influences the size of the inhibition zone theinoculum incubation temperature incubation time and media consistency anddepth are all important factors that influence the size of growth inhibition zones

The NCCLS establishes the interpretive breakpoint criteria from which thesizes of growth inhibition zones are translated into susceptible intermediate andresistant phenotypes These breakpoints are established through error-rate-bounded analysis of disk diffusion and MIC data for hundreds of bacterial iso-lates The ultimate goal of any disk diffusion assay is to achieve 100 correlationbetween the phenotypes determined by lsquolsquogold standardrsquorsquo MIC assays and thoseobtained with the disk diffusion assay Because 100 correlation is not oftenattainable it becomes important to limit or prevent any false susceptibility errorswith the disk diffusion assay False susceptibility errors occur when a bacteriumappears susceptible by disk diffusion assay but is found to be truly resistant byMIC methods This type of error has also been referred to as a lsquolsquofatal errorrsquorsquobecause of the life-threatening problems it can present to patients with seriousinfections What level of false susceptibility error rates is acceptable with thedisk diffusion assay has been a controversial subject for many years

Special Disk Diffusion Assays for Detection of ESBLs Similar to prob-lems encountered with dilution-based quantitative assays the disk diffusion assay

348 Lister

TA

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kd

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is unreliable in detecting ESBL-mediated resistance Up to 75 of ESBL-produc-ing strains appear susceptible to cefotaxime and 43 appear susceptible to ceftaz-idime according to disk diffusion methodology [3195] Three modifications ofthe disk diffusion assay have been shown to provide improved detection ofESBLs in clinical isolates (see Table 6)

The three-dimensional assay [32] is performed much like the standard diskdiffusion assay The one exception is that the bacterial inoculum is introducedinto a slit in the agar as well as on top of the agar Commercial disks containingextended-spectrum cephalosporins or aztreonam are placed in close proximity tothe slit in the agar If the inoculated organism produces an ESBL drug is inacti-vated before it can diffuse through the concentrated culture in the agar slit andtruncated zones of inhibition are observed The three-dimensional assay reliablydetects 90 of ESBL-mediated resistance but is currently not suited for routinelaboratory use

The double-disk diffusion assay [31] is based upon the sensitivity of ESBLsto inhibition by clavulanic acid In this assay disks containing extended-spectrumcephalosporins or aztreonam are placed proximate to a disk containing clavulanicacid The inhibition of ESBLs by clavulanic acid results in an enhancement ofthe inhibition zone around commercial ceftazidime cefotaxime ceftriaxone oraztreonam disks Although this assay substantially improves the detection ofESBLs the optimum distance between disks required for detection varies sub-stantially from strain to strain Therefore multiple assays must be run with vary-ing distances between the clavulanate- and drug-containing disks Of even moreconcern are the reports of ESBLs that also exhibit resistance to clavulanate inacti-vation The increased prevalence of these enzymes threatens the future usefulnessof the double-disk assay and any other ESBL detection assays that require clavu-lanate inhibition of the enzymes

The NCCLS has approved a set of disk diffusion screening assays to detectESBLs in E coli K pneumoniae and K oxytoca [94] For initial screeningzones of inhibition around cefpodoxime ceftazidime cefotaxime ceftriaxoneor aztreonam disks are evaluated for diameters that fall below ESBL-indicativebreakpoints ESBL production is then confirmed by a special assay in which 10microg of clavulanate is added to commercial disks of ceftazidime and cefotaximeand a 5 mm increase in the zone of inhibition around the cephalosporin-clavula-nate disks compared to the cephalosporins alone confirms ESBL production

4 PHARMACODYNAMICS AND ANTIMICROBIAL

RESISTANCE

The field of antimicrobial pharmacodynamics strives to establish relationshipsbetween the pharmacokinetics of a drug its interaction with target bacteria andthe effective treatment of infections As previously discussed the emergence of

350 Lister

resistance can result in therapeutic failure when the original isolate is susceptibleby routine assays Therefore the relationship between antimicrobial pharmacody-namics and suppression of resistant subpopulations is also critical for optimizingtherapy of many infections

Resistance to antimicrobial agents can develop through two distinct path-ways (1) acquisition of new genes encoding resistance-mediating proteins and(2) evolutionary mutations in existing genes The impact of optimizing antimicro-bial pharmacodynamics on each of these pathways is very different Althoughthe optimization of antimicrobial pharmacodynamics can slow the evolution ofmost mutation-mediated resistance mechanisms there are many acquired resis-tance mechanisms that cannot be treated with safe doses of antibiotic Thereforethe goal of pharmacodynamics should be to optimize therapy such that mutationalresistance is prevented This section will differentiate acquired from mutationalresistance and discuss the pharmacodynamic parameters that can impact theemergence of mutational resistance during therapy

41 Acquired Versus Mutational Resistance

411 Acquired Resistance

Acquired resistance as the name suggests represents the development of resis-tance in a bacterium through the acquisition of a new gene or set of genes fromother bacteria in the environment Examples of acquired resistance mechanismsinclude plasmid-encoded β-lactamases plasmid-encoded aminoglycoside-inacti-vating enzymes acquired vancomycin resistance and methicillin resistance me-diated through mecA The common theme with these resistance problems is thatthey are passed from bacterium to bacterium on transferable genetic elementsie plasmids or transposons Once a bacterium acquires one of these resistancemechanisms the level of resistance that is expressed usually exceeds the levelsof antibiotic that can be safely achieved in serum or other sites of infection Forexample acquisition of the TEM-1 β-lactamase by an E coli can increase theMIC of ampicillin from 2 to 256 microgmL [96] Similarly acquisition of the genesresponsible for vancomycin resistance can increase the MIC of vancomycinagainst enterococci from 2 to 512 microgmL [97] Although acquisition of new genesusually results in an instantaneous conversion to high-level resistance there areexceptions to this rule It has been well established that penicillin resistanceamong pneumococci is mediated through the acquisition of PBP gene segmentsfrom other resistant streptococci in the environment However development ofhigh-level penicillin resistance does not result from a single transfer event butrather requires multiple steps affecting multiple PBPs

412 Mutational Resistance

In contrast to acquired resistance mutational resistance is mediated through ge-netic changes (point mutations) within a bacteriumrsquos own chromosome These


evolutionary mutations can alter the binding of a drug to its target decrease theaccumulation of a drug at the site of action or disrupt the regulatory genes con-trolling the production of resistance-mediating enzymes Similar to acquired re-sistance mechanisms mutational events can result in instantaneous high-levelresistance or gradual decreases in susceptibility requiring multiple steps toachieve clinically significant resistance Whether high-level resistance is achievedwith a single point mutation or requires multiple mutational events depends uponthe specific drug and the specific target bacterium In fact differences betweendrugs within the same family can be observed This point is best illustrated usingthe example of β-lactam resistance in gram-negative bacteria as mediated throughderepression of the ampC operon

As previously discussed derepression of the ampC results from point muta-tions within a regulatory gene ampD resulting in a loss of the negative controlof ampC transcription [38] Once negative control is lost AmpC production in-creases significantly and the bacteria become resistant to virtually all β-lactamantibiotics Such mutations occur in one out of every 106ndash107 viable bacteriaTherefore one or more highly resistant mutants are likely to be present at thestart of therapy of some infections If these mutants are not eliminated by thehost or killed by the antibiotic they can eventually become the predominantpopulation and lead to therapeutic failure Clinical failure due to the emergenceof this resistance problem has been observed in 19ndash80 of patients infected withbacteria possessing inducible AmpC cephalosporinases [6] Although almost anyβ-lactam antibiotic can select for these mutants there appears to be a particularassociation with the lsquolsquothird generationrsquorsquo cephalosporin ceftazidime [2]

In contrast a single mutational event is not always sufficient to provideresistance to the lsquolsquofourth generationrsquorsquo cephalosporin cefepime especially amongthe Enterobacteriaceae In a 1994 survey of five medical centers in the UnitedStates cefepime was tested against 256 ceftazidime-resistant clinical isolates[98] Among the Enterobacter in this study almost all species were 100 suscep-tible to cefepime The only exception was E cloacae of which 94 of the ceftaz-idime-resistant isolates remained susceptible to cefepime In a separate study100 of Enterobacter strains with proven ampC derepression were susceptibleto cefepime [99] The activity of cefepime against ceftazidime-resistant Entero-bacter spp can be explained by the differing number of mutational steps requiredto achieve resistance with each of these drugs In contrast to the single mutationalevent required for ceftazidime resistance it takes two independent mutationalevents to achieve cefepime resistance among Enterobacter spp [100] First thelevel of AmpC production must be increased through derepression of the ampCoperon and then the penetration of cefepime through the outer membrane mustbe slowed or prevented through a second mutational event Since each of thesemutational steps occurs in one out of every 106ndash107 viable bacteria it is unlikelythat resistance will develop in a ceftazidime-susceptible enterobacter during thecourse of therapy with cefepime This hypothesis is supported by data from a

352 Lister

murine infection model [101] However once an enterobacter mutates to a stateof ampC derepression and bacterial numbers increase above 106 the chances ofselecting the second permeability mutation increases and emergence of cefepimeresistance becomes a real threat

Differences between antibiotics in the number of mutational steps requiredto achieve clinically relevant resistance is not unique to the β-lactam class assimilar observations have been reported with other drug classes as well particu-larly the fluoroquinolones [102] Although appropriate dosing of antibiotics doesnot often impact problems associated with acquired resistance pharmacodynam-ics can play an influential role in slowing the development of mutational resis-tance especially when first step mutations do not result in high-level resistanceTherefore the relationship between antimicrobial pharmacodynamics and theemergence of mutational resistance is essential for developing optimum therapeu-tic strategies

42 Antimicrobial Pharmacodynamics and Emergence

of Resistance

The three best characterized pharmacodynamic parameters influencing the clini-cal efficacy of antibiotics are the time antibiotic concentrations remain above theMIC (T MIC) the ratio of peak concentrations to the MIC (peakMIC) andratio of the area under the concentration curve to the MIC (AUCMIC) Whichpharmacodynamic parameter is most predictive of clinical efficacy depends uponthe class of drugs being used For example with β-lactam antibiotics the T MIC is the pharmacodynamic parameter that most influences clinical outcomewhereas with fluoroquinolones the most important parameter can either be thepeakMIC ratio or the AUCMIC ratio Further complicating therapeutic strate-gies is the observation that the pharmacodynamic parameter that most influencesoverall efficacy may not be the most important parameter impacting the emer-gence of resistance during therapy With the data accumulated to date it appearsthat the peakMIC ratio is the most critical pharmacodynamic parameter dictatingwhether resistance will emerge during therapy with virtually all drug classes

421 PeakMIC Ratio and Emergence of ResistanceDuring Therapy

The likelihood of a bacterium undergoing two mutational events during thecourse of therapy is very small Therefore to prevent the emergence of resistanceduring therapy the pharmacodynamic focus must be on effectively treating boththe original isolate and any first-step mutants present within the initial bacterialpopulation This demands that the concentration of antibiotic at the site of infec-tion exceed the MIC for the first-step mutant Since neither T MIC nor AUCMIC directly addresses this need the peakMIC ratio becomes the pharmacody-


namic parameter that can most impact selection of resistance Indirect evidenceto support this hypothesis has come from studies in an animal model of infection[103] Using a neutropenic rat model of Pseudomonas sepsis Drusano and col-leagues evaluated the effects of dose fractionation of lomefloxacin on survivalAlthough the emergence of resistance was not directly evaluated in this studythe peakMIC ratio was most closely linked to survival when the ratios exceeded101 Their hypothesis to explain these results was that sufficient peakMIC ratiosresulted in suppression of mutant subpopulations thus preventing death due tothe emergence of resistance Similar conclusions were reached in a study of lev-ofloxacin in the treatment of human infections [104]

The best evidence supporting the importance of peakMIC ratios in pre-venting the emergence of resistance comes from studies with in vitro pharmacoki-netic models The flexibility provided by these models allows for the design ofexperiments to specifically address this issue Using a two-compartment pharma-cokinetic model Blaser et al [105] varied the pharmacokinetics of enoxacin andnetilmicin such that bacteria were exposed to varying peakMIC ratios whilemaintaining a constant total drug exposure over 24 h These investigators ob-served the outgrowth of resistant subpopulations when simulated peakMIC ratiosfailed to exceed 81 Similarly Marchbanks et al [106] used a two-compartmentpharmacokinetic model to simulate ciprofloxacin doses of 400 mg TID (peakMIC ratio 41) and 600 mg BID (peakMIC ratio 61) and observed out-growth of resistant subpopulations of P aeruginosa When the same daily doseof 1200 mg was simulated as a single injection the 111 peakMIC ratio pre-vented the outgrowth of resistant subpopulations However in the same studypeakMIC ratios of only 61 were sufficient to prevent the emergence of resistantsubpopulations of S aureus Similarly Lacy et al [107] observed differencesbetween levofloxacin and ciprofloxacin and the emergence of resistance in experi-ments with S pneumoniae Although these investigators simulated peakMICratios of 51 with both fluoroquinolones they observed the emergence of resis-tance only in studies with ciprofloxacin Resistance did not emerge in studieswith levofloxacin despite peakMIC ratios as low as 11 These data suggest thatalthough the peakMIC ratio exerts an important influence on the emergence ofresistance during therapy the minimum peakMIC ratio needed to prevent thisproblem will likely vary depending upon the drug used and the bacterium tar-geted This conclusion is supported with data from my own pharmacodynamicstudies of P aeruginosa

422 Studies with Levofloxacin Against P aeruginosa

A two-compartment pharmacokinetic model was used to simulate the serum phar-macokinetics of a once daily 500 mg dose of levofloxacin and evaluate pharmaco-dynamic interactions against a panel of six P aeruginosa strains Three of thestrains evaluated were fully susceptible to levofloxacin (MIC 05 microgmL) and

354 Lister

three strains were borderline susceptible (MIC 2 microgmL) The peak concentra-tion simulated for the 500 mg dose was 6 microgmL which provided peakMICratios of 131 for the fully susceptible strains and 31 for the borderline-suscepti-ble strains Levofloxacin was dosed into the model at 0 and 24 h The pharmaco-dynamics of levofloxacin against representative susceptible and borderline-sus-ceptible strains of P aeruginosa are shown in Fig 1 In studies with the fullysusceptible strains levofloxacin exhibited rapid and significant bactericidal activ-ity with viable counts falling to undetectable levels (10 CFUmL) within 4ndash12 h after the first dose In studies with the borderline-susceptible strains lev-ofloxacin also exhibited significant bacterial killing However killing was ob-served only over the initial 6ndash8 h with viable counts increasing rapidly thereafterdue to the outgrowth of resistant subpopulations By 24 h viable counts in thelevofloxacin-treated cultures were approaching those in the drug-free control cul-tures The MICs of levofloxacin against the resistant subpopulations ranged from8 to 16 microgmL Therefore emergence of resistance could have been predictedbecause peak concentrations with each dose of levofloxacin never exceeded theMICs for these mutant subpopulations

423 Studies with Ceftazidime and Ticarcillin AgainstP aeruginosa

A two-compartment pharmacokinetic model was used to investigate the relation-ships between mutational frequencies pharmacodynamics and the emergence ofresistance during the treatment of P aeruginosa with ticarcillin and ceftazidimeMutational frequency studies were performed in agar by exposing logarithmicphase cultures of P aeruginosa 164 to ticarcillin and ceftazidime at concentra-tions two- four- and eightfold above their respective MICs The initial MICsfor ticarcillin and ceftazidime against P aeruginosa 164 were 16 microgmL and 2microgmL respectively Analysis of mutational frequencies showed there was oneresistant mutant present among every 106ndash107 parent cells with MICs of ticarcil-lin and ceftazidime against these mutants increasing to 512 microgmL and 32 microgmL respectively Using a two-compartment pharmacokinetic model the kineticsof a 30 g QID dose of ticarcillin and 20 g TID dose of ceftazidime were simu-lated and their pharmacodynamics against P aeruginosa 164 were evaluatedover 24 h With peak concentrations of 260 microgmL for ticarcillin and 140 microgmL for ceftazidime peakMIC ratios were 161 for ticarcillin and 701 for cefta-zidime The observed pharmacodynamic interactions are shown in Fig 2 In stud-ies with ceftazidime viable counts decreased over 3 logs throughout the 24 hexperimental period and no resistant mutants were detected on drug selectionplates The lack of resistance emergence was not unexpected because peak con-centrations achieved with the 20 g dose of ceftazidime were fourfold above the32 microgmL MIC of first-step mutants In contrast outgrowth of a resistant mutantwas observed in studies with ticarcillin despite simulated peakMIC ratios of


FIGURE 1 Pharmacodynamics of (A) levofloxacin against P aeruginosaGB240 and (B) P aeruginosa GB65 in an in vitro pharmacokinetic model Le-vofloxacin MICs were 05 microgmL for P aeruginosa GB240 and 20 microgmL forP aeruginosa GB65 Levofloxacin was dosed at 0 and 24 h and the pharmaco-kinetics of a 500 mg dose were simulated Each datum point represents themean number of viable bacteria per milliliter of culture for duplicate experi-ments Error bars show standard deviations

356 Lister

FIGURE 1 Continued


FIGURE 2 Pharmacodynamics of ticarcillin and ceftazidime against P aerugi-nosa 164 in an in vitro pharmacokinetic model Ticarcillin and ceftazidimeMICs were 16 microgmL and 20 microgmL respectively Ticarcillin was dosed at 06 12 and 18 h and the pharmacokinetics of a 30 g dose were simulatedover each 6 h dose interval Ceftazidime was dosed at 0 8 and 16 h and thepharmacokinetics of a 20 g dose were simulated over each 8 h dose intervalEach datum point represents the mean number of viable bacteria per milliliterof culture for duplicate experiments Error bars show standard deviations

358 Lister

161 against P aeruginosa 164 The outgrowth of these mutant subpopulationscould have been predicted because peak concentrations of ticarcillin never ex-ceeded the MIC against the resistant subpopulation

These pharmacodynamic data and data from other investigators highlightthe importance of the peakMIC ratio in dictating whether emergence of resis-tance will complicate therapy Unfortunately these studies also demonstrate thatthe optimum peakMIC ratio needed to prevent the emergence of resistance variesbetween different drug classes and with different bacteriandashdrug combinationsFurthermore these studies suggest that optimization of therapy cannot just bedirected toward the lsquolsquoparentrsquorsquo isolate Rather optimization of therapy to preventemergence of resistance during therapy requires a greater understanding of thequantitative influence that different resistance mechanisms exert on susceptibilityIn other words the relationship between peak concentrations and the MIC againstfirst-step mutants is more important in preventing the emergence of resistancethan targeting peakMIC ratios based on MICs against the original isolate

5 CONCLUDING REMARKS

Antibacterial resistance is an inevitable consequence of the overuse of antibioticsin the environment The emergence of multidrug resistance among prominentgram-positive and gram-negative pathogens presents serious therapeutic prob-lems not only for clinicians but also for the clinical microbiologists who haveto reliably detect new resistance mechanisms in the laboratory Where assays failto detect resistance new assays are being developed and provide some hope forthe future However the reliable detection of resistance gains nothing for theclinician faced with dwindling therapeutic options How then do we curve theevolution of antibiotic resistance among bacteria and once again take control ofthe ongoing resistance battle

As the number of stronger or truly novel antimicrobial agents achievingFDA approval declines scientists must continue to develop strategies to slow theemergence of resistance in the environment One approach is to control the useof antibiotics and to rotate antibiotics before resistance becomes a problem Thistheory is referred to as antibiotic cycling [108] A further step that can be takenis to pharmacodynamically optimize antimicrobial therapy such that the evolutionof mutational resistance is slowed This requires dosing antibiotics such that peakconcentrations at the site of infection exceed the MICs of the parent and anyresistant subpopulations that might be present Through the combination of judi-cious antimicrobial use and pharmacodynamically based optimization of dosingthe evolution of antibiotic resistance can be slowed and the lsquolsquopostantibiotic erarsquorsquoneed not become a reality


REFERENCES

1 SD Holmberg SL Solomon PA Blake Health and economic impacts of antimicro-bial resistance Rev Infect Dis 198791065ndash1078

2 JW Chow MJ Fine DM Shlaes JP Quinn DC Hooper MP Johnson R RamphalMM Wagener DK Miyashiro VL Yu Enterobacter bacteremia Clinical featuresand emergence of antibiotic resistance during therapy Ann Int Med 1991115585ndash590

3 CE Phelps Bugdrug resistance Sometimes less is more Med Care 198927194ndash203

4 JE McGowan Antimicrobial resistance in hospital organisms and its relations toantibiotic use Rev Infect Dis 198351033ndash1048

5 DCE Speller The clinical impact of antibiotic resistance J Antimicrob Chemother198822583ndash586

6 CC Sanders WE Sanders Jr β-lactam resistance in gram-negative bacteria Globaltrends and clinical impact Clin Infect Dis 199215824ndash839

7 KS Thomson AM Prevan CC Sanders Novel plasmid-mediated β-lactamases inEnterobacteriaceae Emerging problems for new β-lactam antibiotics In JS Rem-ington MN Swartz eds Current Clinical Topics in Infectious Diseases CambridgeMA Blackwell Science 1996151ndash163

8 J Robert C Moellering Enterococcus species Streptococcus bovis and Leuconos-toc species In GL Mandell JE Bennett R Dolin eds Principles and Practice ofInfectious Diseases 4th ed New York Churchill Livingstone 19951826ndash1835

9 GM Eliopoulos Antibiotic resistance in Enterococcus species An update In JSRemington ed Current Clinical Topics in Infectious Diseases Cambridge MABlackwell Science 199621ndash51

10 WW Spink V Ferris Quantitative action of penicillin inhibitor from penicillin-resistant strains of staphylococci Science 1945102221

11 RN Jones EN Kehrberg ME Erwin Prevalence of important pathogens and antimi-crobial activity of parenteral drugs at numerous medical centers in the UnitedStates I Study on the threat of emerging resistances Real or perceived DiagnMicrobiol Infect Dis 199419203ndash215

12 M Barber Methicillin-resistant staphylococci J Clin Pathol 19611438513 BM Hartman A Tomasz Low-affinity penicillin binding protein associated with

beta-lactam resistance in Staphylococcus aureus J Bacteriol 198415851314 RN Jones AL Barry RV Gardiner The prevalence of staphylococcal resistance

to penicillinase-resistant penicillins A retrospective and prospective trial of isolatesfrom 40 medical centers Diagn Microbiol Infect Dis 198912383ndash394

15 K Kiramatsu H Hanaki T Ino K Yabuta T Oguri FC Tenover Methicillin-resis-tant Staphylococcus aureus clinical strain with reduced vancomycin susceptibilityJ Antimicrob Chemother 199740135ndash136

16 CDC Staphylococcus aureus with reduced susceptibility to vancomycinndashUnitedStates 1997 MMWR 199746765ndash766

17 JW Kislak LMB Razavi AK Daly M Finland Susceptibility of pneumococci tonine antibiotics Am J Med Sci 1965250262ndash268

360 Lister

18 PC Applebaum A Bhamjee JN Scragg AJ Hallett AF Bowen RC Cooper Strep-tococcus pneumoniae resistant to penicillin and chloramphenicol Lancet 1977ii995ndash997

19 S Naraqi GP Kirkpatrick S Kabins Relapsing pneumococcal meningitis Isolationof an organism with decreased susceptibility to penicillin G J Pediatr 197485671ndash673

20 VJ Howes RG Mitchell Meningitis due to relatively penicillin-resistant pneumo-coccus Br Med J 19761996

21 JE Mortensen MR Jacobs LM Koeth Susceptibility of Streptococcus pneumoniaeand Haemophilus influenzae in 1997 Results of a hospital-based US study InAbstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemo-therapy Abstr C-19 Washington DC Am Soc Microbiol 1998

22 CG Dowson A Hutchison JA Brannigan RC George D Hansman J Linares ATomasz JM Smith BG Spratt Horizontal transfer of penicillin-binding proteingenes in penicillin-resistant clinical isolates of Streptococcus pneumoniae Proc NatAcad Sci USA 1989868842ndash8846

23 CG Dowson TJ Coffey C Kell RA Whiley Evolution of penicillin resistance inStreptococcus pneumoniae The role of Streptococcus mitis in the formation of alow affinity PBP2B in Streptococcus pneumoniae Mol Microbiol 19939635ndash643

24 PD Lister Multiply-resistant pneumococcus Therapeutic problems in the manage-ment of serious infections Eur J Clin Microbiol Infect Dis 199514(1)18ndash25

25 AL Smith Antibiotic resistance in Haemophilus influenzae In EM Ayoub GHCassell WC Branche TJ Henry eds Microbial Determinants of Virulence andHost Response Washington DC Am Soc Microbiol 1990321ndash343

26 JH Jorgensen GV Doern C Thornsberry Susceptibility of multiply-resistantHaemophilus influenzae to newer antimicrobial agents Diagn Microbiol Infect Dis1988927ndash32

27 CC Sanders JP Iaconis GP Bodey G Samonis Resistance to ticarcillin-potassiumclavulanate among clinical isolates of the family Enterobacteriaceae Role of PSE-1 β-lactamase and high levels of TEM-1 and SHV-1 and problems with false sus-ceptibility in disk diffusion tests Antimicrob Agents Chemother 1988321365ndash1369

28 KS Thomson DA Weber CC Sanders J Eugene W Sanders β-Lactamase produc-tion in members of the family Enterobacteriaceae and resistance of β-lactamndashenzyme inhibitor combinations Antimicrob Agents Chemother 199034622ndash627

29 CJ Thomson SGB Amyes TRC-1 Emergence of a clavulanic acid-resistant TEMβ-lactamase in a clinical strain FEMS Microbiol Lett 199291113ndash118

30 J Blazquez M Baquero R Canton Characterization of a new TEM-type β-lacta-mase resistant to clavulanate sulbactam and tazobactam in a clinical isolate ofEscherichia coli Antimicrob Agents Chemother 1993372059ndash2063

31 V Jarlier M-H Nicolas G Fournier A Philippon Extended broad-spectrum β-lacta-mases conferring transferable resistance to newer β-lactam agents in Enterobacteri-aceae Hospital prevalence and susceptibility patterns Rev Infect Dis 198810867ndash878


32 KS Thomson CC Sanders Detection of extended-spectrum β-lactamases in mem-bers of the family Enterobacteriaceae Comparison of the double-disk and three-dimensional tests Antimicrob Agents Chemother 1992361877ndash1882

33 A Philippon R Labia GA Jacoby Extended-spectrum β-lactamases AntimicrobAgents Chemother 1989331131ndash1136

34 GA Jacoby Genetics of extended-spectrum β-lactamases Eur J Clin MicrobiolInfect Dis 1994(suppl 1)132ndash11

35 PA Bradford C Urban N Mariano SJ Projan JJ Rahal K Bush Imipenem resis-tance in Klebsiella pneumoniae is associated with the combination of ACT-1 aplasmid-mediated AmpC β-lactamase and the loss of an outer membrane proteinAntimicrob Agents Chemother 199741563ndash569

36 GA Jacoby AA Medeiros More extended-spectrum β-lactamases AntimicrobAgents Chemother 1991351697ndash1704

37 SK DuBois MS Marriott SGB Amyes TEM- and SHV-derived extended-spec-trum β-lactamases Relationship between selection structure and function J Anti-microb Chemother 1995357ndash22

38 CC Sanders β-Lactamases of gram-negative bacteria New challenges for newdrugs Clin Infect Dis 1992141089ndash1099

39 CC Sanders Chromosomal cephalosporinases reponsible for multiple resistance tonewer β-lactam antibiotics Ann Rev Microbiol 198741573ndash593

40 Y Yang P Wu DM Livermore Biochemical characterization of a β-lactamasethat hydrolyzes penems and carbapenems from two Serratia marcescens isolatesAntimicrob Agents Chemother 199034755ndash758

41 E-H Lee MH Nicholas MD Kitzkis G Pialou E Collatz L Gutmann Associationof two resistance mechanisms in a clinical isolate of Enterobacter cloacae withhigh-level resistance to imipenem Antimicrob Agents Chemother 1991351093ndash1098

42 WE Sanders CC Sanders Inducible β-lactamases Clinical and epidemiologicalimplications for use of newer cephalosporins Rev Infect Dis 198810830ndash838

43 JP Quinn AE Studemeister CA DiVincenzo SA Lerner Resistance to imipenem inPseudomonas aeruginosa Clinical experience and biochemical mechanisms RevInfect Dis 198810892ndash898

44 WE Sanders CC Sanders Do in vitro antimicrobial susceptibility tests accuratelypredict therapeutic responsiveness in infected patients In V Lorian ed Signifi-cance of Medical Microbiology in the Care of Patients 2nd ed Baltimore MDWilliams and Wilkins 1982325ndash340

45 HF Chambers Methicillin-resistant staphylococci Clin Microbiol Rev 19881173ndash186

46 RM Massanari MA Pfaller DS Wakefield GT Hammons LA McNutt RF Wool-son CH Helms Implications of acquired oxacillin resistance in the managementand control of Staphylococcus aureus infections J Infect Dis 1988158702ndash709

47 S Dutka-Malen S Evers P Courvalin Detection of glycopeptide resistance geno-types and identification to the species level of clinically relevant enterococci byPCR J Clin Microbiol 19953324ndash27

362 Lister

48 N Clark RC Cooksey BC Hill JM Swenson FC Tenover Characterization ofglycopeptide-resistant enterococci from US hospitals Antimicrob Agents Chemo-ther 1993372311ndash2317

49 S Sachiko N Clark D Rimland FS Nolte FC Tenover Detection of vancomycin-resistant enterococci in fecal samples by PCR J Clin Microbiol 1997352325ndash2330

50 JAMvd Klundert JS Vliegenthart PCR detection of genes coding for aminoglyco-side modifying enzymes In DH Persing TF Smith FC Tenover TJ White edsDiagnostic Molecular Microbiology Principles and Applications Washington DCAm Soc Microbiol 1993547ndash552

51 K Ubukata S Nakagami A Nitta A Yamane S Kawakami M Sugiura A KonnoRapid detection of the mecA gene in methicillin-resistant staphylococci by enzy-matic detection of polymerase chain reaction products J Clin Microbiol 1992301728ndash1733

52 K Murakami W Minamide K Wada E Nakamura H Teraoka S Wantanabe Iden-tification of methicillin-resistant strains of staphylococci by polymerase chain reac-tion J Clin Microbiol 1991292240ndash2244

53 G Arlet A Philippon Construction by polymerase chain reaction and use of intra-genic DNA probes for three main types of transferable beta-lactamases (TEMSHV CARB) FEMS Microbiol Lett 19916619ndash25

54 RC Levesque A Medeiros GA Jacoby Molecular cloning and DNA homology ofplasmid-mediated β-lactamase genes Mol Gen Genet 1987206252ndash258

55 GI Carter KJ Towner NJ Pearson RCB Slack Use of a non-radioactive hybridiza-tion assay for direct detection of gram-negative bacteria carrying TEM beta-lacta-mase genes in infected urine J Med Microbiol 198928113ndash117

56 PL Perine PA Totten KK Holmes EH Sng AV Ratman R Widy-Wersky HNsanze E Habte-Gabr WG Westbrook Evaluation of a DNA hybridization methodfor detection of African and Asian strains of Neisseria gonorrhoeae in men withurethritis J Infect Dis 198515259ndash63

57 FC Tenover MB Huang JK Rasheed DH Persing Development of PCR assaysto detect ampicillin resistance genes in cerebrospinal fluid samples containingHaemophilus influenzae J Clin Microbiol 1994322729ndash2737

58 C Mabilat P Courvalin Development of lsquolsquooligotypingrsquorsquo for characterization andmolecular epidemiology of TEM β-lactamases in members of the family Entero-bacteriaceae Antimicrob Agents Chemother 1990342210ndash2216

59 BW Catlin Iodometric detection of Haemophilus influenzae beta-lactamase Rapidpresumptive test for ampicillin resistance Antimicrob Agents Chemother 19757265ndash270

60 J Escamilla Susceptibility of Haemophilus influenzae to ampicillin as determinedby use of a modified one-minute beta-lactamase test Antimicrob Agents Chemother19769196ndash198

61 CH OrsquoCallaghan A Morris SM Kirby AH Shingler Novel method for detectionof β-lactamase by using a chromogenic cephalosporin substrate Antimicrob AgentsChemother 19721283ndash288

62 GV Doern HH Jorgensen C Thornsberry DA Preston T Tubert JS Redding LAMaher National collaborative study of the prevalence of antimicrobial resistance


among clinical isolates of Haemophilus influenzae Antimicrob Agents Chemother198832180ndash185

63 KGH Dyke Beta-lactamases of Staphylococcus aureus In JMT Hamilton-MillerJT Smith eds Beta-Lactamases London Academic Press 1979291ndash310

64 MC Roberts CD Swenson LM Owens AL Smith Characterization of chloram-phenicol resistant Haemophilus influenzae Antimicrob Agents Chemother 198018610ndash615

65 P Azemun T Stull M Roberts AL Smith Rapid detection of chloramphenicolresistance in H influenzae Antimicrob Agents Chemother 198120168ndash170

66 HW Matthews CN Baker C Thornsberry Relationship between in vitro suscepti-bility test results for chloramphenicol and production of chloramphenicol acetyl-transferase by Haemophilus influenzae Streptococcus pneumoniae and Aerococcusspecies J Clin Microbiol 1988262387ndash2390

67 L de la Maza SIL Miller MJ Ferraro Use of commercially available rapid chlor-amphenicol acetyl-transferase test to detect resistance in Salmonella spp J ClinMicrobiol 1990281867ndash1869

68 D Amsterdam Susceptibility testing of antimicrobials in liquid media In V Lorianed Antibiotics in Laboratory Medicine Baltimore Williams and Wilkins 199153ndash105

69 D Greenwood In vitro veritas Antimicrobial susceptibility tests and their clinicalrelevance J Infect Dis 1981144380ndash385

70 CC Sanders ARTs versus ASTs Where are we going J Antimicrob Chemother199128621ndash622

71 KS Thomson JS Bakken CC Sanders Antimicrobial susceptibility testing withinthe clinic In MRW Brown P Gilbert eds Microbiological Quality Assurance AGuide Towards Relevance and Reproducibility of Inocula New York CRC Press1995

72 JH Jorgensen Antimicrobial susceptibility testing of bacteria that grow aerobicallyIn JA Washington ed Infectious Disease Clinics of North America LaboratoryDiagnosis of Infectious Diseases Philadephia WB Saunders 1993393ndash409

73 CC Sanders KS Thomson PA Bradford Problems with detection of β-lactam resis-tance among nonfastidious gram-negative bacilli Infect Dis Clin N Am 19937411ndash425

74 C Thornsberry Antimicrobial susceptibility testing General considerations In ABalows WJ Hausler KL Herrmann HD Isenberg HJ Shadomy eds Manual ofClinical Microbiology Washington DC Am Soc Microbiol 19911059ndash1064

75 RB Thomson TM File RA Burgoon Repeat antimicrobial susceptibility testingof identical isolates J Clin Microbiol 1989271108ndash1111

76 WA Craig B Suh Protein binding and the antimicrobial effects Methods for deter-mination of protein binding In V Lorian ed Antibiotics in Laboratory MedicineBaltimore Williams and Wilkins 1991367ndash402

77 R Wise AP Gillet B Cadge Influence of protein binding on the tissue levels of6 β-lactams J Infect Dis 198014277

78 M Barza H Vine L Weinstein Reversibility of protein binding of penicillins Anin vitro study employing a rapid diafiltration process Antimicrob Agents Chemo-ther 19721427ndash432

364 Lister

79 J Blaser MN Dudley D Gilbert SH Zinner Influence of medium and method onthe in vitro susceptibility of Pseudomonas aeruginosa and other bacteria to ci-profloxacin and enoxacin Antimicrob Agents Chemother 198629927ndash929

80 NX Chin HC Neu Ciprofloxacin a quinolone caraboxylic acid compound activeagainst aerobic and anaerobic bacteria Antimicrob Agents Chemother 198425319ndash326

81 JF Chantot A Bryskier Antibacterial activity of ofloxacin and other 4-quinolonederivatives In vitro and in vivo comparison J Antimicrob Chemother 198516475ndash484

82 T Kumada HC Neu In vitro activity of ofloxacin a quinolone carboxylic acidcompared to other quinolones and other antimicrobial agents J Antimicrob Chemo-ther 198516563ndash574

83 J Child J Andrews F Boswell N Brenwald R Wise The in-vitro activity of CP99219 a new naphthyridone antimicrobial agent A comparison with other fluoro-quinolone agents J Antimicrob Chemother 199535869ndash876

84 MN Dudley CH Nightingale R Quintiliani RC Tilton In vitro activity of cefo-nicid ceforanide and cefazolin against Staphylococcus aureus and Staphylococcusepidermidis and the effect of human serum J Infect Dis 1983148178

85 HF Chambers J Mills TA Drake MA Sande Failure of a once-daily regimen ofcefonicid for treatment of endocarditis due to Staphylococcus aureus Rev InfectDis 19846 (suppl 4)S870ndashS874

86 D Andes R Walker S Ebert WA Craig Increasing protein binding of cefonicidenhances its in-vivo activity in an animal infection model Abstr A81 Programand Abstracts of the 34th Interscience Conference on Antimicrobial Agents andChemotherapy Washington DC Am Soc Microbiol 1994146

87 National Committee for Clinical Laboratory Standards Methods for dilution anti-microbial susceptibility tests for bacteria that grow aerobically Approved StandardM7-A4 Villanova PA National Committee for Clinical Laboratory Standards1997

88 PA Bradford CC Sanders Use of a predictor panel for development of a new diskfor diffusion tests with cefoperazone-sulbactam Antimicrob Agents Chemother199236394ndash400

89 FH Kayser G Morenzoni F Homberger Activity of cefoperazone against ampicil-lin-resistant bacteria in agar and broth dilution tests Antimicrob Agents Chemother19822215ndash22

90 M Rylander JE Brorson J Johnsson R Norrby Comparison between agar andbroth minimum inhibitory concentrations of cefamandole cefoxitin and cefuro-xime Antimicrob Agents Chemother 197915572ndash579

91 JM Casellas M Goldberg Incidence of strains producing extended spectrum β-lactamases in Argentina Infection 198917434ndash436

92 DF Sahm L Olsen In vitro detection of enterococcal vancomycin resistance Anti-microb Agents Chemother 1990341846ndash1848

93 BM Willey BN Kreiswirth AE Simior G Williams SR Scriver A Phillips DELow Detection of vancomycin resistance in Enterococcus species J Clin Microbiol1992301621ndash1624


94 National Committee for Clinical Laboratory Standards Performance Standards forAntimicrobial Susceptibility Testing Ninth Informational Supplement M100-S9Wayne PA National Committee for Clinical Laboratory Standards 1999

95 A Philippon SB Redjeb G Fournier AB Hassen Epidemiology of extended spec-trum β-lactamases Infection 199517347ndash354

96 PA Bradford CC Sanders Development of test panel of β-lactamases expressedin a common Escherichia coli host background for evaluation of new β-lactamantibiotics Antimicrob Agents Chemother 199539308ndash313

97 DM Schlaes A Bouvet C Devine JH Schlaes S Al-Obeid R Williamson Induc-ible transferable resistance to vancomycin in Enterococcus faecalis A256 Antimi-crob Agents Chemother 198933198ndash203

98 RN Jones SA Marshall Antimicrobial activity of cefepime tested against Bushgroup 1 β-lactamase-producing strains resistant to ceftazidime A multi-laboratorynational and international clinical isolate study Diagn Microbiol Infect Dis 19941933ndash38

99 AF Ehrhardt CC Sanders β-lactam resistance amongst Enterobacter species JAntimicrob Chemother 199332(suppl B)1ndash11

100 CC Sanders Cefepime The next generation Clin Infect Dis 199317369ndash379101 B Marchou M Michea-Hamzehpour C Lucain J-C Pechere Development of β-

lactam-resistant Enterobacter cloacae in mice J Infect Dis 1987156369ndash373102 KS Thomson CC Sanders Dissociated resistance among fluoroquinolones Antimi-

crob Agents Chemother 1994382095ndash2100103 GL Drusano DE Johnson M Rosen HC Standiford Pharmacodynamics of a flu-

oroquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sep-sis Antimicrob Agents Chemother 199337483ndash490

104 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornsief V ReichlJ Natarajan M Corrado Pharmacodynamics of levofloxacin A new paradigm forearly clinical trials JAMA 1998279125ndash129

105 J Blaser BB Stone MC Groner SH Zinner Comparative study with enoxacin andnetilmicin in a pharmacodynamic model to determine importance of ratio of antibi-otic peak concentration to MIC for bactericidal activity and emergence of resis-tance Antimicrob Agents Chemother 1987311054ndash1060

106 CR Marchibanks JR McKiel DH Gilbert NJ Robillard B Painter SH ZinnerMN Dudley Dose ranging and fractionation of intravenous ciprofloxacin againstPseudomonas aeruginosa and Staphylococcus aureus in an in vitro pharmacoki-netic model Antimicrob Agents Chemother 1993371756ndash1763

107 MK Lacy W Lu X Xu PR Tessier DP Nicolau R Quintiliani CH NightingalePharmacodynamic comparisons of levofloxacin ciprofloxacin and ampicillinagainst Streptococcus pneumoniae in an in vitro pharmacokinetic model Antimi-crob Agents Chemother 199943672ndash677

108 CC Sanders WE Sanders Cycling of antibiotics An approach to circumvent resis-tance in specialized units of the hospital Clin Microbiol Infect 19981223ndash225

16

Basic Pharmacoeconomics

Mark A Richerson

Medical Service Corps United States Navy and Department of DefensePharmacoeconomic Center Fort Sam Houston Texas

Eugene Moore

Department of Defense Pharmacoeconomic Center Fort Sam Houston Texas

1 INTRODUCTION

Changes in the economic structure of the US health care delivery system havedriven the need for pharmacists physicians and others in health care to developexpertise in evaluating the economic implications of selecting among competinghealth care programs and drug therapies With the enactment of the Tax Equityand Fiscal Responsibility Act (TEFRA) of 1983 most hospital departments in-cluding pharmacy laboratory and radiology have transitioned from revenue-generating departments to cost centers within their institutions [1] Finite eco-nomic resources for health care combined with the increasing prevalence of man-aged care rapidly emerging new medical technologies and increasing demandon the part of health care consumers has resulted in a need for decision makersto carefully allocate these limited resources to those programs and products thatprovide the most value in terms of health benefits

All views expressed are those of the authors and do not necessarily represent those of the Depart-ments of the Army Navy Air Force or those of the Department of Defense

367

368 Richerson and Moore

The medical community the pharmaceutical industry nursing and otherallied health fields have increasingly discovered the usefulness of various healtheconomic methodologies as they relate to making informed health care allocationdecisions As a result readers must understand how to interpret the ever-increas-ing amount of economic data appearing in the medical literature In a briefingto the Office of Health Economics (OHE) Prichart [2] described the Health Eco-nomics Evaluations Database (HEED) and the growth of health economics evalu-ations appearing in the literature In 1998 the HEED database contained over14000 references Between 1992 and 1996 there was a near doubling of thereferences added each year to the database [2]

So how is a chapter concerning itself with pharmacoeconomic analysisrelevant to a book about the pharmacodynamic properties of antimicrobials In-terestingly when the subject matter of published economic evaluations was ex-amined it was discovered that diseases of the circulatory system neoplasm andinfectiousparasitic diseases were the top three disease states studied between1992 and 1996 Furthermore in examining drug expenditures by therapeutic cate-gory it was found that annual total spending on antimicrobials rose 4 between1999 and 2000 and was ranked fourth within the 16 therapeutic categories evalu-ated (exceeded only by spending on cardiovascular alimentary and central ner-vous system agents) Patients with complaints associated with respiratory tractinfection constitute a large portion of those visiting primary care providers in theUnited States The 1995 National Ambulatory Medical Care Survey (NAMCS)identified acute respiratory tract infection as the most frequent principal diagnosis(31856976 in 1995) provided by office-based physicians [3] Taking into consid-eration the unique pharmacodynamic characteristics of various antimicrobials canlead to economic efficiencies in the treatment of a variety of infectious diseasestates

The purpose of this chapter is to provide the reader with an understandingof the basic principles of pharmacoeconomic analysis Specifically we will de-scribe the elements that define a pharmacoeconomic evaluation and the majortypes of analysis and attempt to demonstrate how these forms of analysis havebeen applied to the decision-making process

2 CHARACTERISTICS OF A PHARMACOECONOMIC

ANALYSIS

Pharmacoeconomics is a discipline concerned with comparing the costs and con-sequences of two or more competing alternatives The four major types of analy-sis include the evaluation of cost minimization cost effectiveness cost vs bene-fit and cost vs utility Most often pharmacoeconomic analysis is concerned withthe selection of particular drugs treatment protocols or therapeutic options Inaddition to the four basic types of analysis other frequently encountered types

Basic Pharmacoeconomics 369

of analysis include cost analysis and costndashconsequence analysis These types ofevaluations are frequently referred to as partial evaluations The basic differencebetween complete and partial evaluations is that partial analyses focus on eithercost or consequence and may or may not describe a comparison between compet-ing products or services The focus of this chapter will be on the complete formsof pharmacoeconomic analysis Although the major types of analysis differ inhow the therapeutic outcomes are quantified they all share one common elementthe inclusion of a cost measure

21 Cost and Perspective

Costs in health care economics studies are generally described as being directindirect or intangible Direct costs are further divided between direct medicalcosts and direct non-medical costs Direct medical costs are the costs of thoseitems that are used up in the course of providing medical services In the caseof pharmacoeconomic studies direct medical costs are typically associated withthe cost of drugs medical supplies laboratory tests hospitalizations and patientvisits Other direct medical costs that are particularly relevant when comparingcompeting antimicrobials include the additional costs associated with therapeuticdrug concentration monitoring treatment failure and adverse drug events

Direct non-medical costs include out-of-pocket expenses incurred by thepatient or by the patientrsquos family in the course of obtaining health care Forexample lost wages that a patient experiences while taking time away from workto attend a doctorrsquos appointment is considered a direct non-medical cost Indirectcosts from an economistrsquos viewpoint are those costs that result from lost produc-tivity The inclusion of indirect costs is controversial for a variety of reasonsparticularly the difficulty in placing a precise dollar value on productivity Whatis the dollar value of lost productivity associated with treatments directed towardchildren or stay-at-home spouses Including indirect costs assumes that a pa-tientrsquos medical condition by itself would not have resulted in lost productivityThere are a number of methods each associated with trade-offs to address theseissues No single method is universally accepted and a tremendous amount ofcontroversy remains about when and how to include indirect cost in health careeconomic studies

Intangible costs refer to those costs incurred outside the medical systemDrummond et al [4] describe a scenario where an occupational health programchanges the production cost of automobiles The increased costs associated withthe program are ultimately passed on to the purchasers of cars far removed fromthe companyrsquos new health program These costs are typically omitted from phar-macoeconomic evaluations because it is generally understood that they are usu-ally insignificant and that there is little to be gained by attempting to includethem in an analysis


The perspective of the ultimate user of the analysis should determine whichtypes of costs are included in the study From a managed care organizationrsquosperspective direct medical costs (cost of providing medical personnel equip-ment and supplies) may be of primary importance In addition to direct medicalcosts an analysis completed from a governmental or societal perspective mayneed to include patientsrsquo out-of-pocket expenses and lost productivity The majormessage for the reader is that the perspective of the analysis and the costs includedin the study must be fully understood before generalizing the results to any oneparticular setting

Evaluations of long-term drug therapies such as those for hypertension anddyslipidemia require that health care costs incurred over the multiple years ofthe study be discounted and reported in the current yearrsquos value Discounting inaddition to accounting for inflation considers the opportunity costs associatedwith investment in one option versus another As a rule of thumb discountingis required when completing an analysis that spans more than a one-year timeframe The evaluation of competing therapy options for acute infectious diseaseprocesses is somewhat straightforward in that the entire episode spans periodsranging from a few days to a few weeks and discounting is generally not requiredOn the other hand in an analysis of a more chronic infection human immunedeficiency infection for example the economic evaluation is likely to requirediscounting in order to express future expenditures in the most current yearrsquosdollar values [5]

22 Major Types of Pharmacoeconomic Evaluation

As stated previously the four major types of pharmacoeconomic evaluation in-clude cost-minimization cost effectiveness cost-benefit and cost-utility analysisEach type is unique in the way in which consequences or outcomes are measuredand compared A cost-minimization pharmacoeconomic evaluation refers to thecomparison of two or more drug alternatives where the choice of any one agentwill result in exactly the same outcome as selection of any of the others Clearlythe focus in this type of analysis becomes cost and the alternative associatedwith the lowest cost is generally the preferred agent This does not mean that theanalysis is concerned with drug acquisition cost only Other costs to considerdepending on the perspective taken in the analysis might include drug adminis-tration cost cost associated with therapeutic drug monitoring and the cost oftreating an adverse drug event Comparison of a brand name drug product andan FDA-approved AB rated generic product serves as a classic example of a cost-minimization study The comparison of two or more antimicrobials for whichrandomized clinical trials have demonstrated equal efficacy serves as anotherexample It cannot be overemphasized however that sufficient information mustbe available to demonstrate equal treatment outcomes with all the agents being


compared Too often equal efficacy is assumed without sufficient supportiveevidence

Cost-effectiveness studies employ an analytical technique that comparesboth costs and outcomes of competing agents or services This form of analysisdiffers from the others in that outcomes are measured in some form of naturalisticunit For example when comparing two drugs used to treat hypertension theoutcome of interest may be reduction in systolic and diastolic pressure In a cost-effectiveness analysis comparing two antimicrobials used to treat acute sinusitisthe outcome of interest is likely to be cure Quenzer et al [6] used complication-free cure as the outcome measure of interest when comparing the cost effective-ness of various antimicrobials used to treat lower respiratory tract infectionsThey contended that from the perspective of a managed care organization patientsatisfaction indirectly measured by a complication-free episode of care was animportant outcome of interest Life years saved is a frequently encountered out-come measure particularly when comparing drugs used for the treatment ofchronic diseases

The term lsquolsquocost-effectiversquorsquo has a very specific set of meanings As Lee andSanchez [7] point out the term has frequently been misused in the literature Itis commonly misinterpreted to mean that the least costly product (frequentlybased on drug acquisition cost only) or decision is the product or decision ofchoice From a pharmacoeconomic decision-making perspective however thereare three different situations that arise in which one decision is more cost-effec-tive than another The most intuitive situation occurs when product A is preferredover product B because it results in lower costs while also being clinically moreeffective than product B Product A would be the decision of choice and wouldbe considered dominant to product B Another fairly straightforward example isa situation where product B is discovered to be equally as effective as productA yet product A results in lower costs Again product A would be preferredThe final description of cost effectiveness is frequently overlooked In a situationwhere product A is both more costly and more effective than product B productA may still be the most cost-effective choice because the extra clinical benefitassociated with product A may be judged to be worth the additional incrementalcosts This illustrates the point that although pharmacoeconomic analysis cancontribute to more informed decisions value judgments will continue to be neces-sary in many situations

Cost-benefit analysis expresses the benefits (or consequences) of a decisionchoice in terms of dollar value By measuring the cost and benefits in the sameunits (currency) it forces decision makers to make explicit decisions aboutwhether the benefits of a drug or program choice are worth the investment (cost)This form of analysis possesses the advantage of allowing comparison of anycombination of drugs and program options available in a particular institutionCost-benefit analysis is particularly useful in situations where the health care


system is funding multiple competing programs or where treatment options arecompeting for the same finite amount of money As long as the benefits of eachchoice can be expressed in currency any combination of programs services ortherapies can be compared One of the most difficult barriers to using this formof analysis is the need to place an acceptable dollar value on a particular levelof health

Cost-utility is a form of analysis where the outcome of interest is the pa-tientrsquos preference or that of a population of patients for a particular state ofhealth status or improvement in health status Utility scores are further multipliedby expected life years to yield quality-adjusted life years (QALY) gained Interms of methodologies a cost-utility analysis is conducted in much the samefashion as a cost-effectiveness analysis Unlike cost-effectiveness analysis wheretwo or more alternatives are compared for the same condition cost-utility analysisallows the comparison of different programs or treatment options directed towarddifferent medical conditions as long as those programs or treatment options resultin changes in patient quality of life The role of cost-utility analysis is controver-sial and the measurement methods continue to evolve Although there are a vari-ety of methods and measure tools designed to collect utility scores none havebeen universally accepted

3 OUTCOMES IN PHARMACOECONOMIC ANALYSIS

Pharmacoeconomics is a discipline concerned with the costs and consequencesof selections of particular drugs treatment protocols or therapeutic options [8]Frequently the consequences of such selections in health care are termed out-comes An outcome in the strictest definition is the final consequence of anaction [9] In health care economic research and decision making there are usu-ally different types of outcome measures describing different markers of interestthat can be studied or analyzed for a given disease state [1011] Outcome mea-sures can describe the efficacy of a drug or treatment option the effectivenessor the economic benefit Outcome measures can be surrogate or intermediate(eg a laboratory value) or absolute (eg absence of disease) The best or mostvalid outcome to study varies with the disease state being analyzed and with theinterests (perspective) of the persons conducting the study Outcomes may beclinical economic or humanistic [134]

31 Outcome Measures in the Treatment

of Infectious Disease

Outcome measures of drug therapy in any disease state are generally driven bypharmacodynamic considerations In the case of infectious disease pharmacody-namics refers to the relationship between the concentration of the drug at its


target tissues and its microbiological effect [12] The microbiological effect ofinterest is of course the eradication of the pathogenic invasion and lsquolsquocurersquorsquo ofthe infection For any given antimicrobial drug administration will result in itsselective distribution to tissues in concentrations that vary according to pharma-cokinetic properties of the drug and the drugndashhostndashdisease interaction [5] Thereis also a specific minimum concentration at which the drug will produce its anti-microbial action with respect to a given pathogen In vitro this is called the mini-mum inhibitory concentration (MIC) and is a measure of the drugrsquos efficacy [5]A drugrsquos MIC is inversely related to its antimicrobial activity The lower a drugrsquosMIC the greater the drugrsquos activity against the given pathogen An antimicrobialdrugrsquos effectiveness is determined by the interplay between its pharmacokineticsand its pharmacodynamics The drug must distribute to the target tissues (site ofinfection) in concentrations that meet or exceed the MIC be capable of killingthe pathogen and do so without causing serious adverse events in the patientThe result is a measurable outcome for the treatment of the infectious diseaseExamples of outcome measures in infectious disease are provided in Table 1

In general outcomes measurement in pharmacoeconomic analysis of infec-tious disease therapy is fairly straightforward The acute nature of most infectiousdiseases (HIV being the exception) makes it easy to define a desired outcomeAn acute infection is a short-duration event with obvious measurable signs of

TABLE 1 Types of Outcomes in Pharmacoeconomic Analysis

Type of outcome Examples applicable to infectious disease

Clinical Cure of infection (confirmed by culture)Absence of opportunistic infection (in immunocom-

promise)Resolution of febrile illnessResolution of pneumonia symptoms (decreased sputum

production lsquolsquoclearingrsquorsquo of sputum color)Economic Decreased length of stay (LOS)

Decreased total health care costDecreased drug acquisition costDecreased intensity of care (nursing and ancillary ser-

vices supplies)Decreased resource utilization

Humanistic Years of life addedQuality-adjusted life years savedDecreased incidence of adverse drug reactionDecreased pain and sufferingQuality of life


morbidity and in many cases the potential for death or serious complications ifinadequately treated The treatment goal is resolution of the infection and in thecase of hospitalized patients discharge or stepdown to a less intensive therapyThe outcome measure that provides the most valuable information in a pharma-coeconomic analysis is cure of the infection Indeed most research studies indrug therapy of infectious diseases measure efficacy andor effectiveness by theprobability that the therapy will result in elimination of the infection In a sensecure of the infection can be seen as the effectiveness measure that is of greatestinterest to the patient Itrsquos frequently the reason for the encounter with the healthcare system in the first place

The health care system or the payer while concerned with clinical out-comes may also be interested in economic outcomes [1] An economic analysisdone from the payerrsquos perspective may focus on decreasing hospital lengths ofstay or intensity of care These variables are directly related to the drugrsquos clinicaloutcomes (ie drugs with greater efficacy might produce more desirable eco-nomic outcomes) but the decision makerrsquos focus might be on the economic out-come for obvious reasons In general payers look to increase economic efficien-cies so that they can provide more care for the same costs or provide the samecare at a greater lsquolsquoprofitrsquorsquo (ie less cost) [11314]

Both the patient and the health care system (or payer) are concerned withhumanistic outcomes Humanistic outcomes are those that describe the impactof a therapy on some aspect of human quality of life longevity pain and suffer-ing or emotional satisfaction [1] Quality of life can be defined as an individualrsquosoverall satisfaction with life and general personal well-being [15] Health-relatedquality of life (HRQOL) includes those aspects of quality of life that are affectedby onersquos health From the patientrsquos perspective drug therapy of infection shouldpreserve or improve HRQOL by maximizing positive humanistic outcomes andminimizing negative humanistic outcomes In acute infection the obvious payoffoccurs when the infection is resolved and the patient is discharged from the hospi-tal with no permanent sequelae In chronic infection such as that which occurswhen an individual has contracted the human immunodeficiency virus (HIV)discussion of humanistic outcomes take on more than one dimension Currenttreatment regimens such as HAART (highly active antiretroviral therapy) do noteradicate the infection but instead suppress viral activity to the point where itsimpact on the bodyrsquos T-helper cells is minimal This comes at a price that pricebeing that the patient must be willing to tolerate side effects that range fromannoying to uncomfortable to life-threatening The patient and physician mustattempt to find the trade-off between the HAART combination that offers thebest promise for longevity with the least impact on quality of life

Humanistic outcomes can be difficult to use in a pharmacoeconomic analy-sis Although most health care providers administrators and researchers agreethat it is important to assess humanistic aspects of care there is no consensus


on standard measurement techniques [8] Because of the subjective nature ofquality of life it is difficult to correlate improvements in HRQOL to clinicalimprovements [8] In the HAART example above for instance the patientrsquos viralload may be brought to undetectable levels (clinical improvement) while the pa-tient reports a decrease in HRQOL (from side effects) Another difficulty withapplying humanistic outcomes to pharmacoeconomic analysis is that it is fre-quently difficult to place an economic value on them Although everyone canagree that prolonging life is mostly desirable it is nonetheless very difficult toanswer the question What is the dollar value of an extra year of life The answeragain depends on the perspective of the answerer A third-party payer might valuethe extra year of life differently than a patient or a health care provider Entiresubdisciplines in health economics are devoted to the issue of assessing and plac-ing value on humanistic and quality of life outcomes in health care Althoughthe subjects are beyond the scope of this chapter two such areas of study arecontingent valuation methodology (CVM) [16] and time trade-off (standard gam-ble) [17] analysis Readers desiring to learn more on these subjects are directedto the vast body of information in the medical literature

4 METHODOLOGICAL CONSIDERATIONS IN

CONDUCTING PHARMACOECONOMIC ANALYSES

The validity of a pharmacoeconomic analysis is influenced a great deal by themethodology one chooses to employ in doing the analysis To be valid and usefulan analysis must use methods that are most appropriate to the disease state inquestion in the health setting of interest Selection of the type of analysis andsecuring the data inputs on which the analysis is based are critical steps in con-ducting a good sound pharmacoeconomic analysis

As stated elsewhere in this chapter there are at least four major types ofpharmacoeconomic analyses cost minimization cost-benefit cost-utility andcost effectiveness are all analysis types [18] In analyzing the pharmacoeconom-ics of an infectious disease agent or protocol one of the four types is usuallymore suitable than the others However in some cases two or more methods areequally valid Some methods may in theory produce results with greater validitybut may be too difficult to put into practice or may be judged unethical

Choice of the analysis type should be based on the goals of the analyst ororganization doing the analysis A government entity concerned with formulatingpublic policy or allocating scarce resources will have different goals and require-ments than a managed care organization desiring to enhance economic efficiencyin its hospitals Researchers seeking to advance the body of knowledge in thediscipline will have still different goals and requirements Organizations seekingto formulate public policy or allocate resources are likely to find cost-benefitanalysis to be more useful [18] If two treatment choices are deemed to be equal


in outcomes an organization is likely to find cost minimization analysis to beuseful [18] In most cases the organization is seeking to provide the most effec-tive treatment for the least expenditure In this case it is likely that cost-effective-ness analysis is most useful

Regardless of which overall analysis type is chosen there remains the ques-tion of which methodology to use in conducting the analysis The analyst has anumber of methodological techniques to choose from each with its own optimalapplications strengths and weaknesses These techniques include cost compari-sons mathematical models simulations and decision analysis

Cost comparison is the simplest methodological technique and it is mostapplicable to cost-minimization analysis This technique is also sometimes calledcost identification When all the costs of therapy are known or identified theyare tabulated and compared Because all therapies are deemed to have equal out-comes the therapy with the lowest identified cost is considered to have an eco-nomic advantage

Mathematical models and simulations are very frequently used in costeffectiveness and cost-benefit analyses [18] A mathematical model is a modelthat seeks to precisely describe in mathematical terms all of the costs and conse-quences of a particular therapeutic option Simulations are quite similar to mathe-matical models except in one critical aspect Generally speaking mathematicalmodels represent static points in time whereas simulations attempt to showchange over a time course [11] Hence a lsquolsquosimulation clockrsquorsquo (ie a mechanismto account for time change in regular increments) is a critical part of a simulationSimulations when done on a computer allow changing events to be representedgraphically perhaps making it easier for the intended audience to lsquolsquoseersquorsquo thedifferences among competing alternatives Mathematical models and simulationsare more alike than different in that they both require precise identification ofcosts and consequences in mathematical terms Generally speaking mathematicalmodels are used more often in pharmacoeconomics than simulations though afew exceptionally well done simulations can be found in the literature [19] Thereader seeking to learn more about simulations and their application to medicaldecision making should consult the references at the end of this chapter [18ndash22]

As stated above the mathematical model expresses the costs and conse-quences of treatment choices in mathematical terms When applied to cost-effec-tiveness analysis this means that the analyst must develop an equation that ex-presses as precisely as possible the relationship between the variables (inputs)that influence the costs and outcomes of each treatment course Variables willof course differ with each disease state or drug type being analyzed Some poten-tially important variables to consider when developing a mathematical modelinclude the acquisition cost of the drug the cost of administering the drug nursingcosts the probability of incurring additional visits additional visit costs the prob-ability of hospitalization hospitalization costs the probability of adverse drug


TABLE 2 Advantages and Disadvantages of Modeling

Advantages Disadvantages

Allows evaluation of multiple op- It may be difficult to validate find-tions and prediction of outcomes ings

Can be used to predict outcomes as- Decision makers unfamiliar with thesociated with rare events technique may be skeptical

Does not endanger or inconve- Data may be difficult to get andornience patients or providers apply

Costs are much lower than an obser- It usually requires assumptionsvational study

Source Adapted from Ref 18

events (ADE) the cost of ADE the probability of treatment failure the cost oftreatment failure the probability of additional laboratory monitoring the costsof additional laboratory monitoring the probability of treatment success andbiological measures of outcome

Mathematical models can be further categorized as stochastic or determin-istic [11] If none of a modelrsquos inputs involve probabilities the model is saidto be deterministic If one or more of the modelrsquos inputs involve probabilitydistributions the model is said to be stochastic For example in a pharmacoki-netic model the mathematical relationships describing the drugrsquos absorption dis-tribution and elimination are known and can be represented by specific equationsand the model is considered deterministic In a model such as one for the treat-ment of community-acquired pneumonia several variables may involve probabil-ity distributions The likelihood of successful resolution of infection of seekingretreatment after treatment failure of experiencing an ADE severe enough torequire treatment or of presenting with an infection caused by a resistant organ-ism are all governed by probabilities of occurrence and the model is consideredstochastic A stochastic model in which the inputs from one stage are dependenton the outputs from another stage is termed a Markov chain or Markov model[11]

Still another type of mathematical model which can be either stochasticor deterministic is the analytical hierarchy model This type of model is alsocalled a multiattribute utility theory (MAUT) model [2021] The key feature ofthis model is the fact that all categories of model inputs are weighted by relativeimportance to the decision maker prior to running the model For example ahealth care system choosing among new antibiotics for formulary inclusion mayplace a higher weight on the attribute lsquolsquocoverage of resistant organismsrsquorsquo thanon the attributes of price probability of ADE or cost of administration In thiscase the lsquolsquoutilityrsquorsquo or health systemrsquos valuation will have additional influence


TABLE 3 Types of Mathematical Models

Type Description

Stochastic Involves probability distributions uncertainty in val-ues of variables

Deterministic No probabilities involved all variables specificallystated and all mathematical relationships known

Analytical hierarchy Involves valuation weighting of specific model attri-butes by end users

on the modelrsquos behavior and results The types of mathematical models and theircharacteristics are described in Table 3

41 Decision Analytic Modeling

Another type of modeling that is very useful in pharmacoeconomics is decisionanalytic (DA) modeling Decision analytic models seek to quantify the expectedvalue of a particular course of action by examining the probabilities of eventsoccurring after the decision and the outcomes (payoff) that result from the event[1122] These models involve the construction of a decision tree with eachbranch representing an outcome probability The analyst can compare competingchoices and select the decision that offers the best expected value for the healthsystem In cost-effectiveness analysis the lsquolsquowinnerrsquorsquo is usually the treatment paththat yields the lowest expected value in dollars In cost-benefit analysis the lsquolsquowin-nerrsquorsquo is usually the treatment path that yields the highest expected value in dollarsA simplified example of a decision tree is shown in Fig 1

The case in Fig 1 illustrates the basic principles of decision trees In stan-dard notation a square box represents a decision node (eg treat with A or B)circles represent chance (probability) nodes and triangles represent terminal

FIGURE 1 Antibiotic A vs antibiotic B in acute infection


(payoff) nodes Multiplying the probabilities at each chance node by the payoffsand summing the results gives the expected value of each choice In the exampleantibiotic B has a 5 greater probability of cure but costs a little more for eachcourse of therapy Although it is not necessarily obvious the most cost-effectivechoice in the above example is antibiotic B Although it would appear that B ismore expensive by $25 per course of therapy when one factors in the greaterprobability of cure (assuming an equal cost of failure of $1100) the expectedvalue of B becomes $380 [(90 $300) (10 $1100)] versus an expectedvalue of $399 [(85 $275) (15 $1100)] for A Thus one can say that onaverage over a large number of cases B will actually work out to be less expensivethan A by approximately $19 per case In practice decision trees are usuallymore complex than the example shown above There may be many branchesrepresenting probabilities and associated payoffs for ADEs complications re-treatment etc Decision trees are nonetheless very useful when one can obtainthe proper data (probabilities and the associated costs) with which to construct themodel A more detailed example of a decision analysis applied to the economicevaluation of competing antibiotic regimens can be seen in a recent study byRicherson et al [23]

42 Data Sources for Pharmacoeconomic Studies

The decision on which type of model to use in a pharmacoeconomic analysis isdependent upon the data available for inputs In turn the data available for inputsdepends in many cases on the pharmacoeconomic question one is trying to an-swer Certain data lend themselves to answering some questions more accuratelythan others Certain data are inappropriate for answering some types of questionsThus it is wise for the analyst not to approach the matter of data considerationstoo cavalierly when planning a pharmacoeconomic study

In order to increase the scientific quality and integrity of pharmacoeco-nomic studies consensus guidelines have been developed by some countries andby professional and academic organizations Australia and Canada adopted na-tional guidelines on health economic analysis [2425] and the International So-ciety for Pharmaceutical Outcomes Research has developed an internationalconsensus statement on pharmacoeconomic methodology [26] Each of these doc-uments addresses in detail the issue of data validity and applicability of data tovarious types of analyses

There are a number of data sources available to use in constructing a phar-macoeconomic study or model Data can generally tell the researcher three thingsthat may be used in a pharmacoeconomic study the likelihood of outcomes asso-ciated with the treatment or nontreatment of disease the treatment patterns associ-ated with the therapy and the costs associated with the therapy andor diseasestate [27] Some researchers have coined the term lsquolsquotransition probabilitiesrsquorsquo to


refer to the first of these The data must be incorporated into the analysis in amanner that is rational and appropriate to the assumptions of the analysis

Table 4 shows the data types that may be used in conducting a pharmaco-economic analysis or constructing a pharmacoeconomic model The data typeswill vary in their applicability to different types of pharmacoeconomic analysesObviously one of the best sources for data in a health care system is local research[8] An advantage of using local research data is that a well-designed local re-search project can tell the analyst more about the probable costs and consequencesof treatment decisions at his or her hospital than data from other facilities that mayor may not have had similar patients and prescribing practices A disadvantage tousing local research data is that it takes considerable time and resources to con-duct a good study [828] By the time the study is designed and implementedand the findings analyzed market conditions may have changed and renderedthe results nonapplicable Also many hospitals are under budgetary pressuresthat do not allow the staffing or other resources needed to conduct a well-designedstudy

43 Evaluating Pharmacoeconomic Studies

Often those in health care will need to evaluate other pharmacoeconomic studieseither for the purpose of making a formulary or treatment decision or for thepurpose of gathering data for their own models In those cases it is importantto make sure that the pharmacoeconomic study is valid and applicable to thehealth care system in question There are several key questions that one mustask in evaluating a pharmacoeconomic analysis these are listed in Table 5

The first question listed in Table 5 is especially important when reviewinga study or analysis that makes conclusions about the pharmacoeconomics of agiven treatment option A clearly defined clinical question that is scientificallyrelevant and capable of being answered by the study design is a must It simplydoes not make sense to attempt to ascertain the economic advantage of a therapyif the study was not properly designed to determine a relevant clinical outcomeFor example it is common to find articles in which one antibiotic is claimed tobe economically superior to another but the data supporting the conclusion comefrom analyzing a subgroup that just happened to respond better to treatment be-cause they had a particular variant of the infecting organism that was unantici-pated at the start of the trial Similarly it is also important to know whether allof the interventions compared have been shown to be clinically effective Forexample it may be invalid to determine that a very low cost antibiotic is lsquolsquomorecost-effectiversquorsquo than another when in fact the low cost antibiotic is no longerconsidered clinically effective because of increasing microbial resistance

Other important questions surround economic models First and foremostthe modelrsquos assumptions must be rational and a realistic representation of real-

Basic Pharmacoeconomics 381T

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TABLE 5 Key Questions for Evaluating Pharmacoeconomic Studies

Is the analysis based on a study that answers a clearly defined clinicalquestion about an economically important issue

Does the type of analysis match the objectives of the organization orperson conducting the study Is it appropriate

Does the analysis seek to answer questions for which it is not structuredIs the quality of the data appropriate to the conclusions of the analysisIs there real or perceived bias in the way in which the analysis was

structuredIf an economic model was the model tested for sensitivity of its inputsWere the modelrsquos assumptions realistic in comparison to lsquolsquoreal lifersquorsquoWhose viewpoint are costs and benefits being considered fromHave all the interventions compared been shown to be clinically effectiveHow were costs and benefits measured

Source Adapted from Refs 14 and 22

life occurrences [715] Failure and retreatment rates probability of laboratorymonitoring and likelihood of additional visits are some areas where modelerssometimes make unrealistic assumptions that may bias a modelrsquos results Anotherimportant consideration for economic models is sensitivity testing [151829]The modelrsquos inputs should be tested around a range of values to determine howchanging these values affect the modelrsquos results If the modelrsquos results appear tobe similar across the likely range of possible input values then the model is saidto be robust On the other hand if the modelrsquos results vary greatly when smallbut realistic changes are made in its inputs then it is less valid A final consider-ation that should be given to economic models is whether or not the model istransparent and reproducible All of the major governmental and academic bodiesthat have developed guidelines have included recommendations that the modelrsquosinputs and mathematical relationships be transparent (ie equations and variablesclearly shown) They also recommend that the model be constructed in a mannerthat would make it possible for other researchers or analysts to duplicate themodel and its findings [16ndash18] Models that have hidden (lsquolsquoblack boxrsquorsquo) relation-ships may be considered of questionable validity

The rapid development of new drug technologies combined with direct-to-consumer advertising increased patient access to care and finite resources willrequire that decision makers have the analytical tools necessary to make impor-tant health-related resource allocation decisions This chapter has attempted tointroduce the reader to some of the available pharmacoeconomic methodologies


used to assess the cost and outcomes of competing drug therapy options includ-ing effective antimicrobial therapy

REFERENCES

1 Juergens JP Szeinbach SL Smith MC Will future pharmacists understand pharma-coeconomic research Am J Pharm Educ 199256135ndash140

2 Pritchard C Trends in economic evaluation Office of Health Economics BriefingNo 36 April 1998

3 Vital and Health Statistics Series 13 No 1294 Drummond MF Stoddart GL Torrance GW Methods for the Economic Evaluation

of Health Care Programmes New York Oxford Univ Press 19875 Eisenberg JM Clinical economics A guide to the economic analysis of clinical

practice JAMA 19892622879ndash28866 Quenzer RW Pettit KG Arnold RJ Kaniecki DJ Pharmacoeconomic analysis of

selected antibiotics in lower respiratory tract infection Am J Managed Care 199731027ndash1036

7 Lee JT Sanchez LA Interpretation of lsquolsquocost-effectiversquorsquo and soundness of economicevaluations in the pharmacy literature Am J Hosp Pharm 199148622ndash627

8 Bootman L Pharmacoeconomics (lsquolsquoThe Green Bookrsquorsquo)9 Webstersrsquo New Riverside University Dictionary Boston Riverside 1988

10 Rupp MT Kreling DH The impact of pharmaceutical care on patient outcomesWhat do we know Drug Benefit Trends 19979(2)35ndash47

11 Tse CS Outcomes measurement across the continuum of care Med Interface 199710(11)103ndash107

12 Kenreigh CA Wagner LT Medscape Pharmacists Treatment Update HillsboroMedical Education Collaborative Inc June 1999

13 McDonald RC Coons SJ An Introduction to Health Economics Indianapolis EliLilly 1993

14 Greenhalgh T Papers that tell you what things cost (economic analysis) Br Med J1997315596ndash599

15 Jones AJ Sanchez LA Pharmacoeconomic evaluation Applications in managedhealth care formulary decision making Drug Benefit Trends 19957(9)121519ndash2232ndash34

16 Morrison GC Gyldmark M Appraising the use of contingent valuation Health Econ19921(4)233ndash243

17 Dolan P Gudex C Kind P Williams A The time trade-off method Results froma general population study Health Econ 19965(2)141ndash154

18 Dean BS Gallivan S Barber ND Van Ackere A Mathematical modeling of phar-macy systems Am J Health Syst Pharm 1997542491ndash2499

19 Lightwood JM Glantz SA Short-term economic and health benefits of smokingcessation Myocardial infarction and stroke Circulation 1997961089ndash1096

20 Schumacher GE Multiattribute evaluation in formulary decision making as appliedto calcium channel blockers Am J Hosp Pharmacy 199148(2)301ndash308


21 McCoy S Chandramouli J Mutnick A Using multiple pharmacoeconomic methodsto conduct a cost-effectiveness analysis of histamine H2 receptor antagonists AmJ Health Syst Pharm 199855(suppl 4)S8ndashS12

22 Summers KH Hylan TR Edgell ET The use of economic models in managed carepharmacy decisions J Managed Care Pharm 1998442ndash50

23 Richerson MA Ambrose PG et al Pharmacoeconomic evaluation of alternativeantibiotic regimens in hospitalized patients with community acquired pneumoniaInfect Dis Clin Pract 19987227ndash233

24 Commonwealth of Australia Dept of Human Services amp Health Guidelines for thepharmaceutical industry on preparation of submissions to the Pharmaceutical Bene-fits Advisory Committee including major submissions involving economic analysisCanberra Australian Govt Pub Service 1995

25 Canadian Coordinating Office for Health Technology Guideline for Economic Eval-uation of Pharmaceuticals Canada Ottawa CCOHTA 1994

26 International Society for Pharmaceutical Outcomes Research Consensus Statementon Pharmacoeconomic Studies Philadelphia ISPOR 2000

27 Nuijten MJC The selection of data sources for use in modeling studies Pharmaco-economics 199813(3)305ndash315

28 Else BA Armstrong EP Cox ER Data sources for pharmacoeconomic and healthservices research Am J Health-Syst Pharm 1997542601ndash2608

29 Brennan A Akehurst R Modelling in health economic evaluation What is its placeWhat is its value Pharmacoeconomics 200017(5)445ndash459

17

Utilizing Pharmacodynamics andPharmacoeconomics in Clinicaland Formulary Decision Making

Paul G Ambrose


Annette Zoe-Powers

Bristol-Myers Squibb Company Plainsboro New Jersey

Rene Russo

Rutgers University Piscataway New Jersey

David T Jones

University of California at Davis Sacramento California

Robert C Owens Jr

Maine Medical Center Portland Maineand University of Vermont College of Medicine Burlington Vermont

1 INTRODUCTION

Managed care organizations (MCOs) community hospitals and university teach-ing hospitals are challenged to control total organizational cost while maintaininghigh quality patient care and services Most health care institutions have imple-

385

386 Ambrose et al

mented processes including pharmacy and therapeutics committees (PampTs) andformularies in an effort to control drug costs In essence a formulary is a listof products that have been reviewed and approved by the Pharmacy and Thera-peutics Committee Moreover the goal of the PampT and formulary process is tooptimize clinical outcomes while controlling or minimizing cost Despite theseefforts drug and organizational expenditures continue to increase Because manyantibiotic regimens could be considered appropriate for the treatment of a giveninfectious disease the challenge is to find the most cost-effective regimens Itshould be realized however that a low drug cost does not necessarily correlateto a low organizational cost For instance although a drug with a high acquisitionprice compared with standard therapy may increase total drug costs it may sig-nificantly decrease the institutional expenditures in other areas Unfortunatelyfew prospective randomized clinical data are available comparing new therapeu-tic modalities and standard regimens under usual care conditions

Consequently it is crucial to develop methods to treat infections that areboth clinically sound and economically effective The application of pharmacody-namic and pharmacoeconomic concepts to issues involving the use of antimicro-bial agents provides a data-driven paradigm to achieve these goals This approachoften identifies the best agent to maximize bacterial killing the appropriate doseand the most cost-effective agent to select for formulary inclusion and clinicaluse In order to understand the discussion contained in this chapter a basic under-standing of pharmacodynamic theory and pharmacoeconomic concepts is essen-tial For a complete review of these concepts the reader is encouraged to reviewChapters 5 7 9 and 16 as they form the basis for the discussion that follows

In this chapter pharmacodynamic and pharmacoeconomic principles areused to guide clinical and formulary decisions Through the use of contemporaryantimicrobial agents as real-life illustrations the reader will become cognizanton how these concepts can be applied to formulary decision making and in theclinical practice of medicine and pharmacy Moreover agents from a variety ofantimicrobial classes (eg cephalosporins fluoroquinolones and macrolides)and modes of bacterial killing (time-dependent versus concentration-dependent)will be used as examples Obviously the following evaluations are not meantto be complete unto themselves but rather are intended to outline the use ofpharmacodynamic and pharmacoeconomic data in the decision-making process

2 CEFEPIME VERSUS CEFTAZIDIME

21 Background

Cefepime and ceftazidime are intravenously administered β-lactam antibioticsthat exhibit a broad spectrum of antibacterial activity that includes aerobic gram-positive and gram-negative bacteria Due to their spectrum of activity these

Clinical and Formulary Decision Making 387

agents have emerged as standard therapy for a number of serious nosocomialinfections such as hospital-acquired pneumonia and the empirical therapy of fe-brile neutropenia

22 Pharmacokinetics

The pharmacokinetic parameters of cefepime and ceftazidime in adults are pre-sented in Table 1 [12] From these data it is apparent that the two agents aresimilar pharmacokinetically in terms of volume of distribution clearance percenturinary excretion and protein binding However cefepime has an approximately25 longer serum half-life and a slightly larger area under the serum concentra-tionndashtime curve (AUC) For this reason cefepime is dosed on a twice-dailyschedule whereas ceftazidime must be dosed three times daily in those patientswith normal or moderately reduced renal function (Clcr 50ndash60 mLmin) Thepharmacokinetics of both agents vary with degree of renal function Followinga 1000 mg dose of cefepime in patients with glomerular filtration rates (GFRs)of less than 60 mLmin serum half-life and AUC increase from 23 to 49 h andfrom 131 to 292 microg sdot hmL respectively [3] Similarly following a 1000 mgdose of ceftazidime in patients with GFRs less than 50 mLmin serum half-lifeand AUC increase from approximately 19 to 40 h and 130 to 270 microg sdot hmLrespectively [14]

23 Microbiology

Comparative antimicrobial activities of cefepime and ceftazidime against variouspathogens are presented in Table 2 [5] From these data it appears that cefepimehas lower minimal inhibitory concentration (MIC) values than ceftazidimeagainst most gram-positive aerobes especially Streptococcus pneumoniae andStaphylococcus aureus This is significant because gram-positive aerobes pri-marily staphylococci are a leading cause of serious nosocomial infection In factS aureus is the second most common cause of hospital-acquired pneumonia

Against aerobic gram-negative microorganisms including Pseudomonasaeruginosa the microbiological activity of cefepime is similar to that of ceftazi-dime however notable exceptions exist Unlike ceftazidime cefepime has beenshown to have enhanced stability in the presence of Bush-Jacoby-Mederios group1 β-lactamases which when induced can render all other currently availablecephalosporins inactive [67] Not unexpectedly therefore cefepime demon-strates significantly lower MICs against isolates such as Enterobacter species andCitrobacter freundii that elaborate this enzyme In addition cefepime exhibitsappreciably more activity against Klebsiella pneumoniae These observationscarry considerable clinical relevance because P aeruginosa K pneumoniaeEnterobacter species and Citrobacter species are the first third fourth and fifthmost likely pathogens in hospital-acquired pneumonia respectively Although it

388 Ambrose et al

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5

390 Ambrose et al

is tempting to conclude from in vitro microbiological observations that cefepimeis a preferential choice over ceftazidime the significance if any of these observa-tions will become apparent in the pharmacodynamic discussion below

24 Pharmacodynamics

An accurate prediction of clinical outcome requires the use of pharmacodynamicconcepts that integrate microbiological and pharmacokinetic data Cefepime andceftazidime like all other β-lactam antimicrobial agents kill bacteria in a time-dependent fashion Once the drug concentration exceeds the MIC of the pathogenkilling occurs at a zero-order rate and increasing drug concentration does notresult in a proportionally increased rate of kill Thus the goal with β-lactamantimicrobial agents is to maintain their duration or time above the MIC for aslong as possible over any one dosing interval As mentioned in Chapter 5 areasonable time period for β-lactam antimicrobial agents to exceed the MIC is40ndash60 of any single dosing interval [8] Earlier comparisons of in vitro activityagainst several clinically relevant pathogens between cefepime and ceftazidimewere made In the following analysis we will explore the potential clinical sig-nificance of these observations

Time versus plasma concentration curves of cefepime and ceftazidime arepresented in Figs 1 and 2 respectively Following a 1000 mg intravenous doseof cefepime every 12 h serum levels remain above the MIC of most organismsover the entire dosing interval Against P aeruginosa however cefepime serumconcentration time above the MIC (T MIC) is approximately 50 of the dosinginterval Following a 1000 mg intravenous dose of ceftazidime every 8 h a some-what different picture emerges The serum concentration of ceftazidime remainsabove the MIC over the entire dosing interval for only E coli and K pneumoniaeAgainst P aeruginosa and S aureus serum ceftazidime T MIC is approxi-mately 50 Moreover against C freundii and E cloacae for only 25 of thedosing interval does the ceftazidime serum concentration remain above the MIC

Based on these observations one would expect similar clinical outcomeswhen either agent is used to treat infections involving E coli or K pneumoniaebecause both agents maintain drug concentrations over the entire dosing intervalHowever against C freundii and E cloacae it would be predicted that cefepimeis the superior agent because cefepime maintains levels over 100 of the dosinginterval compared with 25 for ceftazidime Indeed the literature is replete withcase reports describing failure of ceftazidime when used to treat serious infectionsinvolving these pathogens [9] In fact because these organisms can elaborateBush-Jacoby-Mederios group 1 β-lactamases third generation cephalosporins arenot recommended for infections when these organisms are likely to be involvedOn the other hand cefepimersquos advantage against S aureus would not be expectedto be of clinical relevance because ceftazidime maintains serum levels above the


FIGURE 1 Concentrationndashtime profile of cefepime following a 1 g dose every12 h with MIC values of selected bacteria superimposed

MIC for approximately 50 of the dosing interval Moreover one would predictsimilar outcomes against P aeruginosa for the same reason [10]

25 Pharmacoeconomics

As mentioned earlier few prospective randomized clinical data are availablecomparing new therapeutic modalities and standard regimens under usual careconditions Naturalistic trials are a study design approach that compares one drugregimen such as a new drug with one or more commonly used therapies underusual care conditions In other words naturalistic studies compare clinical andother outcome measures of competing therapeutic alternatives based upon howthe drugs are actually used in clinical practice rather than in the strict confinesof traditional clinical trials This information is crucial especially as new prod-ucts are introduced to the market because health care providers must make clini-cal and economic decisions concerning these agents Two such studies have di-rectly compared cefepime and ceftazidime and are reviewed briefly below

Ambrose et al [1] compared the cost effectiveness of cefepime and ceftazi-dime in intensive care unit patients with hospital-acquired pneumonia The effi-cacy safety and cost effectiveness of each agent were evaluated in a prospective

392 Ambrose et al

FIGURE 2 Concentrationndashtime profile of ceftazidime following a 1 g dose ev-ery 8 h with MIC values of selected bacteria superimposed

noninterventional investigator-blinded study involving 100 patients Clinicalsuccess rates were 60 and 78 for patients treated with ceftazidime and cefe-pime respectively (P 005) Microbiological eradication rates were 55 forceftazidime and 77 for cefepime (P 004) In those patients in whom Paeruginosa were isolated the organism was eradicated in 70 (1420) of cefe-pime patients and 50 (714) of ceftazidime patients Interestingly the authorsnoted that the MICs of P aeruginosa and S aureus were higher for ceftazidimethan for cefepime at the study institution and that the resultant pharmacodynamicdifferences may be the reason for their observations The frequency of concomi-tant antibiotic use was less in the cefepime group [ceftazidime 74 (3750)cefepime 44 (2250) P 0004] particularly with the use of vancomycinCefepime was more cost effective than ceftazidime (ceftazidime $2452810 cef-epime $1999621) Sensitivity analysis of efficacy rates demonstrated that ceftaz-idime would have to be 50 more effective than cefepime to change the eco-nomic outcome The authors concluded that cefepime was clinically moreefficacious and more cost effective than ceftazidime in similar patients withhospital-acquired pneumonia [11]


Owens et al [12] compared the cost effectiveness of cefepime dosed 1000ndash2000 mg every 12 h and ceftazidime dosed 1000ndash2000 mg every 8 h as empiricalmonotherapy for febrile neutropenia The efficacy safety and cost effectivenessof each agent were evaluated in a prospective noninterventional evaluator-blinded study involving 99 patients

Patients were stratified by risk for infection in accordance with the IDSAFDA and HIS guidelines (ie cancer type age and fever) Clinical assessmentswere made at 72 h of therapy and at 7 days post-therapy or at discharge fromthe hospital Clinical success was defined as apyrexia maintained clinical signsand symptoms improved and organism eradicated (when applicable) no relapseor new infection and survival Failure was defined as inability to eradicate thepathogen death due to infection and discontinuation of study agent because ofnonresponse

Clinical success rates were 80 and 96 for patients treated with ceftazi-dime and cefepime respectively (P 0028) Moreover the frequency of CDCIDSA-unsupported vancomycin addition to therapy occurred significantly (P 0001) less often in the cefepime group (14) than in the ceftazidime group(46)

The authors found that cefepime was more cost effective than ceftazidime(ceftazidime $50128 cefepime $31407) Sensitivity analysis of efficacy ratesdemonstrated that cefepime efficacy would have to decrease by 50 to changethe economic outcome The authors also noted that the elimination of ceftazidimefrom the formulary was associated with a favorable impact on Enterobacter cloa-cae susceptibility in the study units

26 Conclusion

Based upon this pharmacodynamic analysis it would be predicted that clinicaloutcomes would be improved if cefepime were chosen over ceftazidime for thetreatment of nosocomial infection This prediction is supported by comparativepharmacoeconomic data obtained from studies naturalistic in design Thereforeit may be reasonable to select cefepime over ceftazidime for formulary inclusion

3 CLARITHROMYCIN VERSUS AZITHROMYCIN

31 Background

Azithromycin and clarithromycin are two of the most commonly prescribed anti-biotics in the United States Because of their convenient dosing schedule andactivity against respiratory tract pathogens azithromycin and clarithromycin areprimarily used to treat infections such as community-acquired pneumonia sinus-itis and bronchitis

394 Ambrose et al

32 Pharmacokinetics

The pharmacokinetics parameters in adults of both macrolides are shown in Table3 The bioavailability and volume of distribution are similar between the twodrugs However azithromycin has a markedly longer half-life (t12) than clarithro-mycin 68 h and 3ndash4 h respectively [1314] As a result azithromycin can bedosed just once daily for 5 days Clarithromycin undergoes metabolism throughoxidative and hydrolytic pathways that produce several metabolites Converselyazithromycin is not highly metabolized with 75 of the parent compound beingexcreted unchanged [15] Because the macrolides rapidly distribute into tissuesresulting in high concentrations within the cells the serum concentration of mac-rolides is markedly lower than that of β-lactam antibiotics

33 Microbiology

The microbiological activity of macrolides against various bacteria is shown inTable 4 In vitro studies have shown that both agents are active against S pneu-moniae and M catarrhalis Based solely on MIC data azithromycin appears tobe more active against H influenzae than clarithromycin However the combina-tion of clarithromycin and its active metabolite 14-hydroxyclarithromycin hasbeen demonstrated to have additive or even synergistic activity against H in-fluenzae [16]

34 Pharmacodynamics

As mentioned earlier macrolides such as erythromycin and clarithromycin andazilides like azithromycin are generally considered concentration-independentkilling agents As with β-lactam agents the duration of time that the drug concen-tration exceeds the MIC of the infecting pathogen at the site of infection is theprimary determinant of efficacy for macrolides and azilides [4] How long thelevels of these agents should remain above the MIC remains controversial asthese agents have a prolonged post-antibiotic effect (PAE) with gram-positivecocci and Haemophilus influenzae [1718] Craig et al [17] postulated that thePAE allows these drugs to yield maximal efficacy in murine thigh infectionswhen concentrations exceed the MIC for significantly less than 50 of the dosinginterval (ie 30ndash35) Due to an extended PAE and long serum half-life theproper pharmacodynamic correlate for azithromycin may be the AUCMIC ratiorather than T MIC

Other investigators suggest that the concentration of the drug at the site ofinfection specifically where bacteria attach to the mammalian cell is significantlyhigher than the concentration in the serum This is due to macrolides and azilidesespecially azithromycin being highly concentrated inside mammalian cells Asthe drug egresses out of the cell a drug concentration gradient exists that is


TA

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etic

Par

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ers

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thro

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ing

500

mg

(Day

1)an

d25

0m

g(D

ays

2ndash5

)D

ose

san

d50

0m

gD

ose

sR

esp

ecti

vely

a

Cm

axH

alf-

life

AU

CV

ssC

lear

ance

U

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ary

P

rote

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t(micro

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)(h

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(L)

(mL

min

)ex

cret

ion

bin

din

g

Azi

thro

myc

in0

2468

21

2160

630

65

7ndash5

1C

lari

thro

myc

in2

854

820

84

191

437

3042

ndash70

aC

max

p

eak

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mco

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ntr

atio

n

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C

area

un

der

the

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ime

curv

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yst

ate

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rote

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ind

ing

vari

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dif

fere

nt

con

cen

trat

ion

s

396 Ambrose et al

TABLE 4 Comparative In Vitro Microbiological Activity of Azithromycinand Clarithromycin

AntimicrobialOrganism (no tested) agent MIC50 MIC90

Gram-positiveStreptococcus pyogenes (105) Azithromycin 003 003

Clarithromycin 0016 0016Staphylococcus aureus BLA neg Azithromycin 013 013

(54) Clarithromycin 006 006

Streptococcus pneumoniae penicil- Azithromycin 003 4lin-susceptible (26) Clarithromycin 0016 2

Penicillin resistant (15) Azithromycin 05 16Clarithromycin 025 16

Gram-negativeHaemophilus influenzae (1137) Azithromycin 2 2

Clarithromycin 8 16Moraxella catarrhalis (723) Azithromycin 006 006

Clarithromycin 006 012

Source Refs 40ndash42

highest at the mammalian cell surface and lowest in the serum In this paradigmthe drug concentration in the serum is a poor correlate for that at the site ofinfection [19]

Azithromycinrsquos long serum half-life (approximately 68 h) which resultsin low serum concentration over an extended period of time has raised concernregarding its propensity to select for macrolide-resistant isolates [20] Leach etal [21] prospectively studied the impact of community-based azithromycin treat-ment on carriage and resistance of Streptococcus pneumoniae Single-doseazithromycin (20 mgkg) was given to children in a remote Aboriginal commu-nity in Australia with trachoma and their household contacts who were childrenCarriage rates of pneumococci resistant to azithromycin immediately before treat-ment with azithromycin and 2ndash3 weeks 2 months and 6 months after treatmentwere 19 545 345 and 59 respectively In this study the authors con-cluded that the selective pressure of azithromycin allowed the growth and trans-mission of pre-existing azithromycin-resistant strains

Similarly azithromycin was evaluated in an open clinical microbiologicaland pharmacokinetic study in hospitalized patients with acute exacerbation ofchronic bronchitis [22] Patients received 500 mg on the first day followed by250 mg once daily for 4 days Cultures of Haemophilus influenzae during andafter treatment showed many infections persisting Geometric mean MICs of


FIGURE 3 Pharmacodynamic concept Darwinrsquos window of natural selectionSchematic illustration of the serum pharmacokinetic profile of two oral drugregimens over one dosing interval If the serum concentrationndashtime curvesof two drugs one each with a short or long serum half-life are compared withan MIC value superimposed a period or lsquolsquowindowrsquorsquo of potential Darwinianselection appears For the agent with a short half-life the duration of timebetween when the drug concentration falls below the MIC and its total elimi-nation from the body is short relative to that of the agent with a much longerhalf-life

azithromycin for these organisms rose from 123 mgL pretreatment to 487 mgL a week after the end of treatment

One explanation for these observations relates to azithromycinrsquos long se-rum half-life and the duration of time subinhibitory concentrations of drug exist[23] If the serum concentrationndashtime curves of two drugs one each with shortand long serum half-lives are compared with an MIC value superimposed aperiod or lsquolsquowindowrsquorsquo of potential Darwinian selection appears (Fig 3) For theagent with a short half-life the duration of time between when the drug concentra-tion falls below the MIC and its total elimination from the body is short relativeto that of the agent with a much longer half-life For an agent with a 68 h half-life such as azithromycin the drug is not totally eliminated from the body for5ndash7 half-lives or 14ndash20 days after the end of therapy Therefore this period ofnatural selection or lsquolsquoDarwinrsquos windowrsquorsquo may be the pharmacodynamic explana-tion for the aforementioned observations

398 Ambrose et al


Health economic models have been used to investigate the cost consequencesof different treatment options in community-acquired respiratory tract infections[112425] Two recent studies evaluated the relationship between cost of careand either first-line antibiotic clinical efficacy or microbiological eradication ratefor the treatment of community-acquired pneumonia and acute exacerbation ofchronic bronchitis [2526] These studies used a Markov model that was popu-lated with health care resource estimates obtained from a panel of physicians(including general practitioners and pulmonology specialists) Cost was deter-mined by multiplying utilized resource items by the price of each item and evalu-ated from the perspective of the third-party payer

Use of these models has led to several significant observations First theleast expensive antibiotic is not necessarily the most cost-effective treatment mo-dality second not unexpectedly treatment failure resulted in the increased utili-zation of health care resources These resources included physician office visitslaboratory tests antibiotics and hospital admissions Consequently clinical effi-cacy rate was found to be a key cost driver due to the high cost of treatmentfailure This was especially true of treatment failures that resulted in hospitaladmissions

These observations are supported by a retrospective review of the medicalliterature since 1990 which investigated the relationship between clinical efficacyand microbiological eradication rates The review found 12 comparative studiesof acute exacerbation of chronic bronchitis in which both clinical and microbio-logical success rates were reported for 27 treatments involving 15 different antibi-otics A significant correlation (r 090) was found between clinical failurerates and microbiological eradication failure rates Hence a key cost driver inthe treatment of acute exacerbation of chronic bronchitis is the eradication rateof the causative bacterial pathogens by the first-line antibiotic Failure to eradicatethe causative pathogens increases the risk of treatment failure with its associatedadditional treatment costs

In a prospective randomized comparative study Weiss [26] evaluated theefficacy and safety of azithromycin clarithromycin and erythromycin in the ther-apy of patients with the clinical diagnosis of acute exacerbation of chronic bron-chitis Although the clinical efficacy rates were higher for azithromycin (80)and clarithromycin (85) compared with erythromycin (70) 40 of theazithromycin group requested and received prescription refills The financial im-pact of this observation is not limited to the mere cost of an additional prescriptionfor azithromycin which is minimal compared to the burden placed on the healthcare system in the form of additional physician office visits and potential hospital-izations


36 Conclusion

Based upon the foregoing pharmacodynamic analysis it would be predicted thatthe use of azithromycin as first-line antibiotic treatment might result in the morerapid selection of resistant strains of respiratory tract pathogens and treatmentfailure Moreover clinical and microbiological failure of first-line therapy re-mains the key cost driver in patients with community-acquired respiratory tractinfections For these reasons it may be reasonable to select clarithromycin overazithromycin for formulary inclusion and clinical use

4 GATIFLOXACIN VERSUS LEVOFLOXACIN

41 Background

Fluoroquinolones such as gatifloxacin and levofloxacin have become popularchoices for the empirical treatment of community-acquired pneumonia acute ex-acerbation of chronic bronchitis and sinusitis These agents are different fromearlier quinolones due to their increased in vitro activity against Streptococcuspneumoniae which is the most common and difficult pathogen associated withcommunity-acquired respiratory tract infection The increased popularity of gati-floxacin and levofloxacin has paralleled the rising resistance rates among pneu-mococci and H influenzae to many traditional antimicrobial agents such as thecephalosporins and macrolides

42 Pharmacokinetics

The pharmacokinetic profiles of gatifloxacin and levofloxacin are presented inTable 5 These two agents are similar with regard to volume of distributionpercent urinary excretion and protein binding Gatifloxacin demonstrates aslightly longer half-life whereas levofloxacin has a slightly larger area under theserum concentrationndashtime curve (AUC) Both agents are administered once dailyBoth agents are primarily excreted as unchanged drug from the kidney with negli-gible hepatic metabolism Both agentsrsquo pharmacokinetics vary with renal func-tion and doses should be decreased accordingly in those patients with renal insuf-ficiency As with many of the newer generation fluoroquinolones the oralbioavailabilities of gatifloxacin and levofloxacin are near 100 making theseideal agents for oral antimicrobial therapy

43 Microbiology

Comparative antimicrobial in vitro activities of gatifloxacin and levofloxacinagainst various pathogens are presented in Table 6 One of the notable differences

400 Ambrose et al

TA

BLE

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oki

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AU

CV

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ary

P

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)(h

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(mL

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28

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140

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rum

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trat

ion

ndashtim

ecu

rve

Vss

volu

me

of

dis

trib

uti

on

atst

ead

yst

ate

TABLE 6 Comparative In Vitro Microbiological Activity of Gatifloxacinand Levofloxacin


Gram-positiveEnterococcus faecalis (100) Gatifloxacin 025 2

Levofloxacin 1 2Enterococcus faecium (40) Gatifloxacin 2 4

Levofloxacin 2 8

Staphylococcus aureus Gatifloxacin 006 013Meth-S (90) Levofloxacin 013 025

Meth-R (63) Gatifloxacin 013 16Levofloxacin 05 16

Staphylococcus epidermidis Gatifloxacin 013 025Meth-S (39) Levofloxacin 05 05


Streptococcus pneumoniae Gatifloxacin 025 1Pen-S (30) Levofloxacin 1 2

Pen-R (15) Gatifloxacin 025 05Levofloxacin 1 2

Streptococcus pyogenes (47) Gatifloxacin 025 05Levofloxacin 1 1

Streptococcus agalactiae (38) Gatifloxacin 025 05Levofloxacin 05 1

Gram-negativeCitrobacter freundii (52) Gatifloxacin 006 1

Levofloxacin 003 05Enterobacter cloacae (63) Gatifloxacin 0016 006

Levofloxacin 003 006Escherichia coli (30) Gatifloxacin 0008 0016

Levofloxacin 0016 003Haemophilus influenzae (46) Gatifloxacin 0008 0016

Levofloxacin 003 006Klebsiella pneumoniae (61) Gatifloxacin 003 013

Levofloxacin 003 013Moraxella catarrhalis (11) Gatifloxacin 0008 016

Levofloxacin 0008 006Morganella morganii (41) Gatifloxacin 006 025

Levofloxacin 003 006Proteus mirabilis (37) Gatifloxacin 013 025

Levofloxacin 003 025Pseudomonas aeruginosa (50) Gatifloxacin 4 32

Levofloxacin 2 32Serratia marcescens (55) Gatifloxacin 025 4

Levofloxacin 025 8Stenotrophomonas maltophilia (50) Gatifloxacin 025 4

Levofloxacin 025 8

Source Refs 43 44

402 Ambrose et al

in these in vitro data involves the pneumococci Gatifloxacin exhibits up to fourtimes greater potency than levofloxacin against isolates of S pneumoniae whichas mentioned earlier is the most common and most virulent pathogen associatedwith community-acquired respiratory tract infection Although it is tempting toconclude from this in vitro microbiological observation that gatifloxacin is a pref-erential choice over levofloxacin the clinical relevance if any of this observationwill become apparent in the following pharmacodynamic discussion

44 Pharmacodynamics

The pharmacodynamic properties of fluoroquinolone antibiotics have been wellelucidated Gatifloxacin and levofloxacin like all fluoroquinolones eliminatebacteria most rapidly when their concentrations greatly exceed the MIC of thetargeted organism This type of activity is referred to as concentration-dependentor time-independent killing [27ndash30] Although peakMIC ratios of greater than10ndash121 correlate with optimal bactericidal activity the AUCMIC ratio is theimportant parameter determining the efficacy of fluoroquinolones

Data obtained from animal models in vitro pharmacodynamic studies andclinical outcome studies indicate that the magnitude of the AUCMIC ratio canbe used to predict response Forrest et al [30] demonstrated that an AUCMICratio of 125 was associated with the best bacterial eradication rates in the treat-ment of infections caused by gram-negative pathogens For gram-positive bacte-ria it appears that the AUCMIC ratio may be lower An in vitro model of infec-tion demonstrated that for levofloxacin and ciprofloxacin an AUCMIC ratio ofapproximately 30 was associated with a 4 log kill while ratios less than 30 wereassociated with a significantly reduced extent of bacterial killing and in someinstances bacterial regrowth [31] In like fashion Lister and Sanders [32] reportedthat for levofloxacin and ciprofloxacin an AUCMIC ratio of 32ndash64 was associ-ated with maximal eradication of S pneumoniae in an in vitro model of infectionThese data are supported by data from animal models of infection where survivalwas associated with an AUCMIC ratio of 25ndash30 against pneumococcus [33]Finally for gatifloxacin and levofloxacin bacteriological eradication was associ-ated with an AUCMIC ratio greater than 33 in patients with community-acquiredpneumonia and acute exacerbation of chronic bronchitis [34]

Clinicians often make such interquinolone comparisons based upon AUCMIC ratios Typically this is accomplished by using mean AUC values usuallyobtained from normal healthy volunteers and MIC90 values from the literatureWhen making comparisons in this manner variability in pharmacokinetic (AUC)and microbiological (MIC) data is not accounted for [35] Thus the metrics devel-oped applies only to a minimum number of clinical instances

Pharmacodynamic modeling using Monte Carlo simulation is one approachthat takes into account variability in microbiological and pharmacokinetic data


Monte Carlo simulation estimates the probability of achieving a pharmacody-namic outcome This is accomplished by selecting an AUC value from a popula-tion of actual patients (in proportion to their probability of occurrence) and pair-ing it with an MIC value from a clinical isolate (also in proportion to theprobability of occurrence) This is done for thousands of iterations resulting ina probability distribution that reflects the chance that a pharmacodynamic targetwill be achieved in a patient treated with a given drug

Ambrose et al performed such an analysis using gatifloxacin and levoflox-acin pharmacokinetic data obtained from adult patients (18 years or older) en-rolled in clinical trials conducted at multiple centers [36] and susceptibility datafor S pneumoniae from almost 2000 isolates [37] The gatifloxacin data set in-cluded 64 acutely ill patients all of whom had community-acquired infectionsthe pharmacokinetic data set for levofloxacin included 172 acutely ill patientsall of whom had community-acquired infections [38] AUC and MIC probabilitydistributions based on these data were then plotted for both agents and a 5000-patient Monte Carlo simulation was performed

Following standard 500 mg doses of levofloxacin the probability of achiev-ing an AUCMIC ratio of greater than 30 against S pneumoniae was 80(Fig 4) With standard 400 mg doses of gatifloxacin the probability of achieving

FIGURE 4 Pharmacodynamics of levofloxacin against Streptococcus pneu-moniae Results of a 5000 patient Monte Carlo simulation based upon thevariability in the pharmacokinetics in the population of patients modeled andthe MIC variability observed in a large susceptibility surveillance programThe dark-colored bars represent the number of simulated patients with AUCMIC ratios less than 30 whereas the light-colored bars represent the numberof simulated patients with AUCMIC ratios of 30 or greater The probabilityof attaining an AUCMIC ratio of at least 30 is approximately 80

404 Ambrose et al

FIGURE 5 Pharmacodynamics of gatifloxacin against Streptococcus pneu-moniae Results of a 5000 patient Monte Carlo simulation based upon thevariability in the pharmacokinetics in the population of patients modeled andthe MIC variability observed in a large susceptibility surveillance programThe dark-colored bars represent the number of simulated patients with AUCMIC ratios less than 30 whereas the light-colored bars represent the numberof simulated patients with AUCMIC ratios of 30 or greater The probabilityof attaining an AUCMIC ratio of at least 30 is approximately 94

an AUCMIC ratio of greater than 30 for S pneumoniae was 94 (Fig 5) Thesedata suggest that in treating patients with community-acquired pneumonia dueto S pneumoniae the pharmacodynamic target hit rate is greater for gatifloxacinthan for levofloxacin Interestingly these pharmacodynamic target hit rates mir-rored the bacteriological eradication rates observed in randomized double-blindmulticenter phase III clinical trials involving these two agents [39] Unfortu-nately currently there are no comparative pharmacoeconomic data available forthese two agents

45 Conclusion

Based upon the foregoing pharmacodynamic analysis it would be predicted thatthe use of gatifloxacin would achieve a greater pharmacodynamic target hit rateagainst pneumococci than levofloxacin In the era of multidrug-resistant pneumo-cocci this may result in the less rapid selection of resistant strains if gatifloxacinis used preferentially over levofloxacin However more clinical data will beneeded to confirm or refute this hypothesis Nonetheless due to the aforemen-tioned pharmacodynamic differences between these two agents it may be reason-able to select gatifloxacin over levofloxacin for the treatment of community-acquired respiratory tract infections


5 SUMMARY

In this chapter pharmacodynamic theory and pharmacoeconomic principles wereused as a guide for rational decision making The use of these principles can beapplied to any drug or drug class and should be considered an aid in a data-driven decision-making paradigm Our purpose was not to convince the readerto select one antimicrobial agent over the other in the examples given but toillustrate how pharmacodynamic and other data can and have been used for deci-sion making

REFERENCES

1 Product information Fortaz (Ceftazidime sodium) Glaxo Pharmaceuticals June1994

2 Product information Maxipime (Cefepime hydrochloride) Bristol-Myers SquibbCompany January 1996

3 Barbhaiya RH Knopp CA Forgue ST Matzke GR Guay DRP Pittman KA Phar-macokinetics of cefepime in subjects with renal insufficiency Clin Pharmacol Ther199048268ndash276

4 Ambrose PG Ceftazidime Antibio Clin 1997144ndash495 Marshall SA Aldridge KE Allen SD Fuchs PC Gerlach EH Jones RN Compara-

tive antimicrobial activity of piperacillin-tazobactam tested against more than 5000recent clinical isolates from five medical centers Diagn Microbiol Infect Dis 199521153ndash168

6 Hiraoka M Inoue M Mitsuhashi S Hydrolytic rate at low drug concentration as alimiting factor in resistance to newer cephalosporins Rev Infect Dis 198810746ndash751

7 Chong Y Lee K Kwon OH In-vitro activities of cefepime against Enterobactercloacae Serratia marcescens Pseudomonas aeruginosa and other gram-negativebacilli J Antimicrob Chemother 199332(suppl 3)21ndash29

8 Quintiliani R Nicolau DP Nightingale CH Clinical relevance of penicillin-resistantStreptococcus pneumoniae Infect Dis Clin Prac 19965S37ndashS41

9 Chow JW Fine MJ Shales DM Quinn JP Hooper DC Johnson MP et al Entero-bacter bacteremia Clinical features and emergence of antibiotic resistance duringtherapy Ann Intern Med 1991115585ndash590

10 Owens RC Ambrose PG Quintiliani R Ceftazidime to cefepime formulary switchPharmacodynamic and pharmacoeconomic rationale Conn Med 1997225ndash227

11 Ambrose PG Richerson MA Stanton ME Bui K Nicolau DP Nightingale CH etal Cost-effectiveness analysis of cefepime compared with ceftazidime in intensivecare unit patients with hospital-acquired pneumonia Infect Dis Clin Prac 19998245ndash251

12 Owens RC Owens CA Holloway WJ Comparative evaluation of the cefepime Q12hour dosing interval as empirical therapy for febrile neutropenic patients Pharmaco-therapy 199919496ndash497

406 Ambrose et al

13 Product information Zithromax (azithromycin) Pfizer Inc June 199614 Product information Biaxin (clarithromycin) Abbott Laboratories August 199415 Shepard RM Fouda HG Johnson RC et al Disposition and metabolism of azithro-

mycin in rats dogs and humans Int Congr Infect Dis Montreal Quebec July 15ndash19 1990 abstr 184

16 Jorgenson JH Maher LA Howell AW Activity of clarithromycin and its principalhuman metabolite against Haemophilus influenzae Antimicrob Agents Chemother1994351524ndash1526

17 Craig WA Rikardsdottir S Watanable Y In vivo and in vitro post-antibiotic effectsof azithromycin Abstracts of the 32nd Interscience Conference on AntimicrobialAgents and Chemotherapy Anaheim September 1992

18 Odenholt-Tornqvist I Lowdin E Cars O Post-antibiotic effects and post-antibioticsub-MIC effects of roxithromycin clarithromycin and azithromycin on respiratorytract pathogens Antimicrob Agents Chemother 199539221ndash226

19 Nightingale CH This volume Chapter 920 Jackson MA Burry VF Olson LC Duthie SE Breakthrough sepsis in macrolide-

resistant pneumococcal infection Pediatr Infect Dis 1996151049ndash105021 Leach AJ Shelby-James TM Mayo M Gratten M Laming AC Currie BJ Mathews

JD A prospective study of the impact of community-based azithromycin treatmentof trachoma on carriage and resistance of Streptococcus pneumonia Clin Infect Dis199724356ndash362

22 Davies BI Maesen FPV Gubbelmans R Azithromycin (CP-62993) in acute exacer-bations of chronic bronchitis An open clinical microbiological and pharmacokineticstudy J Antimicrob Chermother 198823743ndash751

23 Guggenbichler JP Kastner U Influence of antibiotics on the normal flora InfectMed 199815(suppl A)15ndash22

24 van Barlingen H Volmer T Lacey LF The clinical failure rate of first-line antibiotictreatment is the key cost driver in the treatment of patients with severe community-acquired lower respiratory tract infections Abstracts 6th Intl Symp on QuinolonesDenver CO November 1998

25 Lacey LF Volmer T Harris AM The microbiological eradication rate of first-lineantibiotic treatment is a key cost driver in the treatment of acute exacerbation ofchronic bronchitis Abstracts 6th Intl Symp on Quinolones Denver CO November1998

26 Weiss LR A clinical study of macrolide therapy in a select patient population Ab-stracts 4th Intl Conf on the Macrolides Azilides Streptogramins and KetolodesBarcelona Spain January 1998

27 Preston SL Drusano GL Berman AL Fowler CL Chow AT Dornseif B et alLevofloxacin population pharmacokinetics and creation of a demographic model forprediction of individual drug clearance in patients with serious community-acquiredinfection Antimicrob Agents Chemother 1998421098ndash1104

28 Drusano GL Johnson DE Rogan M Standford HC Pharmacodynamics of a fluor-oquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sepsisAntimicrob Agents Chemother 199337483ndash490

29 Dudley MN Pharmacodynamics and pharmacokinetics of antibiotics with specialreference to the fluoroquinolones Am J Med 1991 91(suppl 6A)45Sndash50S


30 Forrest A Nix DE Ballow CH Schentag JJ Pharmacodynamics of intravenous ci-profloxacin in seriously ill patients Antimicrob Agents Chemother 1993371073ndash1081

31 Lacy ML Lu W Xu X Nicolau DP Quintiliani R Nightingale CH Pharmacody-namic comparisons of levofloxacin and ciprofloxacin against Streptococcus pneu-moniae in an in vitro model of infection Antimicrob Agents Chemother 199943672ndash677


33 Vesga O Craig WA Activity of levofloxacin against penicillin-resistant Strepto-coccus pneumoniae in normal and neutropenic mice Abstracts 36th InterscienceConf on Antimicrobial Agents and Chemotherapy New Orleans Sept 15ndash181996

34 Ambrose PG Grasela DM Pharmacodynamics of fluoroquinolones against Strep-tococcus pneumoniae Analysis of phase-III clinical trials Abstracts 40th Inter-science Conf on Antimicrobial Agents and Chemotherapy Toronto Sept 17ndash202000

35 Ambrose PG Quintiliani R Limitations of single-point pharmacodynamic analysisPediatr Infect Dis J 200019769

36 Ambrose PG Grasela DM Effect of pharmacokinetic and microbiological variabil-ity on the pharmacodynamics of gatifloxacin and levofloxacin against Streptococcuspneumoniae Abstracts 39th Interscience Conf on Antimicrobial Agents and Che-motherapy San Francisco Sept 26ndash29 1999

37 Jones RN Pfaller MA Doern GV Beach M Antimicrobial activity of gatifloxacina newer 8-methoxy fluoroquinolone tested against over 23000 recent clinical iso-lates from the SENTRY antimicrobial surveillance program Abstracts 37th Intersci-ence Conf on Antimicrobial Agents and Chemotherapy San Diego Sept 24ndash271997


39 Mayer H Pierce P Ambrose PG Bacteriologic eradication of gram-positive organ-isms Gatifloxacin and levofloxacin Abstracts Am Thoracic Soc Chicago May 5ndash10 2000

40 Fung-Tomc J Huczko E Stickle T Minassian B Kolek K Denbleyker KBonner D Kessler R Antibacterial effects of cefprozil compared with those of13 oral caphems and 3 macrolides Antimicrob Agents Chemother 199539(2)533ndash538

41 Doern GV Brueggemann AB Pierce G Hogan T Holley HP Rauch A Prevalenceof antimicrobial resistance among 723 outpatient clinical isolates of Moraxella cat-tarhalis in the United States in 1994 and 1995 Antimicrob Agents Chemother 199640(12)2884ndash2886

42 Doern GV Brueggeman AB Pierce G Holley HP Rauch A Antibiotic resistanceamong clinical isolates of Haemophilus influenzae in the United Sataes in 1994 and1995 Antimicrob Agents Chemother 199741(2)292ndash297

408 Ambrose et al

43 Bauerfeind A Comparison of the antibacterial activities of the quinolones BAY12-8039 gatifloxacin trovefloxacin clinafloxacin levofloxacin and ciprofloxacin JAntimicrob Therapy 199740639ndash651

44 Goldstein EJC Citron DM Merriam CV Tyrell K Warren Y Activity of gatifloxa-cin compared to those of five other quinolones versus aerobic and anaerobic isolatesfrom skin and soft tissue samples of human and animal bite wound infections Anti-microb Agents Chemother 199943(6)1475ndash1479

409

Index

Aminoglycosides 125clinical use and application 139mechanism of action 126microbiologic spectrum 127PAE 133pharmacodynamics 129pharmacokinetics 128resistance 137synergy 137toxicodynamics 138

Animal models (studies) 10 67beta-lactams 105changing drug elimination 76changing drug input 76design issues 73 81

comparison to humanpharmacokinetics 75

drug assay 74modeling 74sampling 73

endocarditis 86endpoints 68 82mathematical indices 79meningitis model 85models 83

thigh infection 83

[Animal models (studies)]osteomyelitis 86quantifying 72quinolones 163peritonitissepticemia model 84pharmacokinetics 72pneumonia animal model 83pyelonephritiskidney model 84relevance to humans 68resistance 89selection of appropriate PK-PD

models 82streptogramins 236vancomycin 188

Antibacterial resistance 327Antifungals 285

Amphotercin B 287in vitro data 288in vivo data 290

animal models 286antifungal combinations 296azoles 292

in vitro data 292clinical studies 294

clinical data 287flucytosine 294

410 Index

[Antifungals]in vitro data 294in vivo data 295

in vitro data 286Antivirals 259

clinical studies of pharmacodynamics272

in vitro systems 260protein binding 262

Area under the concentration time curve(AUC) 159

AUCquinolones 160

Azalides 205pharmacodynamic model 213pharmacodynamics 207tissue distribution 208

Biofilm-catheter model 54Beta-lactamase inhibitor combinations

111Beta-lactams 99Breakpoints 89 155

Clindamycin 227antimicrobial activity 227chemistry 227in vitro studies 228mechanism of action 227pharmacodynamics 228pharmacokinetics 228PAE 230serum bactericidal titer 229serum inhibitory titer 229

Clinical decision making 385azithromycin 393

microbiology 394pharmacodynamics 394pharmacokinetics 394

clarithromycin 393microbiology 394pharmacodynamics 394pharmacokinetics 394

cefepime 386microbiology 387pharmacodynamics 390

[Clinical decision making]pharmacoeconomics 391pharmacokinetics 387

ceftazidime 386microbiology 387pharmacodynamics 390pharmacoeconomics 391pharmacokinetics 387

gatifloxacin 399microbiology 399pharmacodynamics 402pharmacokinetics 399

levofloxacin 399microbiology 399pharmacodynamics 402pharmacokinetics 399

Clinical efficacy 33Clinical trial data

drug exposure 308endpoints 304human pharmacodynamics 303pathogen identification 304susceptibility determination 304

Clinical pharmacodynamic targets 28160

Clinical practice 35application of pharmacodynamic pa-

rameters 35Clinical studies 106Combination therapy in vitro model 51Concentration-dependent killing 158

aminoglycosides 125metronidazole 225quinolones 155

Continuous infusionbeta-lactams 106

Doxycycline 249Drug combinations 12

Effect of antimicrobial agents on micro-organisms 24

Endocarditis model 86Experimental infection models 235

streptogramins 235

Index 411

Fibrin clot in vitro model 55Formulary decision making 385

azithromycin 393microbiology 394pharmacodynamics 394pharmacokinetics 394


cefepime 386microbiology 387pharmacodynamics 390pharmacoeconomics 391pharmacokinetics 387




Free drug 163

Glycylcyclines 250

Human infections 12Human studies

quinolones 165vancomycin 188

Intracellular pathogens in vitro model57

In vitro models 41bacterial adherence 62beta-lactams 104clindamycin 228dilutionary artifacts 62limitations 59

altered bacterial growthcharacteristics 60

[In vitro models]morphology 60physiology 60sensitivity to serum 61virulence 60

effects of immune system 59microorganism viability 61protein binding 61quinolones 163resistance 113streptogramins 236vancomycin 185

In vitro studies 104

Ketolides 205Killing

bacterial activity 2Kinetic bacterial killing properties 26

LY333328 197

Macrolides 205azithromycin 393 394 398clarithromycin 393 394 398pharmacodynamic model 213pharmacodynamics 207tissue distribution 208

MBC 25Meningitis animal model 85Metronidazole 221

absorption 223antimicrobial activity 222

bacteria 223protozoa 223

bactericidal effect 225chemistry and mechanism of action

222concentration dependent killing 225distribution 224excretion 225metabolism 224PAE 226pharmacodynamics 225pharmacokinetics 223serum bactericidal titers 226minocycline 249

412 Index

MIC 25Microbiology

cefepime 387ceftazidime 387gatifloxacin 399levofloxacin 399

Minimum bactericidal concentration 2Minimum inhibitory concentration 2

New antimicrobials and formulations 15

One-compartment in vitro model 43Osteomyelitis animal model 86Outcome data

quinolones 163

Parameters for microbiological activityof antimicrobial agents 25

Patterns of antimicrobial activity 5Peak concentration ratio (CpMIC) 160PeakMIC

resistance 352quinolones 160

PeritonitisSepticemia animal model 84Pharmacodynamic analysis 169

single point 170Monte Carlo 171

Pharmacodynamic concepts 1 158Pharmacodynamics

aminoglycosides 125 129animal models 82azithromycin 394beta-lactams 99

correlation to in vitro resistance 113cefepime 391ceftazidime 391clarithromycin 394clindamycin 228clinical efficacy 33 35clinical use and application 139gatifloxacin 402human clinical trials 303levofloxacin 402magnitude required for clinical

efficacy 9

[Pharmacodynamics]metronidazole 225new dosage regimens 14parameters 5 29pharmacokinetics versus pharmacody-

namics 100quinolones 167resistance 349 352

peakMIC 352streptogramins 234tetracyclines 251

Pharmacoeconomics 367 368azithromycin 398cefepime 391ceftazidime 391clarithromycin 398cost 369data sources 379decision analysis 378evaluating studies 380methodological considerations

375outcomes 372

measures 372perspective 369types 370

cost benefit 371cost effectiveness 371Pharmacokinetics 28

aminoglycosides 125 128animal models 72 82

comparison to humans 75basic principles 184cefepime 387ceftazidime 387clindamycin 228gatifloxacin 399glycylcyclines 250doxycycline 249levofloxacin 399minocycline 249quinolones 161magnitude required for clinical

efficacy 9metronidazole 223parameters 5

Index 413

[Pharmacokinetics]pharmacodynamics versus pharmaco-

kinetics 100streptogramins 233tissue fluid 28tetracyclines 248

Pneumonia animal model 83Post-Antibiotic Effect (PAE) 3 87

101 133 159clindamycin 230metronidazole 226

Post antibiotic leukocyte enhancement4

Protein binding 89 103Prevention of resistance 14Pyelonephritiskidney animal model

84

Quinolones 155animal models (studies) 163AUC 160human studies 165in vitro models (studies) 163outcome data 163peakMIC 160

Quinolone generation 156

Resistance 89 137 327acquired 350antibacterial 327automated susceptibility systems 344detection in clinical laboratory 333

clinical relevance susceptibilitytests 333

emerging problems 328gram-negative bacteria 329

E coli 330H influenzae 329K pneumoniae 330other enterobacteriaceae 331P aeruginosa 333

gram-positive bacteria 328enterococcus 328S aureus 328S pneumoniae 329

[Resistance]interpretive breakpoints 343methods for detection 334

aminoglycoside 335beta-lactam 335cat 339chloramphenicol acetyltransferase

339direct detection of beta-lactamases

337gene products 337glycopeptide 335

mutational 350qualitative assays 347

disk diffusion 347ESBL disk diffusion 347

quantitative assays 341agar dilution 342e-test 342macrobroth dilution 341microbroth dilutions 342

pharmacodynamics and resistance349 352

ceftazidime 354levofloxacin 353ticarcillin 354

screening assays 344ESBL 346high level aminoglycoside screen

346MRSA screen 346vancomycin screen 346

susceptibility methods phenotypic339

in vivo correlation 339growth in or on artificial media

340inoculum size 340pharmacodynamic correlations

341protein binding 340

Serum bactericidal titers 226clindamycin 229metronidazole 226

Serum inhibitory titer 229

414 Index

Streptogramin 231absorption 232animal models 236antimicrobial activity 231bactericidal effect 234chemistry 231distribution 232excretion 234experimental infection models 235in vitro models 236mechanism of action 231metabolism 233PAE 235pharmacodynamics 234pharmacokinetics 232synergy 137 238

Sub-MIC effects 4Susceptibility breakpoint determinations

15

Teicoplanin 196Tetracyclines 247

doxycycline 249glycylcyclines 250minoclycline 249pharmacokinetics 248pharmacodynamics 251

Thigh infection model 83Time-dependent killing 158

beta-lactams 99macrolides 205

[Time dependent killing]tetracyclines 247vancomycin 178

Tissue distributionazalides 208macrolides 208

Tissue fluid 28Tissue penetration 109Total drug 163Toxicodynamics aminoglycosides

138Transitional therapy 156Two-compartment in vitro model

48

Vancomycin 178adverse drug reactions 193animal studies 188antimicrobial spectrum 178clinical application 189clinical uses 189dosing 193history 178human studies 188inappropriate uses 192in vitro studies 185pharmacokinetics 180pharmacology 179resistance 181therapeutic monitoring 193toxicity 193

Right

Bottom

Marcel Dekker Inc New York bull BaselTM

edited by

Charles H NightingaleHartford Hospital

Hartford Connecticut

Antimicrobial Pharmacodynamics

in Theory and Clinical Practice

Takeo MurakawaFujisawa Pharmaceutical Company Ltd

Osaka Japan

Paul G AmbroseCognigen Corporation

Buffalo New York

Copyright copy 2001 by Marcel Dekker Inc All Rights Reserved

ISBN 0-8247-0561-0

This book is printed on acid-free paper

HeadquartersMarcel Dekker Inc270 Madison Avenue New York NY 10016tel 212-696-9000 fax 212-685-4540

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Copyright 2002 by Marcel Dekker Inc All Rights Reserved

Neither this book nor any part may be reproduced or transmitted in any form or by anymeans electronic or mechanical including photocopying microfilming and recordingor by any information storage and retrieval system without permission in writing fromthe publisher

Current printing (last digit)10 9 8 7 6 5 4 3 2 1

PRINTED IN THE UNITED STATES OF AMERICA

INFECTIOUS DISEASE AND THERAPY

Series Editor

Burke A Cunha

Winthrop-University HospitalMineola and

State University of New York School of MedicineStony Brook New York

1 Parasitic Infections in the Compromised Host edited by Peter DWalzer and Robert M Genta

2 Nucleic Acid and Monoclonal Antibody Probes Applications inDiagnostic Methodology edited by Bala Swaminathan and GyanPrakash

3 Opportunistic Infections in Patients with the Acquired Immuno-deficiency Syndrome edited by Gifford Leoung and John Mills

4 Acyclovir Therapy for Herpesvirus Infections edited by David ABaker

5 The New Generation of Quinolones edited by Clifford Siporin Carl LHeifetz and John M Domagala

6 Methicillin-Resistant Staphylococcus aureus Clinical Managementand Laboratory Aspects edited by Mary T Cafferkey

7 Hepatitis B Vaccines in Clinical Practice edited by Ronald W Ellis8 The New Macrolides Azalides and Streptogramins Pharmacology

and Clinical Applications edited by Harold C Neu Lowell S Youngand Stephen H Zinner

9 Antimicrobial Therapy in the Elderly Patient edited by Thomas TYoshikawa and Dean C Norman

10 Viral Infections of the Gastrointestinal Tract Second Edition Revisedand Expanded edited by Albert Z Kapikian

11 Development and Clinical Uses of Haemophilus b Conjugate Vac-cines edited by Ronald W Ellis and Dan M Granoff

12 Pseudomonas aeruginosa Infections and Treatment edited byAldona L Baltch and Raymond P Smith

13 Herpesvirus Infections edited by Ronald Glaser and James F Jones14 Chronic Fatigue Syndrome edited by Stephen E Straus15 Immunotherapy of Infections edited by K Noel Masihi16 Diagnosis and Management of Bone Infections edited by Luis E

Jauregui17 Drug Transport in Antimicrobial and Anticancer Chemotherapy

edited by Nafsika H Georgopapadakou

18 New Macrolides Azalides and Streptogramins in Clinical Practiceedited by Harold C Neu Lowell S Young Stephen H Zinner andJacques F Acar

19 Novel Therapeutic Strategies in the Treatment of Sepsis edited byDavid C Morrison and John L Ryan

20 Catheter-Related Infections edited by Harald Seifert Bernd Jansenand Barry M Farr

21 Expanding Indications for the New Macrolides Azalides andStreptogramins edited by Stephen H Zinner Lowell S YoungJacques F Acar and Harold C Neu

22 Infectious Diseases in Critical Care Medicine edited by Burke ACunha

23 New Considerations for Macrolides Azalides Streptogramins andKetolides edited by Stephen H Zinner Lowell S Young Jacques FAcar and Carmen Ortiz-Neu

24 Tickborne Infectious Diseases Diagnosis and Management editedby Burke A Cunha

25 Protease Inhibitors in AIDS Therapy edited by Richard C Ogdenand Charles W Flexner

26 Laboratory Diagnosis of Bacterial Infections edited by Nevio Cimolai27 Chemokine Receptors and AIDS edited by Thomas R OrsquoBrien28 Antimicrobial Pharmacodynamics in Theory and Clinical Practice

edited by Charles H Nightingale Takeo Murakawa and Paul G Am-brose

29 Pediatric Anaerobic Infections Diagnosis and Management ThirdEdition Revised and Expanded Itzhak Brook

Additional Volumes in Production

Preface

To use antibiotics appropriately the clinician needs to understand fundamentalpharmacodynamic concepts These concepts are essential for they form the verybasis for therapeutic strategies that maximize clinical benefit while minimizingtoxicity to the patient The objectives of this book are first to review the constel-lation of scientific and medical literature concerning antibiotics and pharmacody-namics The relevance of this complex information is then synthesized into aneasy-to-understand discussion of concept and theory Finally the reader is shownhow to apply these theories and concepts with specific examples to the clinicalpractice of medicine and pharmacy In other words this book takes the readerfrom the test tube through the animal and human volunteer laboratory to thepatientrsquos bedside

The book includes a thorough discussion of the pharmacodynamics of allmajor classes of the antimicrobial armamentarium These include penicillinscephalosporins cephamycins carbapenems monobactams aminoglycosidesquinolones macrolides antifungals antivirals and others Additionally a phar-macodynamic discussion of new classes of antimicrobial agents that are uponthe horizon such as the ketolide antibiotics is included

This book is unique in that no other text of its kind currently exists Theinformation that this book provides integrates medical microbiology clinical in-fectious diseases and pharmacokinetics This book pulls together in one text theessential elements of these disciplines and does so in a very understandable andpractical manner

The infectious disease physicians and pharmacists we selected as contribu-tors are eminently qualified and are recognized experts in their field Moreoverthese authors were chosen on the basis of their ability to convey their perspective

iii

iv Preface

and expertise lucidly which makes them ideal teachers They all agreed that therewas a need for such a book and were excited about joining in this venture

This book will find an audience in a large array of healthcare disciplinesincluding college educators medical pharmacy and microbiology students in-fectious disease physicians and pharmacy specialists medical house staff clinicaland staff pharmacists clinical microbiologists and other healthcare decisionmakers

Charles H NightingaleTakeo MurakawaPaul G Ambrose

Contents

Preface iiiContributors vii

1 Pharmacodynamics of Antimicrobials General Concepts andApplications 1William A Craig

2 Microbiology and Pharmacokinetics 23Charles H Nightingale and Takeo Murakawa

3 In Vitro Antibiotic Pharmacodynamic Models 41Michael J Rybak George P Allen and Ellie Hershberger

4 Animal Models of Infection for the Study of AntibioticPharmacodynamics 67Michael N Dudley and David Griffith

5 β-Lactam Pharmacodynamics 99JoCarol J McNabb and Khanh Q Bui

6 Aminoglycoside Pharmacodynamics 125Myo-Kyoung Kim and David P Nicolau

7 Pharmacodynamics of Quinolones 155Robert C Owens Jr and Paul G Ambrose

v

vi Preface

8 Glycopeptide Pharmacodynamics 177Gigi H Ross David H Wright John C Rotschafer andKhalid H Ibrahim

9 Macrolide Azalide and Ketolide Pharmacodynamics 205Charles H Nightingale and Holly M Mattoes

10 Metronidazole Clindamycin and StreptograminPharmacodynamics 221Kenneth Lamp Melinda K Lacy and Collin Freeman

11 Tetracycline Pharmacodynamics 247Burke A Cunha and Holly M Mattoes

12 Pharmacodynamics of Antivirals 259George L Drusano Sandra L Preston and Peter J Piliero

13 Antifungal Pharmacodynamics 285Michael E Klepser and Russell E Lewis

14 Human Pharmacodynamics of Anti-Infectives Determinationfrom Clinical Trial Data 303George L Drusano

15 Antibacterial Resistance 327Philip D Lister

16 Basic Pharmacoeconomics 367Mark A Richerson and Eugene Moore

17 Utilizing Pharmacodynamics and Pharmacoeconomics inClinical and Formulary Decision Making 385Paul G Ambrose Annette Zoe-Powers Rene RussoDavid T Jones and Robert C Owens Jr

Index 409

Contributors

George P Allen PharmD Postdoctoral Fellow Pharmacy Practice WayneState University and Detroit Receiving Hospital and University Health CenterDetroit Michigan

Paul G Ambrose PharmD Director Infectious Disease Research CognigenCorporation Buffalo New York

Khanh Q Bui PharmD Bristol-Myers Squibb Company Chicago Illinois

William A Craig MD Professor of Medicine and Therapeutics Departmentof Infectious Diseases University of Wisconsin and William S Middleton Me-morial Veterans Hospital Madison Wisconsin

Burke A Cunha MD Chief Infectious Disease Division Winthrop-University Hospital Mineola and Professor of Medicine State University ofNew York School of Medicine Stony Brook New York

George L Drusano MD Professor Department of Medicine and Pharmacol-ogy Clinical Research Institute Albany Medical College Albany New York

vii

viii Contributors

Michael N Dudley PharmD Vice President Department of Pharmacologyand Microbiology Microcide Pharmaceuticals Inc Mountain View California

Collin Freeman PharmD Clinical Science Specialist Department of Scien-tific Affairs Bayer Corporation West Haven Connecticut

David Griffith BS Research Scientist Department of Pharmacology Micro-cide Pharmaceuticals Inc Mountain View California

Ellie Hershberger PharmD Director Infectious Disease Laboratory Re-search Institute William Beaumont Research Institute Royal Oak Michigan

Khalid H Ibrahim PharmD Infectious Diseases Research Fellow Depart-ment of Experimental and Clinical Pharmacology University of Minnesota Min-neapolis Minnesota

David T Jones PharmD MD Department of Cardiology and Internal Med-icine University of California at Davis Sacramento California

Myo-Kyoung Kim PharmD Infectious Disease Fellow Department of Phar-macy Research Hartford Hospital Hartford Connecticut

Michael E Klepser PharmD Associate Professor College of PharmacyUniversity of Iowa Iowa City Iowa

Melinda K Lacy PharmD Assistant Professor Department of PharmacyPractice School of Pharmacy University of Kansas Medical Center Kansas CityKansas

Kenneth Lamp PharmD Medical Science Manager Department of UnitedStates Medicines Bristol-Myers Squibb Plainsboro New Jersey

Russell E Lewis PharmD Assistant Professor Department of Clinical Sci-ences and Administration University of Houston College of Pharmacy HoustonTexas

Philip D Lister PhD Associate Professor Department of Medical Microbiol-ogy and Immunology Creighton University School of Medicine Omaha Ne-braska

Holly M Mattoes PharmD Scientific Communications Manager DesignWriteIncorporated Princeton New Jersey

Contributors ix

JoCarol J McNabb PharmD Assistant Professor College of PharmacyUniversity of Nebraska Medical Center Omaha Nebraska

Eugene Moore PharmD Coordinator Army Ambulatory Care PharmacistProgram Department of Defense Pharmacoeconomic Center Fort Sam HoustonTexas

Takeo Murakawa PhD Supervisor International Development and Regula-tory Affairs Development Division Fujisawa Pharmaceutical Company LtdOsaka and Lecturer (Part) Graduate School of Pharmaceutical Sciences KyotoUniversity Kyoto and Graduate School of Pharmaceutical Sciences TokushimaUniversity Tokushima Japan

David P Nicolau PharmD Division of Infectious Disease Pharmacy Re-search Hartford Hospital Hartford Connecticut

Charles H Nightingale PhD Vice-President for Research and Director ofthe Institute for International Healthcare Studies Hartford Hospital HartfordConnecticut

Robert C Owens Jr PharmD Clinical Specialist Infectious DiseasesMaine Medical Center Portland Maine and University of Vermont College ofMedicine Burlington Vermont

Peter J Piliero MD Assistant Professor Department of Medicine AlbanyMedical College Albany New York

Sandra L Preston PharmD Assistant Professor of Medicine Clinical Re-search Initiative Albany Medical College Albany New York

Mark A Richerson PharmD MS Commander Medical Service CorpsUnited States Navy and Department of Defense Pharmacoeconomic Center FortSam Houston Texas

Gigi H Ross PharmD Infectious Diseases Scientific Liaison Clinical Af-fairs Ortho-McNeil Pharmaceutical Raritan New Jersey

John C Rotschafer PharmD FCCP Professor Department of Experi-mental and Clinical Pharmacology College of Pharmacy University of Minne-sota Minneapolis Minnesota

Rene Russo PharmD Postdoctoral Fellow College of Pharmacy RutgersUniversity Piscataway New Jersey

x Contributors

Michael J Rybak PharmD FCCP BCPS Professor of Pharmacy andMedicine and Director Anti-Infective Research Laboratory Department of Phar-macy Practice Wayne State University and Detroit Receiving Hospital and Uni-versity Health Center Detroit Michigan

David H Wright PharmD Manager Professional Education and ScientificAffairs Ortho-McNeil Pharmaceutical Raritan New Jersey

Annette Zoe-Powers PharmD Director Department of Infectious DiseasesBristol-Myers Squibb Company Plainsboro New Jersey

1

Pharmacodynamics of AntimicrobialsGeneral Concepts and Applications

William A Craig

University of Wisconsin and William S Middleton Memorial Veterans HospitalMadison Wisconsin

1 INTRODUCTION

lsquolsquoPharmacodynamicsrsquorsquo is the term used to reflect the relationship between mea-surements of drug exposure in serum tissues and body fluids and the pharmaco-logical and toxicological effects of drugs With antimicrobials pharmacodynam-ics is focused on the relationship between concentrations and the antimicrobialeffect Studies in the past have focused on pharmacokinetics and descriptions ofthe time course of antimicrobials in serum tissues and body fluids Much lessemphasis has been placed on the time course of antimicrobial activity Studiesover the past 20 years have demonstrated marked differences in the time courseof antimicrobial activity among antibacterials and antifungals [15208488] Fur-thermore the pattern of antimicrobial activity over time is an important determi-nant of optimal dosage regimens [21] This chapter focuses on general conceptsand the application of pharmacodynamics to antimicrobial therapy

1

2 Craig

2 MEASUREMENTS OF ANTIMICROBIAL ACTIVITY

21 Minimum Inhibitory and Minimum Bactericidal

Concentrations

The minimum inhibitory and minimum bactericidal concentrations (MIC andMBC) have been the major parameters used to measure the in vitro activity ofantimicrobials against various pathogens Although the MIC and MBC are excel-lent predictors of the potency of an antimicrobial against the infecting organismthey provide essentially no information on the time course of antimicrobial activ-ity For example the MBC provides minimal information on the rate of bacteri-cidal and fungicidal activity and on whether killing can be increased by higherdrug concentrations In addition the MIC provides no information on growthinhibitory effects that may persist after antimicrobial exposure These persistenteffects are due to three different phenomena the postantibiotic effect (PAE)the postantibiotic sub-MIC effect (PAE-SME) and the postantibiotic leukocyteenhancement (PALE) [176263] The effects of increasing concentrations on thebactericidal and fungicidal activity of antimicrobials combined with the magni-tude of persistent effects give a much better description of the time course ofantimicrobial activity than that provided by the MIC and MBC

22 Killing Activity

Antimicrobials exhibit two primary patterns of microbial killing The first patternis characterized by concentration-dependent killing over a wide range of concen-trations With this pattern higher drug concentrations result in a greater rate andextent of microbial killing This pattern is observed with the aminoglycosidesfluoroquinolones ketolides metronidazole and amphotericin B [9192028] Thesecond pattern is characterized by minimal concentration-dependent killing Withthis pattern saturation of the killing rate occurs at low multiples of the MICusually around four to five times the MIC Drug concentrations above these val-ues do not kill microbes faster or more extensively This pattern is also calledtime-dependent killing because the extent of microbial killing is primarily depen-dent on the duration of exposure This pattern is observed with β-lactam antibiot-ics macrolides clindamycin glycopeptides tetracyclines trimethoprim linezo-lid and flucytosine [48152029]

The different patterns of bacterial killing are illustrated in Fig 1 by showingthe effect of increasing drug concentrations on the in vitro antimicrobial activityof tobramycin ciprofloxacin and ticarcillin against a standard strain of Pseudo-monas aeruginosa [20] Increasing concentrations of tobramycin and ciprofloxa-cin produced more rapid and extensive bacterial killing as exhibited by thesteeper slopes of the killing curves With ticarcillin there was a change in slopeas the concentration was increased from one to four times the MIC However


FIGURE 1 Time-kill curves of Pseudomonas aeruginosa ATCC 27853 with ex-posure to tobramycin ciprofloxacin and ticarcillin at concentrations fromone-fourth to 64 times the MIC (From Ref 20)

higher concentrations did not alter the slope The slight reduction in bacterialnumbers at the higher doses is due to an earlier onset of bacterial killing From2 h on ticarcillin concentrations from 4 to 64 times the MIC produced the samerates of killing

23 Persistent Effects

lsquolsquoPostantibiotic effectrsquorsquo is the term used to describe the persistent suppression ofbacterial growth following antimicrobial exposure [151863] If reflects the timeit takes for an organism to recover from the effects of exposure to an antimicrobialand resume normal growth This phenomenon was first observed in the 1940s inearly studies with penicillin against staphylococci and streptococci [1238] Laterstudies starting in the 1970s extended this phenomenon to newer drugs and togram-negative organisms The postantibiotic effect is demonstrated in vitro byfollowing bacterial growth kinetics after drug removal

Moderate to prolonged in vitro postantibiotic effects are observed for allantibacterials with susceptible gram-positive bacteria such as staphylococci andstreptococci [10] Moderate to prolonged in vitro PAEs are also observed withgram-negative bacilli for drugs that are inhibitors of protein or nucleic acid syn-thesis In contrast short or no postantibiotic effects are observed for β-lactamantibiotics with gram-negative bacilli The only exception has been for carbapen-ems which exhibit moderate PAEs primarily with strains of Pseudomonas aeru-

4 Craig

ginosa [1647] In vitro postantifungal effects (PAFEs) have been observed withvarious yeasts following exposure to amphotericin B and flucytosine but not totriazoles such as fluconazole [3984]

The postantibiotic sub-MIC effect demonstrates the additional effect sub-MIC concentrations can have on the in vitro PAE For example exposure ofstreptococci in the PAE phase to macrolides at drug concentrations of one-tenthand three-tenths of the MIC increased the duration of the postantibiotic effectby about 50 and 100 respectively [1871] The PAE phase can also makestreptococci hypersensitive to the killing effects of penicillin [17] The durationof the postantibiotic sub-MIC effects reported in the literature includes the dura-tion of the PAE plus the enhanced duration due to sub-MIC concentrations Mor-phological changes such as filaments can also be produced by sub-MIC concen-trations [60]

Postantibiotic leukocyte enhancement describes the effects of leukocyteson bacteria during the postantibiotic phase Studies have demonstrated that suchbacteria are more susceptible to intracellular killing or phagocytosis by leuko-cytes [1862] This phenomenon can also prolong the duration of the in vitropostantibiotic effect Antimicrobials that produce the longest postantibiotic ef-fects tend to exhibit the most prolonged effects when exposed to leukocytes

The postantibiotic effect has also been demonstrated in vivo in a varietyof animal infection models [1822] The in vivo phenomenon is actually a combi-nation of the in vitro PAE and sub-MIC effects from gradually falling drug con-centrations The largest number of studies have used the neutropenic mouse thighinfection model [88] When performed in non-neutropenic mice the in vivo PAEwould also include any postantibiotic leukocyte enhancement effects

There are several important differences between the in vivo and in vitroPAEs In most cases in vivo PAEs are longer than in vitro PAEs most likelybecause of the additive effect of sub-MIC concentrations Simulation of humanpharmacokinetics can further enhance the duration of the in vivo PAE by a similarmechanism Prolongation of sub-MIC concentrations of amikacin by simulatingthe human drug half-life (2 h) extended the duration of in vivo PAEs by 40ndash100over values observed with a dose producing the same area under the concentrationversus time curve (AUC) but eliminated with a murine half-life of 20 min [27]In vivo PAEs with some drugs are further prolonged by the presence of leuko-cytes In general the presence of neutrophils tends to double the duration of thein vivo postantibiotic effect for aminoglycosides and fluoroquinolones with gram-negative bacilli [1827] However leukocytes have no major effect on the mini-mal in vivo PAEs observed for β-lactams with gram-negative bacilli

There are also some differences between the in vitro and in vivo PAEs thatquestion the value of measuring the in vitro PAE First the duration of the invitro PAE is not predictive of the duration of the in vivo PAE [40] Secondprolonged PAEs for penicillin and cephalosporins with streptococci are observed


in vitro but not in vivo [228088] Third in vitro studies that suggest that the PAEof aminoglycosides decreases and disappears over a prolonged dosing interval orwith repeated doses have not been confirmed in vivo [3564] Fourth fluconazoleexhibits a postantifungal effect in vivo but not in vitro [384]

3 PATTERNS OF ANTIMICROBIAL ACTIVITY

The pharmacodynamic characteristics described above suggest that the timecourse of antimicrobial activity can vary markedly for different antibacterial andantifungal agents As shown in Table 1 these drugs exhibit three major patternsof antimicrobial activity The first pattern in characterized by concentration-dependent killing and moderate to prolonged persistent effects Higher concentra-tions would kill organisms more rapidly and more extensively than lower levelsThe prolonged persistent effects would allow for infrequent administration oflarge doses This pattern is observed with aminoglycosides fluoroquinolonesdaptomycin ketolides metronidazole and amphotericin B The goal of a dosingregimen for these drugs would be to maximize concentrations The peak leveland the AUC should be the pharmacokinetic parameters that would determinein vivo efficacy

The second pattern is characterized by time-dependent killing and minimalto moderate persistent effects High drug levels would not kill organisms betterthan lower concentrations Furthermore organism regrowth would start very soonafter serum levels fell below the MIC This pattern is observed with β-lactamsmacrolides clindamycin oxazolidinones and flucytosine The goal of a dosingregimen for these drugs would be to optimize the duration of exposure Theduration of time that serum levels exceed some minimal value such as the MICshould be the major pharmacokinetic parameter determining the in vivo efficacyof these drugs

The third pattern is also characterized by time-dependent killing but theduration of the persistent effects is much prolonged This can prevent any re-growth during the dosing interval This pattern is observed with azithromycintetracyclines quinupristin-dalfopristin glycopeptides and fluconazole The goalof a dosing regimen is to optimize the amount of drug administered to ensurethat killing occurs for part of the time and there is no regrowth during the dosinginterval The AUC should be the primary pharmacokinetic parameter that woulddetermine in vivo efficacy

4 PHARMACOKINETICPHARMACODYNAMIC

PARAMETERS

By using the MIC as a measure of the potency of drugndashorganism interactionsthe pharmacokinetic parameters determining efficacy can be converted to

6 Craig

TA

BLE

1T

hre

eP

atte

rns

of

An

tim

icro

bia

lA

ctiv

ity

Pat

tern

1P

atte

rn2

Pat

tern

3

Ph

arm

aco

dyn

amic

char

ac-

Co

nce

ntr

atio

n-d

epen

den

tT

ime-

dep

end

ent

killi

ng

Tim

e-d

epen

den

tki

llin

gte

rist

ics

killi

ng

and

mo

der

ate

toan

dm

inim

alto

mo

der

-an

dp

rolo

ng

edp

ersi

s-p

rolo

ng

edp

ersi

sten

tef

-at

ep

ersi

sten

tef

fect

ste

nt

effe

cts

fect

sA

nti

mic

rob

ials

incl

ud

edA

min

og

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pharmacokineticpharmacodynamic (PKPD) parameters [21] Serum (orplasma) concentrations are used for determining the pharmacokinetic indicesBecause most infections occur in tissues and the common bacterial pathogensare extracellular organisms interstitial fluid concentrations at the site of infectionshould be the primary determinants of efficacy Serum levels are much betterpredictors of interstitial fluid levels than tissue homogenate concentrations Be-cause tissue homogenates mix the interstitial intracellular and vascular compart-ments together they tend to underestimate or overestimate the interstitial fluidconcentration depending on the ability of the drug to accumulate intracellularly[74]

Identification of the primary PKPD parameter that determines efficacy iscomplicated by the high degree of interdependence among the various parame-ters For example a larger dose produces a higher peakMIC ratio a higher AUCMIC ratio and a longer duration of time above MIC If the higher dose producesa better therapeutic effect than a lower dose it is difficult to determine whichPKPD parameter is of major importance because all three increased Howevercomparing the effects of dosage regimens that include different dosing intervalscan reduce much of the interdependence among PKPD parameters Such studiesare often referred to as dose-fractionation studies [3758] For example dividingseveral total doses into 1 2 4 8 and 24 doses administered at 24 12 6 3 and1 h intervals respectively can allow one to identify which PKPD parameter ismost important for in vivo efficacy

Several investigators have used this study design in animal infection modelsto correlate specific PKPD parameters with efficacy for various antimicrobialsagainst gram-positive cocci gram-negative bacilli and Candida species[3492829576187] This is demonstrated graphically in Fig 2 for ceftazidimeagainst Klebsiella pneumoniae and in Fig 3 for temafloxacin against Streptococ-cus pneumoniae In these studies pairs of mice were treated with multiple dosageregimens that varied both the dose and the dosing interval The number of colony-forming units (CFUs) remaining in the thigh after 24 h of therapy was plottedagainst the peakMIC and 24 h AUCMIC ratios and the percentage of time thatserum levels exceeded the MIC that was calculated for each dosage regimen frompharmacokinetic indices As shown in Fig 2 there was a very poor relationshipbetween CFUsthigh and the peakMIC and 24 h AUCMIC ratios On the otherhand an excellent correlation was observed between the number of bacteria inthe thighs and the percentage of time that serum levels exceeded the MIC How-ever the best correlation in Fig 3 was observed with the 24 h AUCMIC ratiofollowed by the peakMIC ratio

The specific PKPD parameters correlating with efficacy in animal infec-tion models for different antibacterials and antifungals are listed in Table 2 Asexpected time above MIC has consistently been the only PKPD parameter corre-lating with the therapeutic efficacy of β-lactam antibiotics Time above MIC is

8 Craig

FIGURE 2 Relationship between three pharmacodynamic parameters (peakMIC ratio 24 h AUCMIC ratio and percentage of time that serum levels ex-ceed the MIC) and the number of K pneumoniae ATCC 53816 in the thighsof neutropenic mice after 24 h of therapy with ceftazidime Each point repre-sents data for one mouse The dotted line reflects the number of bacteria atthe beginning of therapy

FIGURE 3 Relationship between three pharmacodynamic parameters (peakMIC ratio 24 h AUCMIC ratio and percentage of time that serum levels ex-ceed the MIC) and the number of S pneumoniae ATCC 10813 in the thighsof neutropenic mice after 24 h of therapy with temafloxacin Each point repre-sents data for one mouse The dotted line reflects the number of bacteria atthe beginning of therapy


TABLE 2 PKPD Parameters Determining Efficacy for DifferentAntimicrobials

PKPD parameter Antimicrobial

Time above MIC Penicillins cephalosporins aztreonam carbapenemstribactams macrolides clindamycin oxazolidinonesflucytosine

PeakMIC ratio Aminoglycosides fluoroquinolones daptomycin van-comycin teicoplanin amphotericin B

AUCMIC ratio Aminoglycosides fluoroquinolones daptomycin van-comycin ketolides quinupristin-dalfopristin tetracy-clines fluconazole

also the parameter correlating with efficacy of the macrolides clindamycin oxa-zolidinones and flucytosine

The AUCMIC and peakMIC ratios have been the PKPD parameters thatcorrelate with efficacy for aminoglycosides and fluoroquinolones Most studieshave shown slightly better correlation with the AUCMIC ratio than with thepeakMIC ratio PeakMIC ratios appear to be more important in infections wherethe emergence of resistant subpopulations is a significant risk and for drugs thatact on the cell membrane such as daptomycin and amphotericin B [93776]

Although vancomycin tetracyclines azithromycin and quinupristin-dalfopristin do not exhibit concentration-dependent killing the AUCMIC ratiohas been the major PKPD parameter correlating with therapeutic efficacy ofthese drugs in neutropenic animals [2129] A study in normal mice with vanco-mycin and teicoplanin against a strain of S pneumoniae that used mortality asan endpoint demonstrated that the peakMIC ratio was the most important param-eter [55]

5 MAGNITUDE OF PKPD PARAMETER REQUIRED

FOR EFFICACY

Because PKPD parameters can correct for differences in a drugrsquos pharmacoki-netics and intrinsic antimicrobial activity one would expect that the magnitude ofthe PKPD parameters required for efficacy would be similar in different animalspecies Thus results from studies in animal infection models could be predictiveof the activity of drugs in humans This would be especially helpful in designingdosage regimens for both old and new antibacterials in situations where it isdifficult to obtain sufficient clinical data such as with newly emerging resistantorganisms Studies in animals would also allow one to determine if the magnitudeof the PKPD parameter required for efficacy was similar for (1) different drugs

10 Craig

within the same antimicrobial class (2) different dosing regimens (3) differentpathogens and (4) different sites of infection

51 Animal Infection Models

The largest number of studies addressing the magnitude of the PKPD parameterswith various drugs dosing regimens pathogens sites of infection and animalspecies have been performed with β-lactams and fluoroquinolones Time aboveMIC is the PKPD parameter that correlates with the therapeutic efficacy of thevarious β-lactam antibiotics Studies in animal infection models demonstrate thatantibiotic concentrations do not need to exceed the MIC for 100 of the dosinginterval to obtain a significant antibacterial effect [2326285787] In fact an invivo bacteriostatic effect is observed when serum levels exceed the MIC for about30ndash40 of the dosing interval Very similar percentages for time above MIChave been observed in murine thigh and lung infection models for various dosingintervals from 1 to 24 h and with several broad-spectrum cephalosporins againstboth gram-negative bacilli and gram-negative streptococci provided unbounddrug levels were used for highly protein bound cephalosporins such as ceftri-axone

If one uses survival after several days of therapy as the endpoint for efficacyof β-lactams in animal infection models then slightly higher percentages of timeabove MIC are necessary [23070] Figure 4 illustrates the relationship betweentime above MIC and mortality for animals infected with S pneumoniae that weretreated for several days with penicillins or cephalosporins Several studies in-

FIGURE 4 Relationship between the percentage of time serum levels of β-lactams exceed the MIC and survival in animal models infected with S pneu-moniae (Data from Refs 5 30 and 70)


cluded penicillin-intermediate and penicillin-resistant strains The mortality wasclose to 100 if serum levels were above the MIC for 20 or less of the dosinginterval As soon as the percentage of time above MIC reached 40ndash50 or highersurvival was in the order of 90ndash100

The PKPD parameter that best correlates with the efficacy of the fluoro-quinolones is the 24 h AUCMIC ratio [6728] The magnitude of this PKPDparameter required to produce a bacteriostatic effect in animal infection modelsvaried for most organisms from 25 to 50 [2841] These values are equivalent toaveraging one to two times the MIC over a 24 h period [ie (1ndash2) 24 24ndash48] This magnitude was independent of the dosing interval the site of infectionand the fluoroquinolone used provided unbound drug concentrations were usedfor moderate to highly protein bound drugs such as gemifloxacin [7]

The relationship between the 24 h AUCMIC values and outcome for flu-oroquinolones as reported in the literature from studies that treated animals forat least 2 days reported survival results at the end of therapy and provided phar-macokinetic data is illustrated in Fig 5 [28] The infections in these studies in-cluded pneumonia peritonitis and sepsis produced by gram-negative bacilli anda few gram-positive cocci in immunosuppressed mice rats and guinea pigs Ingeneral 24 h AUCMIC ratios less than 30 were associated with greater than50 mortality whereas AUCMIC values of 100 or greater were associated with

FIGURE 5 Relationship between 24 h AUCMIC for fluoroquinolones and sur-vival in immunosuppressed animals infected with gram-negative bacilli anda few gram-positive cocci (From Ref 28)

12 Craig

almost no mortality A value of 100 is equivalent to having serum concentrationsaverage about four times the MIC over a 24 h period (ie 4 times 24 96)

Differences in the magnitude of the PKPD parameter are observed withdifferent classes of β-lactams and with some organisms with both β-lactams andfluoroquniolones For the same types of organisms the percentages for timeabove MIC for a bacteriostatic effect were slightly lower for penicillins than forcephalosporins and even lower for carbapenems [26] These differences are dueto the rate of killing which is fastest with the carbapenems and slowest with thecephalosporins In addition the percentage for time above MIC required for effi-cacy with staphylococci was less than observed with gram-negative bacilli andstreptococci [23] This difference is due to the prolonged in vivo PAEs observedfor β-lactams with staphylococci but not with gram-negative bacilli and strepto-cocci In non-neutropenic mice the magnitude of the 24 h AUCMIC for fluoro-quinolones required for efficacy was about threefold lower for S pneumoniaethan for Klebsiella pneumoniae [728] In vitro models have also demonstrateda lower AUCMIC value for strains of S pneumoniae [5659]

52 Drug Combinations

Very little is known about determining the magnitude of PKPD parameters whendrugs are used in combination Some investigators have suggested that one canadd the magnitude of the 24 h AUCMIC ratios for each of the drugs to estimatethe pharmacodynamic activity of the combination [82] However a study in neu-tropenic mice infected with Pseudomonas aeruginosa demonstrated that the mag-nitudes of the PKPD parameters required when a β-lactam aminoglycoside orfluoroquinolone is used alone are also important in predicting the efficacy ofthese drugs used in combination [66] Thus adding the 24 h AUCMIC ratio ofβ-lactams to that of aminoglycosides or fluoroquinolones has a poor predictivevalue for their activity in combination Instead one must add the effect producedby the percentage of time above MIC for β-lactams to the effect resulting fromthe 24 h AUCMIC ratio of aminoglycosides and fluoroquinolones to accuratelypredict the activity of these combinations Adding the 24 h AUCMIC ratios isappropriate for aminoglycoside-fluoroquinolone combinations because that is theimportant PKPD parameter for both drugs

53 Human Infections

Bacteriological cure in patients with acute otitis media and acute maxillary sinus-itis provides a sensitive model for determining the relationship between outcomeand time above MIC for multiple β-lactam antibiotics A variety of clinical trialshave included pretherapy and repeat sinus puncture or tympanocentesis of middleear fluid after 2ndash7 days of therapy to determine whether the initial organismisolated had been eradicted [25323348547779] Figure 6 demonstrates the


relationship between time above MIC and the bacteriological cure rate formany β-lactams against S pneumoniae and Haemophilus influenzae in patientswith these two infections Several of the recent studies have included penicillin-intermediate and penicillin-resistant strains In general percentages for timeabove MIC greater than 40 were required to achieve an 85ndash100 bacteriologi-cal cure rate for both organisms including resistant pneumococci

Commonly used parenteral doses of ceftriaxone cefotaxime penicillin Gand ampicillin provide free-drug concentrations above the MIC90 for penicillin-intermediate strains of S pneumoniae for at least 40ndash50 of the dosing intervalA variety of clinical trials in severe pneumococcal pneumonia including bacter-emic cases have demonstrated that these β-lactams are as effective against theseorganisms as against fully susceptible strains [435072] Thus the magnitude ofthe PKPD parameter determining efficacy for β-lactams against pneumococciis very similar in animal infection models and in human infections such as pneu-monia sinusitis and otitis media

As illustrated in Fig 5 high survival in animal infection models treatedwith fluoroquinolones was observed with 24 h AUCMIC values of 100 or higherVery similar values were observed in two clinical trials Forrest et al [45] found

FIGURE 6 Relationship between time above MIC and bacteriological cure forvarious β-lactams against penicillin-susceptible (PSSP) penicillin-intermedi-ate (PISP) and penicillin-resistant (PRSP) S pneumoniae and H influenzaein patients with acute otitis media and acute maxillary sinusitis (Data fromRefs 25 32 and 33)

14 Craig

that a 24 h AUCMIC value of 125 or higher was associated with satisfactoryoutcome in seriously ill patients treated with intravenous ciprofloxacin Lowervalues resulted in clinical and microbiological cure rates of less than 50 An-other study in patients with a variety of bacterial infections treated with levoflox-acin found that a peakMIC ratio of 12 or higher and a 24 h AUCMIC ratio of100 or higher resulted in a statistically improved outcome [73] These studiesfurther demonstrate that the magnitude of the PKPD parameter in animal infec-tion models can be predictive of the magnitude of the parameter required foreffective therapy in humans

54 Prevention of Resistance

A peakMIC ratio of 8ndash10 has been shown both in vitro and in vivo to preventthe emergence of resistant mutants during therapy with aminoglycosides and flu-oroquinolones [1331] A 24 h AUCMIC ratio greater than 100 has also beenassociated with a significantly reduced risk for the emergence of resistance duringtherapy [82] The conclusion of this study was dependent almost entirely on theresults with ciprofloxacin in patients with gram-negative bacillary infections pri-marily those due to P aeruginosa A 24 h AUCMIC ratio greater than 100 didnot reduce the risk for the emergence of resistant organisms in patients treatedwith cephalosporins for infections due to gram-negative bacilli producing type1 β-lactamase Much more data are needed on the relationship between PKPDparameters and the development of resistance

6 APPLICATIONS OF PHARMACODYNAMICS

Knowledge of the pharmacodynamics of antimicrobials have proven useful for(1) establishing newer optimal dosing regimens for established drugs (2) devel-oping new antimicrobials or new formulations (3) establishing susceptibilitybreakpoints and (4) formulating guidelines for empirical therapy of infections

61 New Dosage Regimens

Administration of β-lactams by continuous infusion enhances their ability tomaintain serum levels above the MIC Despite many potential advantages of con-tinuous infusion only a few clinical trials have documented the success of thistype of dosage regimen [1424] Recent clinical trials have been designed to deter-mine if continuous infusion will (1) allow for the use of daily dosages of druglower than those required for intermittent administration or (2) improve efficacyagainst bacteria with reduced susceptibility For example equivalent outcomeshave been observed with 3ndash4 gday of ceftazidime by continuous infusion com-pared with 6 gday administered intermittently [5169] Initial results with con-tinuous infusion of large doses of ampicillin have demonstrated success for


moderately ampicillin-resistant strains (ampicillin MIC 32ndash64 mgL) ofvancomycin-resistant Enterococcus faecium [21]

The peakMIC ratio appears to be the major PKPD parameter determiningthe clinical efficacy of aminoglycosides High rates of clinical success in severegram-negative bacillary infections and rapid resolution of fever and leukocytosisin gram-negative bacillary nosocomial pneumonia require a peakMIC ratio of8ndash10 [5365] The once-daily dosage regimen for aminoglycosides was designedto enhance peak serum levels In addition once-daily dosing has the potential todecrease the nephro- and ototoxicity associated with these drugs [85] Uptake ofaminoglycosides into renal tubular cells and middle ear endolymph is more effi-cient with low sustained concentrations than with high intermittent levels[348386]

Most meta-analyses of clinical trials have demonstrated a small but signifi-cant increase in clinical outcome with once-daily dosing and a trend toward de-creased nephrotoxicity [1101144464967] Studies have also demonstratedthat the onset of nephrotoxicity occurs several days later when the drug is admin-istered once daily than when multiple-daily dosage regimens are followed[527581] Nevertheless once-daily dosing may not be ideal for all indicationsStudies in experimental enterococcal endocarditis have shown a greater reduc-tion in bacterial vegetation titers when the aminoglycoside is administered bymultiple-dosing regimens than by once-daily administration [4285]

62 New Antimicrobials and Formulations

Identification of the PKPD parameter and its magnitude required for efficacyhas proven useful for selecting the dosage regimen for phase III clinical trials ofnew antimicrobials For example the dosage regimen for linezolid (600 mg twicedaily) was designed to produce serum levels above 8 mgL for more than 50of the dosing interval [8] New extended release formulations of cefaclor andclarithromycin and the 141 amoxicillin-clavulanate formulation were all de-signed to enhance the duration of time serum levels exceed the MIC

63 Susceptibility Breakpoint Determinations

The Subcommittee on Antimicrobial Susceptibility Testing of the National Com-mittee for Clinical Laboratory Standards (NCCLS) recently incorporated pharma-codynamics as one of the factors to consider when establishing susceptibilitybreakpoints [68] For example pharmacodynamic breakpoints for β-lactam anti-biotics would be defined as the highest MIC that serum concentrations followingstandard dosage would exceed for at least 40 of the dosing interval Table 3compares the old and new susceptibility breakpoints of several oral β-lactamsfor S pneumoniae with the pharmacodynamic breakpoints predicted from serumconcentrations in children and adults The two values for cefaclor reflect the

16 Craig

TABLE 3 Pharmacodynamic and New and Old NCCLS SusceptibilityBreakpoints for Oral β-Lactams with Streptococcus pneumoniae

PharmacodynamicOld NCCLS (T MIC 40) New NCCLS

Drug breakpoint (mgL) breakpoint (mgL) breakpoint (mgL)

Amoxicillin 05 2 2Cefaclor mdash 05ndash1 1Cefuroxime 05 1 1Cefprozil mdash 1ndash2 2Cefpodoxime mdash 05 05

difference in serum levels between standard doses and the extended-release for-mulation and those for cefprozil reflect the difference in serum levels from chil-dren and adults The NCCLS breakpoints are identical to the pharmacodynamicbreakpoints

64 Guidelines for Empirical Therapy

Because the magnitude of PKPD parameters determined in animal infectionmodels can be predictive of antimicrobial efficacy in human infections it is easyto understand why pharmacodynamics is being used more and more in establish-ing guidelines for empirical therapy Recently published guidelines for otitis me-dia acute bacterial rhinosinusitis and community-acquired pneumonia have usedthe ability of antimicrobials to reach the magnitude of PKPD parameters requiredfor efficacy for both susceptible pathogens and those with decreased susceptibilityto rank or select antimicrobials for empirical therapy of these respiratory infec-tions [365078]

7 SUMMARY

Studies over the past 20 years have demonstrated that antibacterials and antifun-gals can vary markedly in their time course of antimicrobial activity Three differ-ent patterns of antimicrobial activity are observed Specific PKPD parameterssuch as the peakMIC and AUCMIC ratios and the time above MIC have alsobeen shown to be major determinants of in vivo efficacy The magnitude of thePKPD parameters required for efficacy are relatively similar in animal infectionmodels and human infections and are largely independent of the dosing intervalthe site of infection the drug used within each antimicrobial class and the typeof infecting pathogen However additional studies are needed to extend currentobservations to other antimicrobials and organisms and to correlate PKPD pa-


rameters with therapeutic efficacy in a variety of animal infection models andhuman infections Pharmacodynamics has many applications including use forestablishing optimal dosing regimens for old drugs for developing new antimi-crobials and formulations for setting susceptibility breakpoints and for providingguidelines for empirical therapy

REFERENCES


2 Andes D Craig WA In vivo activities of amoxicillin and amoxicillin-clavulanateagainst Streptococcus pneumonia Application to breakpoint determinations Anti-microb Agents Chemother 1998 422375ndash2379

3 Andes D van Ogtrop M Characterization and quantitation of the pharmacodynamicsof fluconazole in a neutropenic murine disseminated candidiasis infection modelAntimicrob Agents Chemother 1999 432116ndash2120

4 Andes D van Ogtrop ML In vivo characterization of the pharmacodynamics offlucytosine in a neutropenic murine disseminated candidiasis model AntimicrobAgents Chemother 2000 44938ndash942

5 Andes DR Craig WA Pharmacokinetics and pharmacodynamics of antibiotics inmeningitis Infect Dis Clinics N Am 1999 13595ndash618

6 Andes DR Craig WA Pharmacodynamics of fluoroquinolones in experimentalmodels of endocarditis Clin Infect Dis 1998 2747ndash50

7 Andes D Craig WA Pharmacodynamics of gemifloxacin against quinolone-resistantstrains of Streptococcus pneumoniae with known resistance mechanisms Abstractsof the 39th Interscience Conference on Antimicrobial Agents and ChemotherapyAm Soc Microbiol Washington DC 1999

8 Andes D Van Ogtrop M Craig WA Pharmacodynamic activity of a new oxazolidi-none in an animal infection model Abstracts of the 38th Interscience Conference onAntimicrobial Agents and Chemotherapy Am Soc Microbiol Washington DC 1998

9 Andes D In-vivo pharmacodynamics of amphotericin B against Candida albicansAbstracts of the 39th Interscience Conference on Antimicrobial Agents and Chemo-therapy Am Soc Microbiol Washington DC 1999

10 Bailey TC Little JR Littenberg B Reichley RM Dunagan WC A meta-analysisof extended-interval dosing versus multiple daily dosing of aminoglycosides ClinInfect Dis 1997 24786ndash795

11 Barza M Ioannidis JPA Cappelleri JC Lau J Single or multiple daily doses ofaminoglycosides A meta-analysis Br Med J 1996 312338ndash345

12 Bigger JW The bactericidal action of penicillin on Staphylococcus pyogenes IrishJ Med Sci 1994 227533ndash568

13 Blaser J Stone BB Groner MC et al Comparative study with enoxacin and netil-micin in a pharmacodynamic model to determine importance of ratio of antibioticpeak concentration to MIC for bactericidal activity and emergence of resistanceAntimicrob Agents Chemother 1987 311054ndash1060

18 Craig

14 Bodey GP Ketchel SJ Rodriguez N A randomized study of carbenicillin plus cefa-mandole or tobramycin in the treatment of febrile episodes in cancer patients AmJ Med 1979 67608ndash616

15 Bundtzen RW Gerber AU Cohn DL Craig WA Postantibiotic suppression of bac-terial growth Rev Infect Dis 1981 328ndash37

16 Bustamante CL Wharton RC Wade JC In vitro activity of ciprofloxacin in combi-nation with ceftazidime aztreonam and azlocillin against multiresistant isolates ofPseudomonas aeruginosa Antimicrob Agents Chemother 1990 341814ndash1815

17 Cars O Odenholt-Tornqvist I The post-antibiotic sub-MIC effict in vitro and invivo J Antimicrob Chemother 1993 31(suppl D)159ndash166

18 Craig WA Gudmundsson S Postantibiotc effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams and Wilkins Baltimore MD 1996296ndash329

19 Craig WA Andes D Differences in the in vivo pharmacodynamics of telithromycinand azithromycin against Streptococcus pneumoniae Abstracts of 40th InterscienceConference on Antimicrobial Agents and Chemotherapy Am Soc Microbiol Wash-ington DC 2000

20 Craig WA Ebert SC Killing and regrowth of bacteria in vitro A review Scand JInfect Dis 1991 suppl 7463ndash70

21 Craig WA Pharmacokineticpharmacodynamics parameters Rationale for antibac-terial dosing of mice and men Clin Infect Dis 1997 261ndash12

22 Craig WA Postantibiotic effects in experimental infection models Relationship toin vitro phenomena and to treatment of infections in man J Antimicrob Chemother1993 31149ndash158

23 Craig WA Interrelationship between pharmacokinetics and pharmacodynamics indetermining dosage regimens for broad-spectrum cephalosporins Diagn MicrobiolInfect Dis 1995 211ndash8

24 Craig WA Ebert SC Continuous infusion of β-lactam antibiotics AntimicrobAgents Chemother 1992 362577ndash2583

25 Craig WA Andes D Pharmacokinetics and pharmacodynamics of antibiotics in oti-tis media Pediatr Infect Dis J 1996 15255ndash259

26 Craig WA Ebert S Watanabe Y Differences in time above MIC required for effi-cacy of beta-lactams in animal infection models Abstracts of the 33rd InterscienceConference on Antimicrobial Agents and Chemotherapy Am Soc Microbiol Wash-ington DC 1993

27 Craig WA Redington J Ebert SC Pharmacodynamics of amikacin in-vitro and inmouse thigh and lung infections J Antimicrob Chemother 1991 27(suppl C)29ndash40

28 Craig WA Dalhoff A Pharmacodynamics of fluoroquinolones in experimental ani-mals In J Kuhlman A Dalhoff HJ Zeiler eds Handbook of Experimental Pharma-cology Vol 127 Quinolone Antibacterials pp 207ndash232

29 Craig WA Postantibiotic effects and the dosing of macrolides azalides and strepto-gramins In SH Zinner LS Young JF Acar HC Neu eds Expanding Indicationsfor the New Macrolides Azalides and Streptogramins Marcel Dekker New York199727ndash38

30 Craig WA Antimicrobial resistance issues of the future Diagn Microbiol Infect Dis1996 25213ndash217


31 Craig WA Does the dose matter Clin Infect Dis 2000 in press32 Dagan R Abramason O Leibovitz E Greenberg D Lang R Goshen S Yagupsky

P Leiberman A Fliss DM Bacteriologic response to oral cephalosporins Are estab-lished susceptibility breakpoints appropriate in the case of acute otitis media J InfectDis 1997 1761253ndash1259

33 Dagan R Leibovitz E Fliss DM Leiberman A Jacobs MR Craig W Yagupsky PBacteriologic efficacies of oral azithromycin and oral cefaclor in treatment of acuteotitis media in infants and young children Antimicrob Agents Chemother 2000 4443ndash50

34 De Broe ME Verbist L Verpooten GA Influence of dosage schedule on renal corti-cal accumulation of amikacin and tobramycin in man J Antimicrob Chemother1991 27(suppl C)41ndash47

35 Den Hollander JG Mouton JW van Goor MP et al Alteration of postantibioticeffect during one dosing interval of tobramycin simulated in an in vitro pharmacoki-netic model Antimicrob Agents Chemother 1996 40784ndash786

36 Dowell SF Butler JC Giebink GS Jacobs MR Jernigan D Musher DM RakowskyA Schwartz B Acute otitis media Management and surveillance in an era of pneu-mococcal resistancemdashA report from the drug-resistant Streptococcus pneumoniaetherapeutic working group Pediatr Infect Dis J 1999 181ndash9

37 Drusano GL Johnson DE Rosen M Pharmacodynamics of a fluoroquinolone anti-microbial agent in a neutropenic rat model of Pseudomonas sepsis AntimicrobAgents Chemother 1993 37483ndash490

38 Eagle H Musselman AD The slow recovery of bacteria from the toxic effects ofpenicillin J Bacteriol 1949 58475ndash490

39 Ernst EJ Klepser ME Pfaller MA Postantifungal effects of echinocandin azole andpolyene antifungal agents against Candida albicans and Cryptococcus neoformansAntimicrob Agents Chemother 2000 441108ndash1111

40 Fantin B Ebert S Leggett J et al Factors affecting the duration of in-vivo postanti-biotic effect for aminoglycosides against gram-negative bacilli J Antimicrob Che-mother 1990 27829ndash836

41 Fantin B Leggett J Ebert S Craig WA Correlation between in vitro and in vivoactivity of antimicrobial agents against gram-negative bacilli in a murine infectionmodel Antimicrob Agents Chemother 1991 351413ndash1422

42 Fantin B Carbon C Importance of the aminoglycoside dosing regimen in thepenicillin-netilimicin combination for treatment of Enterococcus faecalisndashinducedexperimental endocarditis Antimicrob Agents Chemother 1990 342387ndash2389

43 Feikin DR Schuchat A Kolczak M Barrett NL Harrison LH Lefkowitz LMcGreer A Farley MM Vugia DJ Lexau C Stefonek KR Patterson JE JorgensenJH Mortality from invasive pneumococcal pneumonia in the era of antibiotic resis-tance 1995ndash1997 Am J Public Health 2000 90223ndash229

44 Ferriols-Lisart R Alos-Alminana M 1996 Effectiveness and safety of once-dailyaminoglycosides A meta-analysis Am J Health Syst Pharmacy 1996 531141ndash1150

45 Forrest A Nix DE Ballow CH Goss TF Birmingham MC Schentag JJ Pharmaco-dynamics of intravenous ciprofloxacin in seriously ill patients Antimicrob AgentsChemother 1993 371073ndash1081

20 Craig

46 Galloe AM Gaudal N Christensen HR Kampmann JP Aminoglycosides Singleor multiple daily dosing A meta analysis on efficacy and safety Eur J Clin Pharma-col 1995 4839ndash43

47 Gudmundsson S Vogelman B Craig WA The in vivo postantibiotic effect of imi-penem and other new antimicrobials J Antimicrob Chemother 1986 18(suppl E)67ndash73

48 Gwaltney JM Savolainen S Rivas P Schenk P Scheld WM Sydnor A KeyserlingC Leigh A Tack KJ Comparative effectiveness and safety of cefdinir and amoxicil-lin-clavulanate in treatment of acute community-acquired bacterial sinusitis Antimi-crob Agents Chemother 1997 411517ndash1520

49 Hatala R Dinh T Cook DJ Once-daily aminoglycoside dosing in immunocompetentadults A meta-analysis Ann Intern Med 1996 124717ndash725

50 Heffelfinger JD Dowell SF Jorgensen JH et al Management of community-acquired pneumonia in the era of pneumococcal resistance Arch Intern Med 20001601399ndash1408

51 Houlihan HH Mercier RC McKinnon PS et al Continuous infusion versus inter-mittent administration of ceftazidime in critically ill patients with gram-negativeinfection Abstracts of the 37th Interscience Conference on Antimicrobial Agentsand Chemotherapy Am Soc Microbiol Washington DC 1997

52 Int Antimicrob Therap Coop Group of EORTC Efficacy and toxicity of single dailydoses of amikacin and ceftriaxone versus multiple daily doses of amikacin and cef-tazidime for infection in patients with cancer and granulocytopenia Ann Intern Med1993 119584

53 Kashuba AD Nafziger AN Drusano GL Bertino JS Optimizing aminoglycosidetherapy for nosocomial pneumonia caused by gram-negative bacteria AntimicrobAgents Chemother 1999 43623ndash629

54 Klein JO Microbiologic efficacy of antibacterial drugs for acute otitis media PediatrInfect Dis 1993 J 12973ndash975

55 Knudsen JD Fuursted K Raber S Espersen F Fridmodt-Moller N Pharmacody-namics of glycopeptides in the mouse peritonitis model of Streptococcus pneumon-iae and Staphylococcus aureus infection Antimicrob Agents Chemother 2000 441247ndash1254

56 Lacy MK Lu W Xu X Tessier PR Nicolau DP Quintiliani R Nightingale CHPharmacodynamic comparisons of levofloxacin ciprofloxacin and ampicillinagainst Streptococcus pneumoniae in an in vitro model of infection AntimicrobAgents Chemother 1999 43672ndash677

57 Leggett JE Fantin B Ebert S Totsuka K Vogelman B Calame W Mattie H CraigWA Comparative antibiotic dose-effect relations at several dosing intervals in mu-rine pneumonitis and thigh-infection models J Infect Dis 1989 159281ndash292

58 Leggett JE Ebert S Fantin B Craig WA Comparative dose-effect relations at sev-eral dosing intervals for beta-lactam aminoglycoside and quinolone antibioticsagainst gram-negative bacilli in murine thigh-infection and pneumonitis modelsScand J Infect Dis 1991 (suppl 74)179ndash184

59 Lister PD Sanders CC Pharmacodynamics of levofloxacin and ciprofloxacin againstStreptococcus pneumoniae J Antimicrob Chemother 1999 4379ndash86

60 Lorian V Effect of low antibiotic concentrations on bacteria Effects on ultrastruc-


ture virulence and susceptibility to immunodefenses In V Lorian ed Antibioticsin Laboratory Medicine 4th ed Williams and Wilkins Baltimore 1991493ndash555

61 Louie A Drusano GL Banerjee P Liu QF Liu W Kaw P Shayegani M TaberH Miller MH Pharmacodynamics of fluconazole in a murine model of systemiccandidiasis Antimicrob Agents Chemother 1998 421105ndash1109

62 McDonald PJ Wetherall BL Pruul H Postantibiotic leukocyte enhancement In-creased susceptibility of bacteria pretreated with antibiotics to activity of leukocytesRev Infect Dis 1981 338ndash44

63 McDonald PJ Craig WA Kunin CM Persistent effect of antibiotics on Staphylococ-cus aureus after exposure for limited periods of time J Infect Dis 1977 135217ndash223

64 McGrath BJ Marchbanks CR Gilbert D Dudley MN In vitro postantibiotic effectfollowing repeated exposure to imipenem temofloxacin and tobramycin Antimi-crob Agents Chemother 1993 371723ndash1725

65 Moore RD Lietman PS Smith CR Clinical response to aminoglycoside therapyImportance of the ratio of peak concentration to minimal inhibitory concentrationJ Infect Dis 1987 15593ndash99

66 Mouton JW van Ogtrop ML Andes D Craig WA Use of pharmacodynamic indicesto predict efficacy of combination therapy in vivo Antimicrob Agents Chemother1999 432473ndash2478

67 Munckhof WJ Grayson JL Turnidge JD A meta-analysis of studies on the safetyand efficacy of aminoglycosides given either once daily or as divided doses J Anti-microb Chemother 1996 37645ndash663

68 National Committee for Clinical Laboratory Standards Development of In-VitroSusceptibility Testing Criteria and Quality Control Parameters Approved Guideline2nd ed Document M23ndashA2 January 2000

69 Nenko AS Cappelletty DM Kruse JA et al Continuous infusion versus intermittentadministration of ceftazidime in critically ill patients with suspected gram-negativeinfection Abstracts of the 35th Interscience Conference on Antimicrobial Agentsand Chemotherapy Am Soc Microbiol Washington DC 1995

70 Nicolau DP Onyeji CO Zhong M Tessier PR Banevicius MA Nightingale CHPharmacodynamic assessment of cefprozil against Streptococcus pneumoniae Im-plications for breakpoint determinations Antimicrob Agents Chemother 2000 441291ndash1295

71 Odenholt-Tornqvist I Lowdin E Cars O Postantibiotic sub-MIC effects of vanco-mycin roxithromycin sparfloxacin and amikacin Antimicrob Agents Chemother1992 361852ndash1858

72 Pallares R Linares J Vadillo M Cabellos C Manresa R Viladrich PF Martin RGudiol F Resistance to penicillin and cephalosporin and mortality from severe pneu-mococcal pneumonia in Barcelona Spain N Engl J Med 1995 333474ndash480

73 Preston SL Drusano GL Berman AL Fowler CL Chow AT Dornseif B ReichiV Natarajan J Corrado M Pharmacodynamics of levofloxacin A new paradigmfor early clinical trials J Am Med Assoc 1998 279125ndash129

74 Redington J Ebert SC Craig WA Role of antimicrobial pharmacokinetics and phar-macodynamics in surgical prophylaxis Rev Infect Dis 1991 13(suppl 10)S790ndashS799

22 Craig

75 Rybak MJ Abate BJ Kang SL Ruffing MJ Lerner SA Drusano GL Prospectiveevaluation of the effect of an aminoglycoside dosing regimen on rates of observednephrotoxicity and ototoxicity Antimicrob Agents Chemother 1999 431549ndash1555

76 Safdar N Andes D Craig WA In-vivo pharmacodynamic characterization of dapto-mycin Abstracts of 37th Infectious Diseases Society of America Washington DC1999

77 Scheld WM Sydnor A Farr B Gratz JC Gwaltney JM Comparison of cyclacillinand amoxicillin for therapy for acute maxillary sinusitis Antimicrob Agents Chemo-ther 1986 30350ndash353

78 Sinus and Allergy Health Partnership Antimicrobial treatment guidelines for acutebacterial rhinosinusitis Otolaryngol Head Neck Surg 2000 123(suppl 1)1ndash32

79 Sydnor A Gwaltney JM Cocchetto DM Scheld WM Comparative evaluation ofcefuroxime axetil and cefaclor for treatment of acute bacterial maxillary sinusitisArch Otolaryngol Head Neck Surg 1989 1151430ndash1433

80 Tauber MG Zak O Scheld WM Hengstler B Sande MA The postantibiotic effectin the treatment of experimental meningitis caused by Streptococcus pneumoniaein rabbits J Infect Dis 1984 149575ndash583

81 Ter Braak EW De Vries PJ Bouter KP Van der Vegt SG Dorrestein GC NorthierJW Van Dijk A Verkooyen RP Verbrugh HA Once-daily dosing regimen foraminoglycoside plus β-lactam combination therapy of serious bacterial infectionsComparative trial with netilmicin plus ceftriaxone Am J Med 1990 8958ndash66

82 Thomas JK Forrest A Bhavnani SM Hyatt JM Cheng A Ballow CH Schentag JJPharmacodynamic evaluation of factors associated with the development of bacterialresistance in acutely ill patients during therapy Antimicrob Agents Chemother 199842521ndash527

83 Tran BH Deffrennes D Aminoglycoside ototoxicity Influence of dosage regimenon drug uptake and correlation between membrane binding and some clinical fea-tures Acta Otolaryngol (Stockholm) 1988 105511ndash515

84 Turnidge JD Gudmundsson S Bogelman B Craig WA The postantibiotic effectof antifungal agents against common pathogenic yeast J Antimicrob Chemother1994 3483ndash92

85 Urban A Craig WA Daily dosing of aminoglycosides Curr Clin Top Infect Dis1997 17236ndash255

86 Verpooten GA Giuliano RA Verbist L Estermans G De Broe ME Once-dailydosing decreases renal accumulation of gentamicin and netilmicin Clin PharmacolTher 1989 4522ndash27

87 Vogelman B Gudmundsson S Leggett J Turnidge J Ebert S Craig WA Correla-tion of antimicrobial pharmacokinetic parameters with therapeutic efficacy in ananimal model J Infect Dis 1988 158831ndash847

88 Vogelman B Gudmundsson S Turnidge J Craig WA The in vivo postantibioticeffect in a thigh infection in neutropenic mice J Infect Dis 1988 157287ndash298

2

Microbiology and Pharmacokinetics



Takeo Murakawa

Fujisawa Pharmaceutical Company Ltd Osaka Kyoto University Kyoto andTokushima University Tokushima Japan

1 INTRODUCTION

Pharmacodynamics as applied to antimicrobial agents is simply the indexing ofmicrobiological data to the drugrsquos concentration in the body (pharmacokinetics)Antimicrobial agents are different from most therapeutic drugs in their mecha-nism of action The major difference between the agents and other drugs involvesthe fact that the target of antimicrobial agents is not the human body but microor-ganisms that live in it A strong understanding of microbiology and pharmacoki-netics under dynamic in vivo conditions is needed to fully understand the topicof microbial pharmacodynamics However it is beyond the scope of this chapterto present such material in detailed survey form Other chapters of this text willdiscuss specific drug classes and illustrate the important pharmacokinetic andmicrobiological properties of those drugs We prefer to discuss the importantaspects of microbiology and pharmacokinetics as they apply to antimicrobialsand the treatment of infections We refer the reader to other chapters of this

23


textbook as well as to standard texts [12] and primary articles on microbiologyand pharmacokinetics for a more basic explanation of these topics

2 MICROBIOLOGY

21 Effect of Antimicrobial Agents on Microorganisms

To understand this topic one must first have a concept of what is meant by theterm antimicrobial agent In this chapter this term refers to a chemical entity (asubstance of particular chemical structure) that has the ability to bind to someimportant site in a microorganism and cause microbial death This important siteis a place in the microorganism where the organism undergoes some biochemicalreaction that is part of its life cycle The binding substance (which we call anantimicrobial agent) blocks or interferes with the biochemical reaction and thusprevents the organism from completely fulfilling those acts necessary to supportits life cycle The results of this lsquolsquointerferencersquorsquo by a chemical (natural or syn-thetic) that we call an antimicrobial agent usually results in cell death if the agentis in high enough concentration for a long enough period of time Assuming thatthis description is fundamentally correct it follows then that three events mustoccur in order for the antimicrobial agent to eradicate the microorganism

1 The antimicrobial agent must reach the action sites and bind to thebinding sites in the microorganism The binding site can be a penicillin-binding protein a DNA gyrase a topoisomerase or a ribosome or anyother point of attachment that interferes with the biochemical reactionsin the microorganism The actual site varies depending upon the chemi-cal structure of the class of the agent and also varies somewhat withinthe same agent class This ability to bind to an active site can be thoughtof as being roughly descriptive (although not completely) of what wewould commonly call the microbiological activity of the antimicrobialagents

2 The antimicrobial agent must occupy a sufficient number of activesites The occupation of a sufficient number of sites is an interestingtopic and is central to the definition of pharmacodynamics The mecha-nism of action of the antimicrobial agent does not have to be fullyelucidated for the purpose of this discussion First it must be recognizedthat the number of active sites that must be occupied is largely un-known Obviously the number is greater than one because we knowat least intuitively that there are many active sites involving manymolecules of active proteins such as enzymes or DNA and we mustdescribe them very qualitatively One term that qualitatively describesthe number of sites is lsquolsquosufficientrsquorsquo The drug must diffuse usually (butnot always) on the basis of the drugrsquos concentration gradient to the


metabolically active site of the microorganism Because concentrationis important one might speculate upon the issue of identifying theimportant place where the drug concentration is favorable to its bindingto active sites Intuitively it is the drug concentration immediately sur-rounding the active site that ultimately controls this process But thatconcentration is in the bacterial cell and is largely unknown

3 The antimicrobial agent must reside on the active site for a period oftime Because antibiotics are not contact poisons but agents that inter-fere with a biochemical process essential for the bacterial life cycle adefinite time must evolve in order for the agent to elicit the desiredresponse The length of time at the active site is related to the strengthof binding to that site

When these three conditions exist the antimicrobial agents will have the desiredeffect on the bacteria

22 Parameters for Microbiological Activity of

Antimicrobial Agents

The killing properties of antimicrobial agents are important for pharmacodynamicconsiderations As described above the killing properties of antimicrobial agentsare dependent upon the antibiotic characteristics such as number of binding sitesand the binding or interfering properties at the active sites ie the drug concen-tration and length of time required to lead to microbial death It is difficult tocompletely characterize these properties However the overall results may besufficient from a practical perspectivemdashthe killing concentration surrounding thebacterial cell should be a good surrogate for the actual drug concentration at theactive site

The following parameters of antimicrobial activity are of common or practi-cal use

1 The minimal inhibitory concentration (MIC) is the lowest drug concen-tration without visible growth for 16ndash20 h at 35degC in the medium

2 The minimal bactericidal concentration (MBC) is the lowest concentra-tion required to kill 999 of the bacterial cells of the inoculum after16ndash20 h exposure to the drug at 35degC

For pharmacodynamic considerations it is important to understand that (1) theseparameters are determined under in vitro conditions and that (2) we cannot useonly one cell of a microorganism for the testsmdashfor example a million cells areused as a unit

Susceptibility of cells is not homogeneous ie each cell of a million cellpopulation has a different MIC or MBC and the total bacterial population showsan MIC distribution that is different for each organismndashdrug combination


23 Kinetic Killing Properties

The killing rate is an important parameter and will vary with the kind of microor-ganism and the strain of the organism as well as the type of antimicrobial agentThe killing properties of antimicrobial agents have two basic forms of relation-ships with drug concentration concentration dependence and time-dependenceIt should be noted that the issues of concentration-dependent and time-dependentbacterial killing are an abbreviated way of describing an antibioticndashbacterial in-teraction This is shown in Fig 1 which illustrates the usual in vitro experimentsto determine whether a drug is static or cidal If we grow bacteria in broth in theabsence of antibiotic the control curve results If we add a small quantity ofdrug the rate of growth still exceeds the rate of death and the organism continuesto grow Obviously that small amount of drug did not have much of an effecton the bacterial life cycle If we add more drug the rate of growth and deathmight be equal and we say that the drug exhibits static properties If we addeven more drug the rate of killing might exceed the rate of living and the drugis now termed a cidal agent If we continue to add increasing amounts of drugthe rate of killing relative to the rate of living continues to increase and if we lookat just this concentration range we conclude that the drug exhibits concentration-dependent killing If we add even more drug we eventually reach a point whereadditional drug does not increase the rate of killing any further and if we justlook at its behavior in that concentration range we say that the drug exhibits time-dependent killing properties In the example illustrated in Fig 1 the same drugexhibited no effect static behavior cidal behavior and concentration-dependentand time-dependent cidal behavior In essence the same drug can have all of

FIGURE 1 Relationship between net effect on bacterial growth and death andtime of exposure to antibiotic at various fixed concentrations


these properties depending on the drugrsquos concentration in relation to its MICTherefore when we say that a drug is a concentration- or time-dependent killerwe seem to be speaking in incomplete sentences A more complete descriptionwould be to add the phrase lsquolsquounder the concentrations achieved after clinicaldosingrsquorsquo The importance of this phenomenon is that although investigators maystudy the properties of a drug and classify it as static or cidal this is arbitrarybecause the same experiment under different conditions would result in a differ-ent classification This is why for the tetracyclines the official US packageinsert has phrases that indicate that these agents are static but exhibit cidal proper-ties against some bacteria

It is important to also realize that the killing property of a drug is relatedto its rate of growth If antibiotics interfere with certain biochemical reactionsin the bacteria that are normally part of their life cycle then the drug needs tobe present bound to the active site at the time the biochemical reaction is tooccur As an example penicillins or cephalosporins show bactericidal activitywhen cell growth is maximal but not for resting cells because the drugs inhibitthe synthesis of the bacterial cell wall and the bacteria must be actively makingcell walls for the antibiotic to have its effect

Sometimes the killing curve shows a reduction in the number of viablecells of an organism even at concentrations lower than the MIC (lsquolsquosubinhibitoryconcentrationsrsquorsquo) This means that some cells in the population are more sensitiveto the drug than the MIC reported for the entire population would indicate Insuch a case the MIC may change as a function of inoculum size

A postantibiotic effect (PAE) is observed with cephalosporins againstgram-negative bacteria When the bacteria are exposed to antibiotics at severaltimes the MIC for a certain period of time and then the antibiotic is removedslow bacterial growth is observed for a certain period of time Possible reasonsfor this phenomenon are that the drug continues to bind to the active sites of thebacteria or that the antibiotics remain in the cell even after the antibiotic hasdisappeared from outside the cell or there may be some methodological problemassociated with the removal of the drug from the test system

The MIC MBC and killing kinetics properties such as concentration-de-pendent and time-dependent properties are commonly used as parameters formicrobiological activity of antimicrobial agents and therefore are of major impor-tance However it is important to understand that these definitions and most dataare obtained in vitro not at the actual sites of infection in the body It is difficultor impossible to obtain the MIC in body fluids and in addition the reported datahave a large amount of variability associated with them due to the twofold dilu-tional techniques commonly used in the field of microbiology This represents100 variability in the reported MIC value As a guide the MIC values are veryuseful but to consider them to be absolute infallible numbers is misleading andconfusing


3 PHARMACOKINETICS

Parameters for the pharmacokinetic properties of antimicrobial agents are thesame as for those of other therapeutic drugs There is no particular issue in phar-macokinetic considerations with antimicrobial agents except that the target siteis located in the infection site where microorganisms are living The infectionsites are distributed throughout the body and an effective concentration of anantimicrobial agent is required to eradicate microorganisms in those infectionsites where they are living

31 Pharmacokinetic Parameters for

Pharmacodynamic Consideration

We do not administer a drug directly into microorganisms but into a humanbody It must be driven to the place where the organism lives by the drug concen-tration in blood Most organisms live in some interstitial fluid (IF) in the bodyalthough some reside inside mammalian cells Assuming we are considering or-ganisms that live in IF it is the drug concentration in this fluid that is importantFortunately drug transfer from the bloodstream into the IF is rather rapid dueto the leakiness of the capillary bed membrane and blood (serum or plasma)concentrations control the IF drug concentration which controls or influencesthe drug concentration around the bacterial active sites In this way we lsquolsquobackintorsquorsquo the realization that drug concentrations in the serum (lsquolsquoserumrsquorsquo will beused in this chapter to mean plasma blood or serum) are important for a sufficientnumber of active sites to be occupied by the drug However it is not true forevery injection site This forms the basis for modern pharmacodynamic theorywhich allows one to index microbiological activity to the serum concentrationof an antibiotic The concentration of the drug in the serum is a major functionof the drugrsquos pharmacokineticsThe pharmacokinetic parameters related to microbiological activity of an antibi-otic are

Peak drug concentration Cp

Half life T12

Area under the drug concentration curve AUCTime period during which drug concentrations exceed MIC or MBC

32 Difference of Pharmacokinetic Parameters in Serum

and Tissues or Fluids

As mentioned above pharmacokinetic parameters should be those based on theantibiotic concentration at the infection site However determination of the drugconcentration at various infection sites is difficult in practice A drug is distributedor penetrated into tissue or tissue fluids of infection sites via the bloodstream


When the drug concentrations at the infection site and in the bloodstream areproportional it is possible to use the serum or blood concentration to index tomicrobiological activity If there is a big difference between the kinetic propertiesin the blood and those at the site of infection (tissuesfluids) then the drug con-centration at the site of infection should be used to index to microbiologicalactivity Serumndashtissue protein binding drug distribution penetration rate anddrug metabolism at these sites will be factors in effecting differences in bloodand infection site drug kinetics

4 CLINICAL ASPECTS

As mentioned before there are major differences between antimicrobial agentsand other therapeutic drugs because the target of antimicrobial agents is not thehuman body but microorganisms that cause infectious diseases The human bodyitself has host defense mechanisms to kill invasive microorganisms Infectiousdiseases are caused by the bacteria creating through the large number of themgrowing in the body a condition that exceeds the bodyrsquos host defense capabilityto recognize and eradicate the invasive microorganism Therefore eradication ofmicroorganisms from the body or infection sites depends upon a combination ofactivities partly provided by the body and partly provided by an antibacterialdrug If host defense activity is normal and the infecting organisms are not verypathogenic clinical efficacy will result even when the activity of the antibioticis modest However when the host defense mechanism is weak or impaired asis the case with immunocompromised patients the antimicrobial agents itselfmust kill and eradicate the infecting microorganisms In such a situation thedosing and activity of the antibiotic must be maximized

5 PHARMACODYNAMICS

51 Pharmacodynamic Parameters

The drug concentration in the serum and the time that it remains there are relatedto the occupation of a sufficient number of binding sites in the bacteria and thelength of time those sites are occupied To produce a pharmacodynamic parame-ter it is only necessary to index a measure of microbiological activity such as theminimum inhibitory concentration (MIC) or minimum bactericidal concentration(MBC) to the area under the curve (AUC) In this chapter we will use the termMIC however the MBC or any other measure of microbiological activity couldalso be used It is interesting that the mathematical product of drug concentrationin the serum (Cp) multiplied by time is termed the drugrsquos area under the serumconcentrationndashtime curve (AUC) A schematic representation of the AUC in-


dexed to the MIC of bacteria is shown in Fig 2 This pharmacodynamic parame-ter AUCMIC is an important and fundamental parameter that relates microbio-logical activity to serum concentration One simple way of thinking about theAUCMIC ratio is that the AUC represents the concentration of drug and timeof exposure that presents itself to the bacteria over and above the minimumneeded to do the job (MIC) The higher this ratio (AUCMIC) the better is thechance of the antibiotic being successful It should also be noted that in natureantibiotics eradicate bacteria (fulfill the three requirements described earlier) byan interaction of microbiological activity and pharmacokinetics not just one ofthe two Decisions related to drug selection should therefore take both of theseproperties into consideration Unfortunately most clinicians because of theirtraining will choose antibiotics on the basis of microbiological activity aloneWe believe that this may result in poor decisions The reason for this belief isthat one can choose the most active antibiotic in the world (lowest MIC againstthe target organism) but if it does not go to where the bacteria reside it will notkill anything The reverse is also true Good pharmacokinetics with very poorantimicrobial activity will also not result in a satisfactory outcome Both needto be considered simultaneously hence the importance of pharmacodynamics

Under certain conditions the AUCMIC [(Cp time)MIC] ratio can besimplified These simplifying assumptions are based upon whether the drug isclassified as a concentration-dependent or time-dependent bacterial killer Thisdetermination is made from in vitro experiments such as those for which data areshown in Fig 3 For the aminoglycoside we can see that as the drug concentrationincreases the rate of killing increases and hence this drug is considered to be aconcentration-dependent killer If one makes the concentration high enough therate of killing is so fast that the contribution of drug exposure to the entire processis insignificant and can be ignored The ratio AUCMIC [(Cp time)MIC] sim-

FIGURE 2 Pharmacodynamic relationship AUCMIC


FIGURE 3 Concentration-dependent vs independent bacterial killing (FromRef 21)

plifies to CpMIC For such a drug the question arises regarding how high the drugconcentration must be in relation to the MIC in order to make this simplifyingassumption The answer is illustrated in Fig 4 where it can be seen that whenone reaches a CpMIC ratio of 10ndash12 1 the simplifying assumption seems tohold Several other investigators have demonstrated the same phenomenon [34]Achieving a CpMIC ratio of 10 or greater forms the basis for high and infrequentdosing of aminoglycosides [5ndash7] If such a ratio is achieved bacterial killing isat a maximum If it is not achieved then the drug can still be quite effectivehowever the simplifying assumption cannot be made and the entire AUC mustbe applied to the ratio A good dosing strategy for the clinical use of such drugsis to use the highest possible dose that achieves this ratio and does not causetoxicity

FIGURE 4 Clinical response vs CpMIC (from Ref 7)


Examination of Fig 3 also reveals that other agents such as the β-lactamsexhibit different bacterial killing attributes It can be seen that once these drugsachieve concentrations of 2ndash4 times the MIC further increases in concentrationdo not yield greater rates of bacterial killing Bacterial killing is at a maximumunder these conditions the contribution of drug concentration to the entire killingprocess is minimal and can be ignored The AUCMIC ratio simplifies to drugexposure of the bacteria and is expressed pharmacodynamically as the time theserum concentrations remain above the MIC of the bacteria (T MIC) For drugsthat display such behavior the clinical dosing strategy is to maximize the T MIC While the minimal T MIC varies with different drugndashbug combinationsit appears from animal [8ndash11] and clinical [12] data that an acceptable workingvalue of T MIC of about 50 of the dosing interval is associated with satisfac-tory outcomes This number is appropriate for patients with intact or functioningimmune systems For immunocompromised patients being 4ndash5 times the MICis related to better outcomes and is roughly equivalent to being above the MICfor the entire dosing interval [13] The working number of 50 T MIC or100 T MIC for immunocompromised patients is just a good approximationof what is necessary to achieve satisfactory clinical response This will vary de-pending upon different conditions such as host factors and drugndashbacteria interac-tions

It is important to also realize that the pharmacodynamic classification ofhow a drug interacts with bacteria is directly related to the bacterial rate ofgrowth If antibiotics interfere with certain biochemical reactions in the bacteriathat are normally part of their life cycle then the drug needs to be present boundto the active site at the time the biochemical reaction would normally occurAlthough this is intuitively obvious what seems to be overlooked is that thepharmacodynamic parameter that correlates best with bacterial eradication willchange with the growth properties of the bacteria A good example of this is arecently published study by Lutsar et al [14] who studied gatifloxacin pharmaco-dynamics in the cerebral spinal fluid (CSF) of rabbits Because gatifloxacin is aquinolone it is expected to be a concentration-dependent bactericidal agent Assuch one would expect that the pharmacodynamic parameters AUCMIC or CpMIC might predominate Whereas these investigators found that AUCcsfMIC didcorrelate with outcomes when the AUCcsf was held constant it was the T MICthat correlated with good outcome not CpcsfMIC This may at first seem counter-intuitive but it can be explained on the basis of the rate of growth of bacteriain CSF If the rate of growth is relatively slow the antibiotic will act only if itis present on the active site when that site is involved in the necessary biochemicalreaction that is part of the bacteriarsquos life cycle When one considers this it is notsurprising that a relationship exists with T MIC rather than high and transitorypeak concentrations


Do bacteria grow more slowly in CSF We believe they do because rapidlygrowing bacteria in the CSF will probably kill the host before therapy can beinitiated We hypothesize that the CSF is a much poorer growth medium forbacteria than either broth or other sites in the body and this slower growth allowstherapy to be initiated but also changes the pharmacodynamic parameter thatcorrelates with effective bacterial eradication

52 Relationship Between Pharmacodynamics and

Clinical Efficacy

The effective treatment of patients suffering from an infectious disease is a multi-faceted process that depends upon many factors for a successful outcome Gener-ally speaking there is an inverse relationship between the MIC of an organismand clinical efficacy ie the lower the MIC the higher the cure rate This isillustrated in Table 1 What can also be seen in this table is that the oppositemdashidentifying the MIC that correlates to an effective clinical cure ratemdashis not soobvious and many MIC values (eg 1 10 20 30 40) can be consideredto result in an acceptable cure rate Table 2 illustrates that eradication of bacteriacan fail to occur even when the bacteria are susceptible to the antibiotic Reportshave been published eg in the treatment of S pneumoniae bacteremia wherethe death rate is substantial in spite of aggressive and appropriate antimicrobialtherapy [1516] Obviously the ability of the antibiotic to kill the organism isonly one factor affecting patient outcome Other factors such as the age of thepatient the vigor of the immune system and psychological factors such as thewill to live and the degree of confidence the patient has in the caregivers andthe health system will affect outcomes Although pharmacodynamic parametersare important and useful in guiding therapy they are not the only issues thataffect outcomes The clinical practice of medicine still remains somewhat of an

TABLE 1 General Relationship BetweenMICs and Clinical Cures (Claforan)

MIC Clinical cures(microgmL) ()

10 9480 90

160 77320 84

640 64

Source Ref 22


TA

BLE

2S

ucc

ess

and

Failu

reo

fR

esis

tan

tan

dS

ensi

tive

Str

ain

so

fH

aem

op

hilu

sIn

flu

enza

Clin

ical

corr

elat

ion

aN

um

ber

of

stra

ins

wit

hIn

dic

ated

MIC

An

tim

icro

bia

lN

um

ber

An

tib

ioti

co

utc

om

eb

of

Pat

ien

ts

025

05

12

48

16

32

Lore

carb

efC

118

64

752

7428

87

6R

F10

18

1A

mo

x

CI

137

753

4225

73

Cla

vR

F11

74

aH

aem

op

hilu

sin

flu

enza

AO

MS

A

MS

A

PE

CB

an

dp

neu

mo

nia

b

CI

cu

reo

rim

pro

ve

RF

re

lap

seo

rfa

ilure

S

ou

rce

Ref

23


art rather than a pure science Pharmacodynamic principles help move clinicalmedicine several steps toward the science side but they are not the only issueof concern in clinical medicine

Issues involving an antibioticrsquos properties such as protein binding PAEand volume of distribution are important properties of the drug however forlicensed or marketed drugs further consideration of these properties is rarelyneeded The rationale behind this statement is related to the process of drug li-censing Using the drug development process the first step is to do preclinicalstudies followed by clinical studies Phase I studies are designed to observe anddetect gross human toxicity and to explore the drug dose and dosing issues Oncethis is completed a value judgment is made concerning a useful and safe clinicaldose The next step phase II is to carry out small-scale clinical trials the mainpurpose of which is to confirm the efficacy of using the dose derived from preclin-ical and phase I studies When it is confirmed that the drug is safe and effectivewith the chosen dosage and dosing regimen the development proceeds to largerscale clinical trials phase III If the dose and dosing regimen are found to beacceptable phase III trials eventually lead to licensing The point of this is thatthe licensed drug has been found to be acceptable useful and nontoxic on thebasis of all its properties For an antibiotic these include its pharmacokineticsand pharmacodynamic properties which involve considerations of volumes ofdistribution half-life PAE protein binding and tissue penetration If the antibi-otic were not acceptable the dose would be changed until it met the criteria forlicensing In other words the observed (empirical) result is due to the dose usedbased upon the agentrsquos overall properties The issues of protein binding PAEand other issues although important have already been taken into account andcontribute to the net effect one observes Further consideration of these issueswith respect to clinical outcomes is rarely necessary Considering a drugrsquos proteinbinding as a separate and unrelated issue does not make an approved agent lessor more effective in clinical use It will change the pharmacodynamic parameterseg AUCMIC and on a comparative basis will affect the rank order of drugsdepending upon their protein binding

53 The Application of Pharmacodynamic Parameters to

Clinical Practice

All pharmacodynamic parameters are simply calculated numbers that have noinherent value by themselves In order for these numbers to have value one mustcorrelate them to an outcome Because most practitioners are interested in treatinginfected patients the most accepted correlation is between the pharmacodynamicparameter and the clinical outcome It is important to realize that if one correlatesthe pharmacodynamic parameter to an outcome that is different than clinical ef-fectiveness then the important or critical value of the pharmacodynamic parame-


ter will change For example if one correlates the AUCMIC to clinical curesone critical number appears such that if one exceeds that parameter one can ex-pect good clinical cures If one correlates the parameter to maximal eradicationof bacteria another AUCMIC number appears that will probably be higher thanthat calculated in the first example If one desires to correlate the pharmacody-namic parameter to the suppression of the emergence of resistance then a thirdcritical pharmacodynamic number will appear

Considering the pharmacodynamic parameter AUCMIC and correlating itto clinical cures when the target organism is S pneumoniae and the drug classis quinolones we can make two important observations Figure 5 represents aschematic hypothetical graph of the relationship between clinical cure rates andAUCMIC ratio This schematic is based upon a variant of a dosendashresponsecurve At some point (to the right of the vertical line) a maximum clinical re-sponse is observed Most clinical studies in humans lie within this plateau be-cause the usual clinical trials are designed for success and not failure Data tocomplete the curve (the data to the left of the vertical line) are generally obtainedfrom animal studies or in vitro experiments The breakpoint (the point where thecurve plateaus) is a value judgment based upon the composite of all of these data[17ndash19] Our value judgment is that an AUCMIC ratio of 30ndash40 is relatedto successful clinical outcomes If one examines Table 3 which is derived fromsuccessful clinical studies one can see that there are a variety of AUCMICratios for total drug that correlate with successful clinical outcomes [20] Thisis expected on the basis of Fig 5 and argues that a single AUCMIC ratio for aparticular drug class against a particular organism does not exist If one performsa similar analysis with the same or different drug classes against different organ-isms a different range of ratios will be found to correlate to acceptable clinicaloutcomes This argues that although pharmacodynamic parameters are useful indetermining proper dosing they should be used as a guide rather than as absolute

FIGURE 5 Dosendashresponse curve for quinoloones vs S pneumoniae


TABLE 3 Oral Quinolone Pharmacodynamics S pneumoniae(Pharmacodynamic Order)

Drug Dose MIC90 AUCMIC

Moxifloxacin 400 mg 025 142Trovafloxacin 200 mg 025 138Grepafloxacin 600 mg 025 908Gatifloxacin 400 mg 05 678Levofloxacin 500 mg 1 476Sparfloxacin 200 mg 05 374

numbers This concept is further strengthened by the realization that the AUCMIC is a ratio with a numerator and a denominator The numerator representsthe AUC but that is usually the AUC of the drug in normal healthy volunteersThe clinical use of antibiotics is in patients and it is the patientrsquos AUC thatshould be used in the ratio Unfortunately this value is not readily available Thenormal volunteerrsquos AUC represents the worst-case situation and this AUC isgenerally smaller than that of an actual patient The denominator has even moreerror and variability associated with it The MIC should be that of the drug andthe bacteria in the host body not in an artificial medium such as broth Brothaffords an ideal growth medium whereas bodily fluids do not The growth ratesof organisms will be different in the body than in broth and the interactionswith the antimicrobial agents will be affected ie the MIC will be different aspreviously discussed Unfortunately it is difficult or impossible to obtain theMIC in bodily fluids therefore we commonly use the incorrect MIC obtained inbroth A large degree of variability is associated with the usual technique ofdetermining the MIC Most MICs are measured using a twofold dilution tech-nique and one dilution to either side of the observed value is not considered tobe an important or real difference A reported MIC of 10 microgmL can be 0510 or 20 microgmL That represents 100 variability in the reported MIC valueIf the numerator is not accurate and the denominator is not accurate then theentire ratio cannot be accurate Reported AUCMIC ratios are too variable to beconsidered absolute numbers and should be used only as guides to therapy Asa guide the MIC is very useful but to consider it to be an absolute infalliblenumber is misleading and confusing

REFERENCES

1 HP Kuemmerle T Murakawa CH Nightingale eds Pharmacokinetics of Antimicro-bial Agents Principles Methods Applications Ecomed 1993


2 PR Murray Editor in Chief and EJ Baron MA Pfaller FC Tenover and RH Yolkeneds Manual of Clinical Microbiology 6th ed ASM Press Washington DC 1995

3 EJ Begg BA Peddie ST Chambers DR Boswell Comparison of gentamicin dosingregimens using an in-vitro model J Antimicrob Chemother 199229427ndash433

4 BD Davis Mechanism of the bactericidal action of the aminoglyocosides MicrobiolRev 198751341ndash350

5 DP Nicolau CD Freeman PP Beleveau CH Nightingale JW Ross R QuintilianiExperience with a once-daily aminoglycoside program administered to 2184 adultpatients Antimicrob Agents Chemother 199539650ndash655

6 MF Keating GP Bodey M Valdivieso V Rodriquez A randomized comparativetrial of three aminoglycosidesmdashcomparison of continuous infusions of gentamicinamikacin and sisomicin combined with carbenicillin in the treatment of neutropenicpatients with malignancies Medicine 197958159ndash170

7 RD Moore PS Lietman CR Smith Clinical response to aminoglycoside therapyImportance of the ratio of peak concentration to minimal inhibitory concentrationJ Infect Dis 198715593ndash99

8 JE Leggett S Ebert B Fantin WA Craig Comparative dose-effect relations at sev-eral dosing intervals for beta-lactam aminoglycoside and quinolone antibioticsagainst gram-negative bacilli in murine thigh-infection and pneumonitis modelsScand J Infect Dis 199174179ndash184

9 JE Leggett B Fantin S Ebert K Totsuka B Vogelman W Calame H Mattie WACraig Comparative antibiotic dose-effect relations at several dosing intervals in mu-rine pneumonitis and thigh-infection models J Infect Dis 1989159281ndash292

10 R Roosendaal IAJM Bakker-Woudenberg JC van den Berghe MF Michel Thera-peutic efficacy of continuous versus intermittent administration of ceftazidime in anexperimental Klebsiella pneumoniae pneumonia in rats J Infect Dis 1985156373ndash378

11 CO Onyeji DP Nicolau CH Nightingale R Quintiliani Optimal times above MICsof ceftibuten and cefaclor in experimental intra-abdominal infections AntimicrobAgents Chemother 1994381112ndash1117

12 GP Bodey SJ Ketchel V Rodriguez A randomized study of carbenicillin plus cefa-mandole or tobramycin in the treatment of febrile episodes in cancer patients AmJ Med 197967608ndash616

13 GP Bodey SJ Ketchel V Rodriguez A randomised trial of carbenicillin plus cefa-mandole or tobramycin in the treatment of febrile episodes in cancer patients Anti-microb Agents Chemother 199236540ndash544

14 I Lutsar IR Friedland L Wubbel CC McCoig HS Jafri WN Ng F Ghaffar GHMcCracken Jr Pharmacodynamics of gatifloxacin in cerebrospinal fluid in experi-mental cephalosporin-resistant pneumococcal meningitis Antimicrob Agents Che-mother 1998422650ndash2655

15 EW Hook III CA Horton DR Schaberg Failure of intensive care unit support toinfluence mortality from pneumococcal bacteremia JAMA 19832491055ndash1057

16 WR Gransden SJ Eykyn I Phillips Pneumococcal bacteremia 325 episodes diag-nosed at St Thomasrsquos Hospital BMJ 1985290505ndash508

17 PD Lister CC Sanders Pharmacodynamics of levofloxacin and ciprofloxacin againstStreptococcus pneumoniae J Antimicrob Chemother 19994379ndash86


18 Lacy M Quintiliani R et al AAC 199943672ndash67719 Lister P Sanders C AAC 1999431118ndash112220 P Ball A Fernald G Tillotson Therapeutic advances of new fluoroquinolones Exp

Opin Invest Drugs 19987761ndash78321 Craig WA et al Scand J Infect Dis 1991suppl 7422 Murry et al Abstract ICCAC meeting 198323 Doern G Pedi Infect Dis J 199514420ndash423

3

In Vitro Antibiotic PharmacodynamicModels

Michael J Rybak

Wayne State University Schools of Medicine and Pharmacy andDetroit Receiving Hospital and University Health Center Detroit Michigan

George P Allen

Wayne State University and Detroit Receiving Hospital andUniversity Health Center Detroit Michigan

Ellie Hershberger

William Beaumont Research Institute Royal Oak Michigan

1 INTRODUCTION

The traditional approach to in vitro assessment of antimicrobial action on organ-ism viability has been through the use of typical susceptibility testing such asagar diffusion assay or broth dilution techniques These tests give either a qualita-tive or quantitative assessment of antimicrobial activity at fixed concentrationsof drug at a single point in time usually 18ndash24 h The killing curve approachallows for periodic or continuous monitoring of viable organisms after antibioticexposure This method significantly improves the ability to assess the interactionbetween antimicrobials and organisms Factors such as the rate and extent of

41

42 Rybak et al

killing as a function of concentration can be determined In addition the impactof combination antibiotics can easily be evaluated However the major limitationof this method is the use of a fixed concentration of antibiotic throughout theexperimental period Therefore it does not represent the clinical setting whereantimicrobial concentrations continuously fluctuate as a function of delivery pen-etration metabolism and elimination

These limitations prompted the development of antibiotic pharmacody-namic models that are capable of simulating antibiotic pharmacokinetics in thepresence of viable bacteria Controlled dilution of media containing the drug andorganism inoculum is the basic function by which these dynamic models simulateantibiotic pharmacokinetics One of the earliest attempts to use an in vitro antibi-otic dynamic model system to simulate human infection was reported by Green-wood and OrsquoGrady [1] These investigators simulated bladder infection by usingglass flasks in which the effect of antibiotics on bacteria in artificial media couldbe studied photometrically Micturition was simulated by alterations in the vol-ume and flow rate of medium so that the concentrations of organism and antibioticin the model varied in a manner similar to in vivo infection Although it was avery novel approach there were a number of problems with this method includ-ing the use of photometric analysis The spectrophotometric method used in thisexperiment proved unreliable because it counted both viable and dead bacteriaand because of its dependence on light transmission through the artificial mediumit was not useful for dense bacterial populations of 106ndash109 CFUmL which areoften more relevant to human infection Another inherent problem was that bacte-ria grew more rapidly in artificial media than was observed in urine In subsequentmodels these limitations were corrected by plating and counting the number ofviable bacteria over the course of the experiment and by using filtered sterilizedurine as the growth medium There remain some mechanical problems with thismodel such as occasional adherence of organisms to the glass culture vesselsduring the washout phase and the inability to simulate cyclical variations in urineantibiotic concentrations due to renal clearance or micturition However thesemodelshave provided realistic simulationsof the responseoforganism andantibiot-ics among patients in clinical trials [2] In addition the bladder model had a muchhigher correlation with a mouse infection protection test than did a variety of stan-dard laboratory tests Furthermore conventional laboratory methods failed to pre-dict antibiotic synergy observed in both the bladder model and mouse models [34]

Over the years there have been a variety of in vitro antibiotic dynamic mod-els described in the literature that have attempted to characterize the pharmaco-kinetic profiles of various antibiotics while simulating an infection site Thesemodels have ranged from simple simulations of bloodstream infections with one-compartment dynamic models to sequestered infections with more sophisticatedmulticompartmental approaches or hybrid systems incorporating infected tissue


or cell lines This chapter presents a description of these models and their inherentadvantages and disadvantages for modeling infection and antimicrobial pharma-cokineticspharmacodynamics

2 ONE-COMPARTMENT MODEL

In the one-compartment model the antimicrobial agent is added to a central com-partment that contains the appropriate culture medium and a known concentrationof the organism The medium inside the central compartment is then displacedvia a peristaltic pump set at a fixed rate to simulate the clearance and half-lifeof the antibiotic (Fig 1) This system represents a simple pharmacodynamicmodel that follows first order pharmacokinetics resulting in an exponential de-crease in the drug concentration Desired pharmacokinetic drug parameters canbe estimated using the equation

c c0eket

FIGURE 1 One-compartment model The bacterial inoculum and antibiotic areintroduced into the central compartment CA concentration of antibiotic ACC central compartment CIA clearance of antibiotic A FM fresh me-dium P peristaltic pump SP sampling and injection port VC volumeof distribution of antibiotic A SB magnetic stir bar WB water bath(375degC) WM waste medium

44 Rybak et al

where

c antibiotic concentrationc0 concentration following drug administrationke elimination rate constantt time

The elimination rate constant is defined by

ke ClVc (1)

and

ke (ln 2)t12 (2)

Therefore

(ln 2)t12 ClVc (3)

Solving for Cl

Cl (ln 2)(Vct12) (4)

where

Cl clearanceVc volume in the central compartmentt12 half-life of the drug

This system has the advantage of being able to simulate human kineticsby simply utilizing sterile flasks connected by tubing passed through a peristalticpump Because of their simplicity one-compartment models have been used ex-tensively throughout the years to evaluate pharmacodynamics of antimicrobialagents Zabinski et al [5] used a modified version of a one-compartment modelpreviously described by Garisson and his colleagues to determine the effect ofaerobic and anaerobic environments on the activities of different fluoroquinolonesagainst staphylococci The system used in this experiment consisted of a 1L glassvessel with inflow and outflow ports silicone tubing a peristaltic pump a freshmedia reservoir and a stir hot plate (to maintain a normal body temperature ofapproximately 37degC) The entire system was then placed in a chamber containingan anaerobic gas mixture

One of the disadvantages of this system is the clearance of bacteria fromthe central compartment along with the antibiotic This clearance may vary sig-nificantly depending on the half-life of the drug For example in an in vitroexperiment using vancomycin (half-life 6 h) against Staphylococcus aureus (start-ing inoculum of 106 CFUmL) an insignificant amount of organism will be lostbecause the doubling time of S aureus is greater than the clearance of vancomy-


cin However utilizing nafcillin (half-life 05 h) against the same isolate mightoverestimate the rate of kill because its clearance rate is greater than the doublingtime of S aureus Previously membranes were used in these models to preventthe elimination of bacteria from the central compartment however accumulationof antibiotic on the surface of the membrane may result in the antibiotic beingretained as well thus confounding interpretation of antibiotic killing effects [6]

In an effort to circumvent the problem of bacterial dilution while preventingconcomitant antibiotic retention Navashin et al [7] used a model that incorpo-rated a modified membrane system that is washed continuously throughout theexperiment Their model is similar in construction to the models described aboveconsisting of a supply of fresh medium that is pumped into a chamber into whichthe bacterial inoculum is injected (Fig 2) Medium is removed from this chamberby a second peristaltic pump however the medium is then eliminated througha filtration device that prevents the loss of bacteria The filtration device includes

FIGURE 2 Two-compartment model with membrane-wash system Mediumis pumped from the working unit (a photometric cell) through a filtration unitdesigned to prevent bacterial loss FM fresh medium FU filtration unitMF membrane filter P peristaltic pump WU working unit of model(Adapted from Ref 7)

46 Rybak et al

a membrane with a pore size of 045 microm To prevent blockage of this membranewith excreted antibiotic outflow medium is continuously run over the surface ofthe membrane before being eliminated to a final reservoir

In the absence of a membrane correction the loss of bacteria becomesimportant when the action of an antibiotic on the bacteria is being evaluatedThis is especially a problem when the activities of two drugs with considerablydifferent half-lives are being compared Haag et al [8] developed differentialequations to describe changes in the bacterial inoculum in the central compart-ment over time Keil et al [9] later examined these equations and developed anextension of them that can be used to compensate for the amount of bacteriabeing lost to the elimination rate

The following are the equations developed by Haag and Keil where N(t)represents the number of colony-forming units per milliliter in the free medium(liquid compartment) at a given time t NB(t) represents the number of CFUsper milliliter in the modelrsquos biofilm layer (solid compartment) at the same timedN(t)dt and dNB(t)dt are the first derivatives of N(t) and NB(t) respectivelykw is the apparent bacterial growth rate constant ka is the rate constant for absorp-tion of bacteria into the biofilm layer and kd is the rate constant for desorptionof cells from the biofilm into the liquid medium [910] The elimination rateconstant is represented by ke

Assuming that kw is the same in broth and biofilm ke ka and kd are constantover the course of the experiment and N(t) is far below the maximum bacterialdensity under nonflowing conditions we have

dN(t)dt

(kw ke ka)N(t) kdNB(t) (5a)

dNB(t)dt

(kw kd)NB(t) kaN(t) (5b)

When the bacterial culture is not diluted

dNprime(t)dt

(kw ka)N(t) kdNB(t) (6)

where dNprime(t)dt describes the change in CFUmL in an undiluted compartmentover time

The total time course of N(t) is described by a second-order differentialequation derived from Eqs (5a) and (5b) Solving this equation (not shown)results in

N(t) Ae m1t Be m2t (7)


and

A B N0 (8)

where N0 is the initial number of CFUmL in the model flask Ae m1t describesthe number of CFUmL coming from the biofilm Be m2t describes the number ofCFUmL coming from the liquid compartment m1 0 and m2 0

The factor Be m2t approaches 0 at high dilution rates and at t infin Assumingthat the development of a biofilm may be neglected (ke ka and kd or ka kd 0 A 0 and B N0) we arrive at

Nprime(t) N(t)e ket (9)

In cases where the desorption of bacteria from the biofilm to the liquid mediumfar exceeds the loss of bacteria due to dilution equation (9) becomes

Nprime(t) N(t) e(12)ket (10)

Comparison of Eqs (9) and (10) shows that the effect of the presence ofa biofilm layer on bacterial loss is compensated for by a factor f with a valuebetween 05 and 1 The value of f approaches 05 as the influence of the biofilmis magnified If ke is constant throughout the course of the investigation Eqs (9)and (10) may be combined to give

Nprime(t) N(t)e f ket (11)

If the antibiotic under study displays two different elimination rate con-stants for the α and β phases the above equations must be altered Assumingthat there is no biofilm in the model flasks manipulation of these equations resultsin

Nprime(t) N(t)esumkei ∆ti (12)

where ∆ti and kei represent any number of time periods ∆t and their correspond-ing elimination rate constants ke If there is a biofilm present we arrive at theequation

Nprime(t) N(t)e(12)sum(kei ∆ti) (13)

Combining Eqs (12) and (13) gives

Nprime(t) N(t)e f sum(kei ∆ti) (14)

Again the value of f is between 05 and 1 Equation (14) is valid only as longas bacteria are in the logarithmic phase of growth For cultures approaching thestationary phase of growth the following equation is valid

48 Rybak et al

Nprime(t) NmaxN(t)

N(t) [Nmax N(t)]eket(15)

Where there is an influence of biofilms and a variable ke the following equationshould be used

Nprime(t) NmaxN(t)

N(t) [Nmax N(t)]efsum(kei∆ti)(16)

where the value of f is again between 05 and 1Keil and Wiedemann [9] evaluated the validity of these equations in a study

of continuous infusions of meropenem with steady-state concentrations of 25ndash75 microgmL against Pseudomonas aeruginosa and Escherichia coli The resultingkill curves were compared to kill curves obtained from incubation of these bacte-ria in a static system with meropenem at constant concentrations of 25ndash75 microgmL Thus the sole difference between experiments was the presence of dilution

Equation (9) can be used to correct for bacterial loss when the duration ofstudy does not exceed 8 h However results of Keil and Wiedemannrsquos studyrevealed that Eq (15) should be used when the duration of the experiment exceeds12 h [9] In addition using a value of 1 for f results in kill curves representingthe worst-case scenario so that the antibacterial effect is not overestimated Un-fortunately this study additionally showed that individual corrections must beperformed for different bacterial species and model apparatuses

Routine application of the above mathematical equations may prove to becumbersome The inclusion of growth control experiments studying bacterialgrowth in the absence of antibiotic can also help to address the severity of theproblem of bacterial dilution A typical growth curve is set to simulate the half-life of the antibiotic under study To compensate for bacterial loss the killingactivity of the antibiotic can be compared to the growth control curve

3 TWO-COMPARTMENT MODEL

The two-compartment model simulates biexponential pharmacokinetics follow-ing intravenous administration Murakawa et al [11] designed a model represent-ing the central and peripheral compartments connected by tubing This modelconsists of a central compartment that contains the antibiotic and organism Me-dium constantly flows into this central compartment Medium is then pumpedfrom the central compartment to a peripheral compartment (free of antibiotic) andis subsequently pumped back into the central compartment via a second peristalticpump Finally it is pumped out of the central compartment to a reservoir forbacterial counts As with the one-compartment model this system allows theorganism to be pumped out of the central compartment therefore dilution of the


organism according to the half-life of the antibiotic is a potential problem Analternative design of the typical two-compartment model places the peripheralcompartment within the central compartment (Fig 3) The bacteria under studyare placed into this inner chamber and antibiotic is administered into the centralcompartment The antibiotic may diffuse into the peripheral compartment butbacteria are trapped within that compartment

In an attempt to mimic drug concentration profiles in tissue versus serumZinner et al [12] devised a model that uses a single capillary unit device as asystem for studying antimicrobial effects (29) Later Blaser et al [13] chose atwo-compartment capillary model that uses a series of artificial capillary unitsrepresenting extravascular infection sites (Fig 4) The Zinner and Blaser modelscan simulate human pharmacokinetics in interstitial fluid for one or two antibiot-ics Each of the models uses an artificial capillary (dialysis) unit connected bysilicone tubing to a peristaltic pump and a culture medium reservoir A samplingport with a bidirectional stopcock is inserted into the tubing and allows injection

FIGURE 3 Two-compartment model Bacteria are introduced into the periph-eral compartment and are unable to pass into the central compartment (illus-trated by heavy arrow) Antibiotic is injected into the central compartmentAB antibiotic B bacteria CC central compartment FM fresh me-dium PC peripheral compartment SB magnetic stir bar SP samplingand injection port WM water medium (Adapted from Ref 22)

50 Rybak et al

FIGURE 4 Two-compartment capillary model The capillary device (dialyzer)is pictured at the bottom of the illustration Inset shows a cross section ofthe capillary bundle Medium flow through the bundle is represented by theheavy arrow Bacteria are trapped in the outer compartment of the bundlewhile antibiotic (light arrows) flow through the dialysis membranes B bac-teria CB capillary bundle OC outer compartment P peristaltic pumpR reservoir SP sampling and injection port SS sampling site (septumfittings) (Adapted from Ref 12)

of antibiotics either toward the capillary unit (bolus injection) or toward the me-dium reservoir (for continuous infusion dosing simulations)

Each capillary unit consists of two chambers an outer chamber or culturetube and an inner chamber that comprises a bundle of 150 hollow polysulfonefibers Each fiber (lsquolsquocapillaryrsquorsquo) has an internal diameter of 200 microm and a wallthickness of 50ndash75 microm In the study reported by Zinner and his colleagues thecapillaries used retained proteins of a size greater than 10000 daltons Howeveradditional capillary units are available that retain proteins of sizes greater than50000 or 100000 daltons The entire capillary bundle is secured by siliconeO-rings which seal the outer chamber (extracapillary space) from the lumens ofthe capillary tubes

An inoculum of bacteria is introduced into the outer chamber of the capil-lary system Meanwhile the antibiotic under study is injected into the silicone


tubing and allowed to diffuse either directly to the capillary unit (to simulateintravenous bolus dosing) or indirectly through the medium reservoir (thus simu-lating a continuous infusion dosing strategy) Because bacteria do not diffuse outof the extracapillary space this model simulates an infected tissue site Dependingon the molecular size of the antibiotic used and the permeability characteristicsof the capillary tubing antibiotic diffuses into the extracapillary space from thecapillary tubing Again because the concentration of antibiotic obtained at thesite where bacteria are present will be lower than that in the capillary tubingthis system allows simulation of the treatment of infections at tissue sites

This model allows the study of a variety of pharmacokinetic and pharmaco-dynamic parameters including the effects on bacterial killing of bolus dosingversus continuous infusions of antibiotics In addition the effects of single orcombined agents can be assessed and the model allows simulation of the fluctua-tions in concentrations that typically occur after dosing in humans Furthermorethe model avoids the problem of bacterial dilution as the volume of bacteriain the extracapillary space is not significantly changed during the experimentHowever controlled variation of antibiotic concentrations in the two chambersof this model may be difficult given the variability inherent in relying on thediffusion of drugs through the walls of the capillary tubes

Blaser et al [13] compared the pharmacokinetics of netilmicin obtained ina two-compartment capillary model to those obtained in an experimental skinsuction blister model in healthy volunteers Netilmicin was administered ac-cording to two dosage schemes in the capillary model in order to achieve peakconcentrations of 16 or 24 microgmL In the human blister model netilmicin wasgiven as an intramuscular injection of 2 mgkg (lean body weight) The in vitromodel demonstrated linear two-compartment pharmacokinetics Peak concentra-tions obtained were higher than nominal for the central compartment and lowerthan nominal for the peripheral compartment One hour after the end of a 60 mininfusion netilmicin concentrations were equivalent in the central and peripheralcompartments Overall concentrations obtained in the in vitro model approxi-mated those obtained in the human blister model (Fig 5) Thus this capillarymodel successfully simulates human pharmacokinetics

4 COMBINATION THERAPY MODEL

In vitro models can also be used to simultaneously study the activity of two drugswith different half-lives in combination Blaser [14] described the procedures forsimulating combination therapy using both one- and two-compartment modelsIn these models both drugs are placed in the central compartment and the clear-ance pump rate is set to simulate the half-life of the drug with the shorter half-life (ClA drug A) (Fig 6) A supplemental compartment is used to replace the

52 Rybak et al

FIGURE 5 Comparison of in vitro and human models Netilmicin concen-trations in the central and peripheral compartments of an in vitro two-compartment capillary unit model are shown The 2 sets of curves representnetilmicin concentrations after administration of infusions attaining peakconcentrations of either 16 or 20 mgL The insert graph illustrates serum() and suction blister () netilmicin pharmacokinetics after administrationin healthy human volunteers Comparison of the main and insert graphs illus-trates the similarity in pharmacokinetics between the in vitro and humanmodels (Adapted from Ref 7)

antibiotic with the longer half-life (drug B) which has been overeliminated Thepump rate for the supplemental compartment (ClS) is set to make up the differencebetween the clearance of the two drugs

ClS ClA ClB

To maintain the same concentration of drug B in both compartments the centraland supplemental compartments should be dosed in a similar manner

Meanwhile drug-free medium is pumped into the central compartment sothat the total amount of medium pumped into the system (M in) equals the totalvolume of medium being pumped out of the system (Mout)


FIGURE 6 Combination therapy model In this example drug B is added froma supplemental chamber to the central reaction flask CA concentration ofdrug A CB concentration of drug B CC central compartment ClA clear-ance of drug A ClB clearance of drug B FM fresh medium Min rateof inflow of fresh medium P peristaltic pump SC supplemental compart-ment WM waste medium (Adapted from Ref 14)

M in ClA ClS

Therefore the pump rate for the drug free medium should be set as ClBThe advantage of this system is that it can be used to determine synergistic

additive or antagonistic effects of combinations of antibiotics simulating the ac-tual pharmacokinetics of each agent For example Houlihan et al [15] evaluatedthe activity of vancomycin alone and in combination with gentamicin againstmethicillin-resistant Staphylococcus aureusndashinfected fibrinndashplatelet clots in aninfection model Vancomycin was dosed at intervals of 6 12 and 24 h andgentamicin was given either as a single daily (every 24 h) dose or every 12 hIn plots of the bacterial inoculum versus time synergism was defined as a 2log increase in killing associated with a combination regimen in comparison tothe most active single agent

In preliminary fractional inhibitory concentration (FIC) testing the combi-

54 Rybak et al

nation of vancomycin and gentamicin displayed indifference However in thepharmacodynamic model several combinations (eg vancomycin every 24 h gentamicin every 24 h) demonstrated a synergistic effect Thus results from useof a static system (FIC testing) were not predictive of results obtained from amodel where simulation of human pharmacokinetics is achieved Blaser et al[16] obtained similar results in a study of ceftazidime-netilmicin combinationsagainst Pseudomonas aeruginosa Using a two-compartment capillary modelsynergism was noted for the combination against certain strains in the initial (46 and 8 h) and final (24 26 and 28 h) periods of evaluation Synergism testingusing the FIC method was predictive of the response seen in the in vitro modelduring the initial period only This serves to reinforce the fact that results obtainedfrom traditional susceptibility methodologies may not correlate with relationshipsseen with the use of pharmacodynamic models where in vivo pharmacokineticsare simulated

5 BIOFILM-CATHETER MODEL

Nickel et al [17] used a modified Robbins device to study the ability of tobra-mycin to penetrate a biofilm layer of Pseudomonas aeruginosa The model con-sisted of a 415 cm long acrylic block with a 2 10 mm inner lumen and 25evenly distributed sampling ports The block was connected via tubing to a reser-voir that was immersed in a 37degC water bath Medium containing the bacteriumunder study was pumped from this reservoir (simulated bladder) through the mod-ified Robbins device (simulated catheter) at a rate of 60 mLh The medium usedconsisted of artificial urine supplemented with nutrient broth Discs cut from acommercially available catheter were suspended within the model and could beremoved for analysis

In the study performed by Nickel et al a biofilm of Pseudomonas aerugi-nosa was formed by passing medium containing logarithmic-phase organismsthrough the model for 8 h Formation of the biofilm was verified by examinationof catheter surfaces using scanning electron microscopy (SEM) and epifluores-cence microscopy After the 8 h colonization period medium was removed andtransferred to flasks for subsequent exposure of these organisms to varying con-centrations of tobramycin for a period of 8 or 12 h Antibiotic-containing mediumwas then pumped through the model and discs of catheter material were removedat 8 and 12 h intervals for determination of colony counts and microscopic exami-nation Thus the killing of organisms in the sessile (biofilm) and planktonic statescould be compared as could the MICs and MBCs of these organisms This modeltherefore allows study of the development of resistance among organisms in abiofilm and of the ability of antibiotics to penetrate the biofilm layer and exertan antimicrobial effect

Walker et al [18] developed an alternative form of the above model that


consisted of a typical one-compartment vessel with plugged catheter segments(127 cm each) suspended within the chamber Catheter material was inoculatedwith 5 106 CFUmL of each organism and counts of sessile and planktonicbacteria were performed using standard plate dilution and SEM This model wasused to test the antiadherence characteristics of various catheter coatings andimpregnations

Prosser et al [19] developed a simplified adaptation of these models Inthis model 05 cm2 discs composed of silicon latex catheter material were spottedwith 80 microL of a bacterial suspension (confirmed to possess an optical density of08 at 540 nm) Discs were then incubated for 1 h washed with a buffer solutionand then transferred to broth-containing Petri dishes and incubated for an addi-tional 20ndash22 h The discs were then rewashed in buffer and placed in plain orantibiotic-containing broth incubated for 4 h washed and resuspended in broth(with or without antibotic) This procedure was repeated at 8 and 24 h At eachtime interval surface film on all discs was scraped off into 5 mL of saline andsamples were plated for colony count determinations Samples were also exam-ined by light microscopy and SEM One advantage of this model lies in the factthat planktonic bacteria are not contained within the model so inactivation ofantibiotic by these planktonic organisms does not occur In addition this modelmay be less cumbersome to operate than the modifed Robbins device

6 INFECTED FIBRIN CLOT MODEL

One modification of a one-compartment model is the infected fibrin clot modeldeveloped by Rybak et al [1520] which uses fibrin clots to simulate a deepsequestered infection such as those associated with cardiac vegetations (Fig 7)Fibrin clots made of human cryoprecipitate organisms and platelets (250000ndash500000 platelets per clot) are placed in 15 mL Eppendorf tubes Bovine throm-bin (5000 units) is then added to each tube after insertion of a sterile monofila-ment line into the mixture Each clot is then inserted into a sterile reaction vesseland removed at various time points for determination of bacterial counts Thismodel has been used to evaluate the activity of antimicrobial agents against or-ganisms embedded in a simulated human tissue With the incorporation of plate-lets and high inoculum of bacteria (109 CFUg) the fibrin clots represent condi-tions of simulated endocardial vegetations (SEVs) The system does not consist ofan airtight compartment therefore it requires two peristaltic pumps to pump themedium into and out of the central compartment The SEVs have been shown tobe viable in the models for up to 144 h

Several studies have been performed to compare this model to an in vivorabbit model of endocarditis McGrath et al [20] indirectly compared the resultsobtained from this model to those of a rabbit model of endocarditis done previ-ously They evaluated the effect of teicoplanin on colony-forming units (CFUs)

56 Rybak et al

FIGURE 7 Fibrin clot model Simulated fibrin clots or endocardial vegetationsare suspended within the central compartment The bacterial inoculum isintroduced into this central compartment FC simulated fibrin clots FM fresh medium ML monofilament lines P peristaltic pump SB mag-netic stir bar SP sampling and injection port WB water bath (375degC)WM waste medium (Adapted from Ref 20)

per gram of SEV in the model using human pharmacokinetics to the CFUg ofrabbit vegetations This study demonstrated that the CFUg of SEV were similarto those of the rabbit vegetations however the pharmacokinetics of teicoplaninvaried between the two models and samples were obtained at different timepoints for analysis

A more recent study has retrospectively compared the simulated endocar-dial vegetation model to four different rabbit studies [10] In this study authorscompared the activities of four different fluoroquinolonesmdashciprofloxacin cli-nafloxacin sparfloxacin and trovafloxacinmdashagainst various Staphylococcusaureus and enterococcal isolates simulating pharmacokinetics obtained in therabbit model Models were conducted in the same fashion as the rabbits weretreated and SEVs were evaluated at the same time points as those at which therabbit vegetations were assessed The bacterial inocula in the SEVs were similarto those achieved in the rabbitsrsquo vegetations prior to and following treatmentsuggesting that this model may have a role in initial studies of antibiotics in thetreatment of bacterial endocarditis (Fig 8) To further define the modelrsquos role inthe treatment of endocarditis prospective studies comparing the in vitro modelto rabbit models of endocarditis using various pathogens and antimicrobial agentsare necessary


FIGURE 8 Comparison of in vitro and animal models The activity of clina-floxacin against strains of methicillin-resistant (MRSA-494) and -susceptible(MSSA -1199) Staphylococcus aureus is illustrated in the graph Bacterial kill-ing observed with in vitro (lsquolsquomodelrsquorsquo) and animal (lsquolsquorabbitrsquorsquo) models of endo-carditis are shown for each organism Similarities between the animal andin vitro models in terms of the change in bacterial inoculum and final inocu-lum value (at 98 hours) may be seen (Adapted from Ref 16)

7 IN VITRO MODEL OF INTRACELLULAR PATHOGENS

The previously mentioned models evaluate the activity of antibiotics against ex-tracellular pathogens However many pathogens such as Salmonella spp Shi-gella spp Campylobacter spp Escherichia coli Proteus mirabilis Listeriamonocytogenes Haemophilus influenzae group B streptococci and Helicobacterpylori can survive intracellularly thereby escaping antimicrobial treatment Cer-tain antimicrobial agents such as azithromycin and clarithromycin possess intra-cellular activity and an in vitro model can be a valuable tool in determining suchactivity Several in vitro models using different cell types have been employedto determine intracellular activity of antimicrobial agents however continuousexposure to a fixed concentration of antibiotic does not reflect human pharmaco-kinetics

58 Rybak et al

Hulten et al [21] have designed an in vitro model to study the intracellularactivity of antimicrobial agents by simulating human pharmacokinetics (Fig 9)In this model a human epithelial cell line is grown in the appropriate mediumand seeded into cell culture inserts containing a 045 microm membrane Inserts areexposed to a fixed concentration of the bacteria and incubated for several hoursto allow the bacteria to penetrate into the cells Extracellular bacteria are thenremoved and inserts are placed in a metal rack that is fixed on a glass chamberconnected to a pump simulating pharmacokinetics of the antibiotic Inserts areremoved at various time points and cells are processed and lysed prior to deter-mination of bacterial inoculum This model was used previously to determinethe intracellular activity of amoxicillin clarithromycin and amoxicillin againstH pylori This model was also used to evaluate the activity of different agentsagainst Mycobacterium tuberculosis [21]

FIGURE 9 Intracellular pathogen model Medium is pumped through the cen-tral compartment (glass chamber) at a rate to simulate antibiotic pharmacoki-netics Inset shows a cross section of the central compartment with cell cul-ture inserts suspended within a metal rack B bottom section of centralcompartment CC cell culture insert FM fresh medium HP hot plate(375degC) P peristaltic pump SB magnetic stir bar SP sampling andinjection port SR stainless steel rack WM waste medium (Adapted fromRef 21)


8 LIMITATIONS OF IN VITRO MODELS

Although the in vitro models described in this chapter are widely used in the studyof antimicrobial pharmacokinetics and pharmacodynamics certain limitations totheir use must be noted The applicability of results obtained from in vitromodels to the treatment of infections in vivo is limited by the factors describedbelow

81 Effects of the Immune System

The majority of in vitro models commonly used are devoid of immune systemfactors that is antimicrobial effects are studied in an environment that does notcontain the host defense factors The antimicrobial effect may thus be underesti-mated because organisms are free from the inhibitory action of such immunesystem factors as leukocytosis phagocytosis and immunoglobulins

Shah [22] employed a model that incorporated fresh human blood fromhealthy volunteers in an effort to replicate in vivo immune system effects Themodel consisted of a glass chamber (similar to the one-compartment model previ-ously described) with an inner cell suspended within The inner cell composedof plexiglass was enclosed at each end with membrane filters designed to allowdiffusion of antibiotics Heparinized human blood was incorporated into the innerchamber and the outer chamber contained a nutrient medium The bacterial inoc-ulum was injected into the inner chamber and antibiotics were administered intothe outer vessel

Use of this model in a study of imipenem against Escherichia coli Kleb-siella pneumoniae and Pseudomonas aeruginosa revealed that in the presenceof blood alone the bacterial count was reduced by 90ndash99 but in all casesbacterial regrowth was noted As expected addition of antibiotic decreased thetime to achieve 99 reduction of the bacterial inoculum and the final bacterialcount was decreased in comparison to that obtained in antibiotic-free modelsAlthough the use of blood as a medium allowed incorporation of some aspectsof the human immune system the full range of host defense factors was notincluded in the model

Incorporation of blood as a medium into in vitro models has not beenwidely employed most likely due to the logistical concerns of working withhuman blood However it should be noted that the lack of an immune systemin typical in vitro models is advantageous in some respects in that the effectof antibiotic exposure alone on the development of bacterial resistance may bemeasured Also results from in vitro models lacking immune system effects maybe satisfactorily applied to infection occurring in neutropenic hosts althoughagain not without some concerns over the clinical applicability of these results

60 Rybak et al

82 Altered Bacterial Growth Characteristics

Multiple investigations have confirmed that the behavior of bacteria in an in vitroenvironment is not equivalent to that in vivo Microorganisms grown in vitrodiffer from those causing in vivo infection with respect to factors discussed inthe following paragraphs

821 Virulence

It has been noted in numerous experiments that bacterial virulence is progres-sively diminished with serial in vitro passage or cultivation [23] The cause ofthis is unknown but it may be due in part to adherence factors or derangementsin biochemical pathways The result of this loss of pathogenicity is potentiallyan overestimation of the effect of a given antimicrobial in an infection modelHowever this reduction in virulence may be strain-specific so the impact onclinical application of in vitro experimental results may be unclear

822 Morphology

Bacteria adapt to life in a blood environment and undergo such morphologicalchanges as encapsulation and mucus production [24] Thus bacteria in vivo pos-sess an enhanced resistance to killing by serum factors In vitro these alterationsin morphology are unstable and organisms rapidly revert to a normal morphol-ogy Thus the effect of an antimicrobial in an infection model may be overesti-mated because the above-described morphological defense factors are absent

823 Physiology

Microorganisms are known to rapidly develop adaptively to a variety of environ-ments possibly as a result of metabolic changes In an in vitro environmentbacteria differ from those cultivated in vivo in terms of amino acid compositionthe synthesis of toxic metabolites and metabolic rate [24] Overall bacteria aremore metabolically active in vivo than in vitro However replication generallyoccurs more rapidly in an in vitro environment possibly due to the lack of im-mune system effects For example it has been shown that the typical growth rateof microorganisms in an in vivo animal model of infection may be on the orderof 002ndash005 h1 wheras the growth rate in an in vitro batch culture may approach2ndash3 h1 [25] Continuous culture of microorganisms which allows precise controlof growth rate may allow maintenance of microorganisms in a state of growthmore closely approximating that of organisms in the natural state [25]

In addition to changes in growth characteristics differences in expressionandor structure of outer membrane proteins may occur such that microorganismsin vitro display reduced permeability These effects are dependent on the compo-sition of the nutrient medium used in model experiments For example the struc-ture and composition of the outer membrane proteins of Escherichia coli may


be altered by changes in growth medium and temperature as well as by the molec-ular size and osmolarity of sugars contained in the growth medium [24]

The overall effect of the above physiological changes on the applicabilityof results obtained from in vitro models is variable and may be difficult to deter-mine These alterations may be highly strain-specific so general conclusions re-garding methods to account for these changes are lacking

824 Sensitivity to Serum Factors

As noted above morphological changes of bacteria in vivo lead to a reducedsusceptibility of those organisms to serum immune factors For example bacteriacultured in vitro display an enhanced susceptibility to phagocytosis [26] There-fore use of models incorporating for example human blood as a medium mayfail to adequately account for immune effects because the bacterial strain maybe more susceptible to the effects of immune mediators than it would be in vivo

83 Microorganism Viability

Certain organisms are difficult to work with in in vitro systems because of viabil-ity problems that are not apparent in vivo For example it is difficult to keepStreptococcus pneumoniae viable for periods longer than 12 h because of thestationary-phase autolytic process that is common to this species For this reasonin vitro killing curves obtained in test tubes do not usually exceed 6ndash12 h soorganism death can be correctly attributed to antibiotic effects rather than to auto-lytic processes

In an in vitro infection model investigation Cappelletty et al [27] demon-strated that the viability of S pneumoniae during growth control experiments isdirectly affected by the clearance of growth medium through the model Simula-tions of antibiotics with longer half-lives were more likely to result in a slowerrate of medium turnover thus increasing the likelihood of autolysis inductionThis serves to reinforce the idea that growth control experiments without antibi-otic must always be performed to ensure that organism viability is optimal [27]

84 Effects of Protein Binding on Antimicrobial Action

Because plasma protein is usually not included in the nutrient broth medium theeffects of protein binding of antimicrobials may not be measured For examplethe antibacterial effect of drugs that are highly bound by plasma proteins maybe overestimated in models where such protein binding is not replicated becauseconcentrations of the drug in the model will exceed those obtained in vivo How-ever incorporation of protein such as human albumin may allow simulation ofthe free-drug concentrations that will be obtained after dosing in humans Forexample Garrison et al [28] assessed the effects of physiological concentrations(38ndash42) of human albumin on the activity of daptomycin and vancomycin

62 Rybak et al

against Staphylococcus aureus Albumin was directly incorporated into the two-compartment in vitro model It was found that the presence of albumin signifi-cantly diminished the bactericidal activity (as measured by kill rate and time to99 kill) of both daptomycin (low- and high-dose regimens) and vancomycinRather than include protein in a model if one knows the degree to which anantibiotic is bound to proteins one may simply dose in such a way as to simulatefree concentrations that will be obtained in the presence of protein This strategyis desirable because of the excessive cost associated with the use of albumin andother proteins in pharmacodynamic models

85 Bacterial Adherence

In a study conducted by Haag et al in vitro models with no dilution andorelimination of growth medium (growth control) were compared to models wherethe addition and elimination of medium took place (addition and elimination wereset to occur at the same rate) The investigators expected that given a dilutionrate higher than the bacterial growth rate an artificial decrease in bacterial countsdue to dilution of organisms would be observed However they instead notedthat an increase in colony-forming units occurred after 1ndash2 h of simulation [8]This phenomenon was ascribed to the presence of a subpopulation of bacteriathat adhered to the inner surfaces of the model in a biofilm layer It is thoughtthat bacteria residing within the biofilm resist dilution but that a certain proportionof bacteria diffuse into the medium at a quantifiable rate

As a result of this phenomenon the effect of an antibiotic on cells in theliquid medium may be measured only in the early stages of an experiment Afterthis time as the biofilm layer forms the effect of the agent on the nondilutableadherent population is measured instead Because organisms in this subpopula-tion typically display reduced antimicrobial susceptibility the effect of the agentunder study may be underestimated [8] On the other hand the effect of an agenton bacteria residing within sequestered sites of infection may be more reliablydetermined

Attempts have been made to prevent bacterial adherence to model surfacesFor example Gwynn et al [29] found that siliconization of their model preventedthe eventual increase in bacterial counts It was postulated that the siliconersquosprevention of the formation of an adherent population was responsible for thiseffect However others have been unable to replicate these findings Bacterialadherence may also depend on the geometry of vessels making up the model andon the material from which models are constructed

86 Dilutionary Artifacts

Although in vitro models incorporating dilution (ie addition and elimination ofmedium at a rate to simulate the half-lives of antibiotics) more closely replicate


the dosing of drugs in humans dilution of the bacterial inoculum is a problemassociated with these models Such dilution (elimination) of microorganisms willresult in an overestimation of the killing effect of an agent being studied Asdescribed previously investigators have employed a variety of strategies to over-come this effect including construction of models that prevent elimination ofbacteria the use of static models in which dilution does not occur and compensa-tion for the effects of dilution by the use of mathematical correction [9]

87 Other Effects

In addition to the above limitations associated with in vitro modeling problemsmay also arise during attempts to interpret results obtained from models incorpo-rating either very highly or minimally bactericidal agents With highly bacteri-cidal antibiotics possessing an extended elimination half-life complete eradica-tion of bacteria to the limit of assay detection will rapidly occur In this scenariothe extent of measurable effects of varying drug dosages will be minimized Incontrast in models in which an agent with minimal bactericidal activity and ashort elimination half-life is used organism regrowth will rapidly overwhelm themodel The effect of regrowth is unknown especially in light of the fact thatorganisms that regrow do not undergo changes in antimicrobial susceptibilityAgain the result of this phenomenon is that effects of the antimicrobial understudy are difficult to measure

9 SUMMARY

In vitro antibiotic pharmacodynamic models represent a significant advancementin the assessment of antimicrobial pharmacodynamics The ability to simulatehuman pharmacokinetics while evaluating the effect of an antibiotic on viablebacteria is clearly superior to more traditional assessments such as basic suscepti-bility testing or kill-curve experiments performed in test tubes These models areadaptable to a variety of conditions allowing for evaluations of different organ-isms environmental conditions dosing schemes combination therapies or resis-tance mechanisms Preliminary information suggests that results obtained fromcertain dynamic models correlate with those found in vivo Although there area number of limitations to the use and applicability of these models they continueto evolve and are likely to add to our overall understanding of antimicrobial andmicroorganism interactions

REFERENCES

1 D Greenwood F OrsquoGrady Potent combinations of beta-lactam antibiotics using thebeta-lactamase inhibition principle Chemotherapy 197521330ndash341

64 Rybak et al

2 JD Anderson KR Johnson MY Aird Comparison of amoxicillin and ampicillinactivities in a continuous culture model of the human urinary bladder AntimicrobAgents Chemother 198017554ndash557

3 JD Anderson Relevance of urinary bladder models to clinical problems and to anti-biotic evaluation J Antimicrob Chemother 198515(suppl A)111ndash115

4 JD Anderson F Eftekhar R Cleeland MJ Kramer G Beskid Comparative activityof mecillinam and ampicillin singly and in combination in the urinary bladder modeland experimental mouse model J Antimicrob Chemother 19818121ndash131

5 RA Zabinski KJ Walker AJ Larsson JA Moody GW Kaatz JC Rotschafer Effectof aerobic and anaerobic environments on antistaphylococcal activities of fivefluoroquinolones Antimicrob Agents Chemother 199539507ndash512

6 J Blaser BB Stone MC Groner SH Zinner Comparative study with enoxacin andnetilmicin in a pharmacodynamic model to determine importance of ratio of peakantibiotic concentration to MIC for bactericidal activity and emergence of resistanceAntimicrob Agents Chemother 1987311054ndash1060

7 SM Navashin IP Fomina AA Firsov VM Chernykh SM Kuznetsova A dynamicmodel for in-vitro evaluation of antimicrobial action by simulation of the pharmaco-kinetic profiles of antibiotics J Antimicrob Chemother 198923389ndash399

8 R Haag P Lexa I Werkhauser Artifacts in dilution pharmacokinetic models causedby adherent bacteria Antimicrob Agents Chemother 198629765ndash768

9 S Keil B Wiedemann Mathematical corrections for bacterial loss in pharmacody-namic in vitro dilution models Antimicrob Agents Chemother 1995391054ndash1058

10 E Hershberger EA Coyle GW Kaatz MJ Zervos MJ Rybak Comparison between arabbit model of bacterial endocarditis and an in vitro infection model with simulatedendocardial vegetations Antimicrob Agents Chemother 2000441921ndash1924

11 T Murakawa H Sakamoto T Hirse M Nishida New in vitro kinetic model forevaluating bactericidal efficacy of antibiotics Antimicrob Agents Chemother 198018377ndash381

12 SH Zinner M Husson J Klastersky An artificial capillary in vitro kinetic model ofantibiotic bactericidal activity J Infect Dis 1981144583ndash587

13 J Blaser BB Stone SH Zinner Two compartment kinetic model with multiple arti-ficial capillary units J Antimicrob Chemother 198515(suppl A)131ndash137

14 J Blaser In-vitro model for simultaneous simulation of the serum kinetics of twodrugs with different half-lives J Antimicrob Chemother 198515(suppl A)125ndash130

15 HH Houlihan RC Mercier MJ Rybak Pharmacodynamics of vancomycin aloneand in combination with gentamicin at various dosing intervals against methicillin-resistant Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infectionmodel Antimicrob Agents Chemother 1997412497ndash2501

16 J Blaser BB Stone MC Groner SH Zinner Impact of netilmicin regimens on theactivities of ceftazidime-netilmicin combinations against Pseudomonas aeruginosain an in vitro pharmacokinetic model Antimicrob Agents Chemother 19852864ndash68

17 JC Nickel JB Wright I Ruseska TJ Marrie C Whitfield JW Costerton Antibioticresistance of Pseudomonas aeruginosa colonizing a urinary catheter in vitro Eur JClin Microbiol 19854213ndash218

18 KJ Walker KJ Kelly AJ Larsson JC Rotschafer DRP Guay K Vance-Bryan Adhe-


sion of Staphylococcus epidermidis (SE) Staphylococcus aureus (SA) and Pseu-domonas aeruginosa (PA) to six vascular catheter materials (abstr) In Program andAbstracts of the 33rd Interscience Conference on Antimicrobial Agents and Chemo-therapy New Orleans LA 1993

19 BL Prosser D Taylor BA Dix R Cleeland Method of evaluating effects of antibiot-ics on bacterial biofilm Antimicrob Agents Chemother 1987311502ndash1506

20 BJ McGrath SL Kang GW Kaatz MJ Rybak Bactericidal activities of teicoplaninvancomycin and gentamicin alone and in combination against Staphylococcusaureus in an in vitro pharmacodynamic model of endocarditis Antimicrob AgentsChemother 1994382034ndash2040

21 K Hulten R Rigo I Gustafsson L Engstrand A new pharmacokinetic in vitro modelfor studies of antibiotic activity against intracellular microorganisms AntimicrobAgents Chemother 1996402727ndash2731

22 PM Shah Activity of imipenem in an in-vitro model simulating pharmacokineticparameters in human blood J Antimicrob Chemother 198515(suppl A)153ndash157

23 DL Watson Virulence of Staphylococcus aureus grown in vitro or in vivo MicrobiolImmunol 197923543ndash547

24 A Dalhoff Differences between bacteria grown in vitro and in vivo J AntimicrobChemother 198515(suppl A)175ndash195

25 P Gilbert The theory and relevance of continuous culture J Antimicrob Chemother198515(suppl A)1ndash6

26 A Dalhoff G Stubner Comparative analysis of the antimicrobial action of polymor-phonuclear leucocytes in vitro ex vivo and in vivo J Antimicrob Chemother 198515(suppl A)283ndash291

27 DM Cappelletty MJ Rybak Bactericidal activities of cefprozil penicillin cefaclorcefixime and loracarbef against penicillin-susceptible and -resistant Streptococcuspneumoniae in an in vitro pharmacodynamic infection model Antimicrob AgentsChemother 1996401148ndash1152

28 MW Garrison K Vance-Bryan TA Larson JP Toscano JC Rotschafer Assessmentof effects of protein binding on daptomycin and vancomycin killing of Staphylococ-cus aureus by using an in vitro pharmacodynamic model Antimicrob Agents Che-mother 1990341925ndash1931

29 MN Gwynn LT Webb GN Rolinson Regrowth of Pseudomonas aeruginosa andother bacteria after the bactericidal action of carbenicillin and other β-lactam antibi-otics J Infect Dis 1981144263ndash269

4

Animal Models of Infection for the Studyof Antibiotic Pharmacodynamics

Michael N Dudley

David Griffith

Microcide Pharmaceuticals Inc Mountain View California

1 INTRODUCTION

Animal models of infection have had a prominent place in the evaluation ofinfection and its treatment From early experiments demonstrating transmissionof infectious agents to satisfy Kochrsquos postulates to evaluation of chemotherapyin the antibiotic era animal models have proven useful for understanding humandiseases

More recently animal models for the study of infectious disease have beenfurther refined to consider the importance of pharmacokinetic factors in theoutcome of infection This allows for the study of the relationship betweendrug exposure (pharmacokinetics) and anti-infective activity (pharmacodynamics)Consideration of these aspects has enhanced the information provided by animalmodels of infection and made the results more predictive of the performance ofdrug regimens in humans This has largely been accomplished through a morethorough understanding of the importance of pharmacokinetics in the outcomeof an infection advances in quantitation of drug concentrations in biological ma-trices and the development of metrics to quantify antibacterial effects in vivo

67


This chapter will review approaches for design analysis and application of theseapproaches in animal models for the study of optimization of anti-infective ther-apy and the discovery and development of new agents

Use of animal models of infection to study the relationship between drugexposure in vivo and antibacterial effects dates back to the very early studies inpenicillin These early investigators noted that the duration of efficacy of penicil-lin in the treatment of streptococcal infections was dependent on the length oftime for which serum concentrations exceeded the MIC [1ndash3] How these earlyobservations ultimately impacted on penicillin therapy in humans is difficult toascertain however it is clear that pharmacodynamic studies in animal modelshave a crucial role in the preclinical evaluation of new anti-infectives optimiza-tion of marketed agents and the assessment of drug resistance

2 RELEVANCE OF ANIMAL MODELS TO INFECTIONS

IN HUMANS

It is largely assumed that efficacy of a drug in an animal model will correspondto that in humans However in many cases the induction and progression ofinfection in small animals does not correspond to that seen in the human settingFor example humans rarely suffer from large bolus challenges of bacteria bythe intravenous route A much more clinically relevant model for pathogenesisin humans involving translocation of bacteria from gastrointestinal membranesdamaged by cytostatic agents has been developed [4] Despite this major differ-ence the model of sepsis is a mainstay for early preclinical evaluation of anti-infectives in rodent models

A few animal models have been widely accepted for prediction of effectsin humans The rabbit model of endocarditis (see below) has proven faithful inmimicking damage to valvular surfaces similar to that reported in human patientsand colonization of these vegetations by bacteria and growth corresponds closelyto that observed in humans For several years prophylaxis regimens for endocar-ditis were based largely on experiments in this model

3 ENDPOINTS IN ANIMAL MODELS OF INFECTION

Examples of major endpoints used in pharmacodynamic studies in animal modelsof infection are shown in Table 1 For lethal infections the proportion of animalssurviving at each doseexposure level are determined to generate typical dosendashresponse curves With greater awareness of the potential for suffering in testanimals during the later stages of overwhelming infection from a failing drugregimen most investigators have substituted rapid progression to a moribundcondition as a more humane alternative endpoint Both endpoints require carefulmonitoring by the investigator and consistent application of these definitions to


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ensure reproducible results Further only the potency of the antimicrobial is esti-mated because the maximum response is generally bounded (100 survival)However given that death from infection often arises due to complex interplaybetween host factors organ damage etc or even other factors that may be unre-lated to antibacterial effects (eg a dropped cage) the investigator must be cau-tious in interpretating results

In contrast quantitation of bacteria in tissues at various time points allowsfor measurement of changes in bacterial numbers over time The antibacterialeffects often correlate with meaningful clinical outcomes in patients (eg relationbetween time to sterilization of CSF in meningitis and residual neurological ef-fects in children) Quantification of bacterial counts in tissues or fluids detectsmore subtle changes in bacterial killing with dosage regimens In additionchanges in susceptibility of the pathogens with treatment can be measured Thebest approach is validation of the number of pathogens in tissues or fluids thatcorrelates with survival in treated or untreated animals Identification of an earlierendpoint that predicts survival in both treated and untreated animals meets themost rigorous definition for a true surrogate marker (ie a marker that predictssurvival in treated and untreated groups)

31 Metrics for Quantifying Anti-Infective Effects In Vivo

Quantitation of antimicrobial effects in vivo usually measures the tissue or hostburden of organism at specified intervals after the initiation of treatment In viewof the differences among drugs in in vitro pharmacodynamic properties (egrate of bacterial killing and postantibiotic and subinhibitory effects) as well aspharmacokinetic properties several approaches have been developed to measurethe time course of antibacterial effects in vivo These metrics tend to focus eitheron the overall extent of bacterial killing over time or on the rate of bacterialkilling either by measuring the time to reach a particular level of bacteria (egtime to 3 log decrease) or by calculation of a killing rate (eg reduction in logCFUh) Table 2 summarizes several representative metrics used for describingthe pharmacodynamic effects on bacteria in vivo

4 PHARMACOKINETIC CONSIDERATIONS IN ANIMAL

MODELS OF INFECTION

As described in earlier chapters one objective of pharmacodynamic modeling isto relate the in vivo exposure of an anti-infective to the observed effects listedabove This requires careful study of antimicrobial pharmacokinetics in the testspecies

Our approach is to measure the pharmacokinetic properties of readily avail-able drugs in the infected animals Although literature data are often available


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the results in a preclinical pharmacokinetic study (where the goal is to carefullymeasure pharmacokinetic properties) may differ from the values obtained in sickanimals or with multiple doses For novel compounds the pharmacokinetics isnot known and studies in both infected and uninfected animals are needed tocharacterize the pharmacokinetic properties of the agent as well as to determinethe actual exposure to drug in the experimental model Nonlinear changes inexposure vs dose (due to changes in drug clearance or bioavailability for extra-vascular administration) should be probed When studying combinations of drugsone must ensure that pharmacokinetic drug interactions do not occur pharmaco-kinetic interactions could cause false interpretations of combination experiments(eg increased effects from antimicrobial synergism) where the effect was reallydue to elevated concentrations of an active component We have also found thatthe formulation used for a second agent may also influence the pharmacokineticproperties of antibiotics (eg PEG plus levofloxacin in mice unpublished obser-vations)

41 Design Issues in Pharmacokinetic Studies for

Animal Models

411 Sampling

Appropriate blood or tissue sampling strategies are key to getting robust estimatesof pharmacokinetic parameters in animals In mice it is usually necessary toeuthanize the animal using CO2 or another AVMA-approved method to obtainadequate volumes of blood for drug assay Cardiac puncture in a dead animalusually yields the greatest volume of blood Harvest of tissues or other samplesand immersion in a suitable matrix for sample processing (eg homogenization)can also be done to quantify tissue levels In larger animal species (eg ratsrabbits) an indwelling intravenous or intra-arterial catheter allows collection ofmultiple samples over time In these cases placement of two cannulas in separatevessels should be undertaken to avoid possible carryover of drug-containing infu-sate into samples

One can use d-optimality criteria to select sampling points [16] Howeverwe rarely use this because the pharmacokinetics of drugs are rapid in smallanimals and the slight advantage of narrowing sampling times is easily lost Fur-ther for novel compounds there is no information upon which to base selectionof sampling times The maximal volume of blood to be collected over a pharma-cokinetic study session should be estimated and approved upon consultation witha veterinarian and the Institutional Animal Care and Use Committee (IACUC)

After collection samples can be centrifuged to separate cells from plasmaor serum and aliquots of the sample transferred to screw-capped storage tubesPilot studies should consider possible loss of drug in polypropylene or other mate-rials that may result in falsely low levels of drug Administration of plasma ex-


panders or volume replacement should be considered for prolonged studies insmall animals Patency of catheters can be maintained by gentle flushing of linesusing heparin (10ndash100 UmL) in 09 saline but overzealous use can result ina systemic anticoagulant effect

412 Drug Assay

Assay of serum and tissue concentrations of drug in the test animal species can bedone using high performance liquid chromatographic (HPLC) or microbiologicalassays The more widespread availability precision sensitivity and rapidity ofHPLC methods [particularly those using detection by tandem mass spectrometry(MSMS)] has resulted in a shift toward the use of these methods particularlyin the early discovery or preclinical setting Metabolites and their antimicrobialactivity should be considered in the final pharmacokineticndashpharmacodynamicanalyses Preparation of standards in the appropriate biological matrix (egserum tissue homogenate) should be undertaken and the sensitivity speci-ficity reproducibility and ruggedness be validated to ensure reproducibility ofresults

413 Modeling Pharmacokinetic Data

Serum or tissue concentration vs time data can be analyzed using a variety ofmethods Although noncompartmental (SHAMmdashslope height area moment)methods are used by many investigators we have found that a more informativeanalysis results when compartmental models are used In addition full parameter-ization allows for better simulations Critical analysis of the selection of the ap-propriate pharmacokinetic model and generation of pharmacokinetic parametersand their confidence intervals is also helpful for performing simulations to calcu-late indices of pharmacokinetic parameters related to the MIC Compartmentalmodeling is also particularly important for estimating pharmacokinetic parame-ters from studies that generate a single composite lsquolsquopopulationrsquorsquo curve of singledata points gathered from destructive sampling (eg serum from mice) Manyinvestigators have adopted WinNONLIN as the fitting program but several othersuitable programs include ADAPT II and MKMODEL Even population model-ing programs (eg NOMEM NPEM) may be useful with some data sets

After estimation of pharmacokinetic parameters simulation of serum levelsfor various dosage regimens can be undertaken Unbound drug concentrationsshould be simulated using protein binding values generated in the same speciesIt is particularly important to correlate effects with unbound drug exposure forstudies where development of pharmacokinetic-pharmacodynamic relationshipswill be extended to other species particularly humans As will be discussed be-low several studies have shown that the free concentration of drug in serum islinked to the efficacy of several anti-infectives Differences in serum protein bind-ing between preclinical species and humans could lead to errors in conclusions


concerning target levels of total drug to obtain acceptable levels of efficacy Ex-amples include experience with cefonicid in the treatment of endocarditis due toS aureus [17]

42 Animal vs Human Pharmacokinetics

The pharmacokinetics of drugs are known to differ markedly between preclinicalanimal species (particularly small animals used in pharmacokinetic studies) andhumans These differences largely arise due to well-described allometric relation-ships that relate physiological variables measured within species to differencesin body weight [18] However the differences in pharmacokinetic properties be-tween animals and humans may arise due to differences in metabolism biliaryor renal transport or a combination of these factors For example although mero-penem is resistant to hydrolysis by human renal dehyropeptidases it is highlysusceptible to inactivation to a deydropeptidase produced in mouse tissues [19]Table 3 depicts examples of differences in pharmacokinetic properties betweenhumans and small animals for several classes of agents These differences areoften most marked for β-lactam agents

In conducting pharmacokinetic-pharmacodynamic investigations in animalmodels with drugs used in humans it is crucial that the differences betweenhumans and animals be considered in the design and interpretation of experi-ments This is particularly important for compounds whose activity in vivo isdependent upon the length of time concentrations exceed the MIC (eg β-lactamsand Pseudomonas aeruginosa) When pharmacokinetic differences between hu-mans and small animals are ignored the results from animal models of infectioncan underestimate the efficacy of compounds in humans

A vivid example of the importance of consideration of human vs mousepharmacokinetics was shown in studies with ceftazidime treatment of Pseudo-monas aeruginosa infection in the neutropenic mouse thigh infection model by

TABLE 3 Comparison of Pharmacokinetic Parameters of SelectedAntimicrobials in Small Animals and Humans

Clearance [L(h sdot kg)] Elimination half-life (h)

Drug Human Rat Mouse Human Rat Mouse Ref

Ceftizoxime 012 116 367 127 026 015 18 20Vancomycin 189 036 075 15 28 063 21 22Ofloxacin 015 045 NA 70 184 NA 23 24Azithromycin 129 385 333 659 68 072 25ndash27

NA not available


Gerber et al in a neutropenic mouse model of infection (see Fig 1) [28] Whenhuman pharmacokinetics were simulated in the mice the bacterial killing wasmuch more sustained than that observed under normal mouse pharmacokineticconditions

Generally two methods exist for simulation of human pharmacokineticparameters in animals In the simplest terms they involve changing drug inputor drug elimination

421 Changing Drug Input

Several investigators have used multiple frequent administration of drugs withrapid drug clearances and short serum elimination half-lives in mice or rats Thisapproach uses the principle of superposition of successive lsquolsquobolusrsquorsquo doses givenwhen concentrations are expected to have declined below target levels Thefrequency of administration may be as short as every 30 min around the clock(Fig 1)

The availability of computer-controlled pumps for delivery of intravenousfluids has enabled the use of these devices to use decreasing rates of drug deliveryto simulate concentrations in humans [29] Two approaches may be used contin-uous infusion of diluent into the infusate which is delivered as an infusion intothe animal (much like what is done in in vitro pharmacodynamic models) or acontinuously changing intravenous infusion rate of a fixed concentration of drugin infusate The former approach has particular usefulness if one wishes to simu-late human concentrations of two drugs in the animal Both of these approacheshave been used in larger animal species (rats rabbits) in the evaluation of treat-ment of endocarditis peritonitis pneumonia and meningitis

The theoretical advantage associated with continuous infusion of antibioticswith certain in vitro pharmacodynamic properties has been studied in animalmodels of infection Continuous infusion studies are considerably simpler thanintermittent administration One simply needs to divide the desired steady-statedrug concentration by the drug clearance in the animal species to determine theinfusion rate Studies have evaluated delivery by this route for up to 5 daysContinuous infusion of drugs may also be obtained in mice using Alzet peritonealor subcutaneous micro-osmotic pumps

422 Changing Drug Elimination

An alternative (and perhaps more direct) way to mimic human drug pharmacoki-netics in small animals is to slow drug elimination For drugs largely excretedunchanged in urine inducing renal impairment by administration of a nephro-toxin can result in slow excretion and allow for close simulation of human dosageregimens in mice Several investigators have administered a single dose of uranylnitrate (eg 10 mgkg given 2ndash3 days prior to antibiotic treatment) to inducetemporary renal dysfunction for studies of short-term duration (eg 24ndash30 h)


FIGURE 1 Comparison of the effects of a single dose of ceftazidime against astrain of Pseudomonas aeruginosa in the neutropenic mouse thigh infectionmodel When a single dose was given to a mouse bacterial killing occurredonly transiently followed by regrowth In contrast when multiple frequentdoses of drug were given to simulate the pharmacokinetic profile in humansmuch more sustained bacterial killing was observed (From Ref 28)


[8] Although convenient this substance is considered to have low-level radia-tion and some states require special permits for its handling and disposal Othersubstances have been reported to cause renal impairment in rodents (eg glyc-erol) [30] however we did not find that it significantly altered the pharmacokinet-ics of aztreonam in mice (Tembe Chen Griffith and Dudley unpublished obser-vations)

Figure 2 compares the serum pharmacokinetic profile for amoxicillin inmice pretreated with a single dose of uranyl nitrate with concentrations in hu-mans When proper doses are administered the serum concentration vs timeprofile is comparable to that observed in humans Simulation of human serumdrug levels allowed for testing of the relationship between amoxicillin MIC andeffects in a mouse model using drug exposures comparable to those observed inhumans [8]

Other approaches for reducing drug clearance include administration ofdrugs that block renal tubular secretion This requires that the agent of interestbe excreted to a high degree by net renal tubular secretion in the animal speciesto be tested Probenecid is one example however the magnitude of the effectmay be variable and highly dose-dependent In addition probenecid may haveeffects on other nonrenal pathways for elimination including phase II metabo-lism

FIGURE 2 Comparison of serum amoxicillin concentrations found in renallyimpared mice with those in human volunteers (From Ref 8)


5 PHARMACODYNAMIC MODELING USING

MATHEMATICAL INDICES FOR RELATING

PHARMACOKINETIC DATA WITH THE MIC

51 Pharmacokinetic-Pharmacodynamic Indices and

Relationship to Dose MIC and Drug Half-Life

As described in earlier chapters several indices for integrating pharmacokineticdata with the MIC have been applied for the study of anti-infective pharmacody-namics Figure 3 depicts several indices that have been used to express theserelationships As shown in the figure all of the parameters are highly correlatedeg an increase in dose results in an increase in all parameters However themagnitude of changes in each parameter with different doses dosing intervalsdrug clearance and MIC will not increase proportionately with all the pharmaco-kinetic-pharmacodynamic (PK-PD) indices This is especially important in thedesign and interpretation of studies of drugs with short half-lives in small animalswhere the percent of the dosing interval drug concentrations that exceed the MICis the most important parameter for describing in vivo antimicrobial effects

A brief consideration of the effects of dose on PK-PD parameters is helpfulin recognizing the relative importance of each of these variables For the ratiosCmaxMIC or AUCMIC a change in dose results in proportional and linear (as-suming linear pharmacokinetics) changes in AUCMIC Similarly the MIC af-fects these parameters (inversely) but the proportion of change is linear

FIGURE 3 Examples of (a) pharmacokinetic parameters and the MIC and (b)pharmacodynamic relationship between a PK-PD parameter and effects invivo


The picture is considerably more complicated in calculation of the numberof hours drug concentrations exceed the MIC For a one compartment pharmaco-kinetic model and bolus drug input

Hours concn exceeds MIC (T MIC) ln doseV ln MIC

ClV

In contrast to the ratio metrics CmaxMIC and AUCMIC the magnitude ofchange in T MIC with dose or MIC is not directly proportional but changesas the natural logarithm of these values Changes in drug clearance (half-life)produce the most marked change in this parameter

The practical implications of this relationship for the design of studies andthe evaluation of PK-PD data in animal models of infection for drugs whereactivity in vivo is dependent upon T MIC are profound Figure 4 depictschanges in T MIC according to MIC or drug dose When drug half-life is veryfast (ClV is large) even high doses of drug will produce only minor changesin T MIC Similarly differences in in vitro potency (MIC) will have littleeffect on T MIC Failure to incorporate these considerations into experimentsresults in erroneous conclusions concerning the effects of changes in MIC ordrug dose and in vivo response for drugs where in vivo activity is linked toT MIC

FIGURE 4 Relationship between MIC and T MIC and dose for vancomycinadministered subcutaneously in mice


52 Designing Experiments to Identify Relationships

Between Pharmacokinetics MIC and In Vivo Effects

In planning experiments in which many regimens can be evaluated (eg short-term studies in mouse models) simulations of serum drug concentrations vstime using pharmacokinetic parameters derived from infected animals should beperformed The parameters MICs and dosage regimens can easily be pro-grammed into a spreadsheet to generate PK-PD data One can assess a numberof lsquolsquowhat ifrsquorsquo scenarios for several doses dosage regimens and MICs and gener-ate two- or three-dimensional plots showing the correlation for each regimen Anexample of uncorrelated PK-PD parameters for several vancomycin regimens ina mouse is shown in Fig 5 One can finalize selection of dosage regimens thatwill minimize the covariance among different PK-PD parameters In selectingregimens it is also important to be mindful of the relationships of dose and MICwith the number of hours serum concentrations will exceed the MIC In somecases it may be virtually impossible to design dosage regimens that produce arange of PK-PD parameters that correspond to those possible in humans In these

FIGURE 5 Lack of correlation between T MIC and AUCMIC ratio for severalregimens of vancomycin administered every 2ndash12 h in a mouse model ofinfection


cases the use of techniques to alter drug input or retard drug clearance may berequired

6 ANIMAL MODELS USED IN PK-PD STUDIES

61 Selection of Appropriate Models for Study

of PK-PD Issues

For the study of pharmacokinetics and pharmacodynamics (PK-PD) in an animalmodel one must take into consideration that the time course of antimicrobialactivity will vary between different antimicrobial agents For example β-lactamantibacterials exhibit very little concentration-dependent killing and in the caseof staphylococci long in vivo postantibiotic effects (PAEs) For these antibacteri-als high drug levels will not kill bacteria more effectively or more rapidly thanlower drug levels because these agents should be bactericidal as long as the drugconcentrations exceed the MIC In contrast fluoroquinolones and aminoglyco-sides show concentration-dependent killing For these agents the peakMIC orAUCMIC ratios should be the PK-PD parameters that most effectively describetheir efficacy [1] With these caveats in mind the choice of an animal model tostudy PK-PD parameters will depend on the organism one wishes to study thepharmacokinetics of the antimicrobial agent and the type of infection one ulti-mately intends to treat in humans

Of the animal models used to study PK-PD relationships the most com-monly used is the neutropenic mouse thigh model fully described by Gerber andCraig in 1982 [20] Other animal models used to study PKndashPD relationshipsinclude animal models of pneumonia [21ndash23] kidney infectionspyelonephritis[24] peritonitissepticemia [25 26] meningitis [27 31] osteomyelitis [32] andendocarditis [33ndash35] In fact almost any model can be used as long as drugexposure can be correlated with organism recovery or response

62 Factors Influencing Endpoints

Factors that affect the results of all endpoints include the test strain and initialchallenge inoculum treatment-free interval (ie interval between bacterial inocu-lation and initiation of treatment) immunocompetence of test animal speciesand administration of adjuvants These all can contribute to outcome and theconclusion concerning the magnitude of the PK-PD parameter required for effi-cacy Gerber et al [36] showed that the efficacy of aminoglycosides and β-lac-tams was markedly affected by delaying treatment requiring a higher dose Influoroquinolone treatment of pneumococcal infection in mice the AUCMIC ra-tio associated with the static effect is similar in immunocompetent or neutropenicmice In contrast AUCMIC is considerably different between neutropenic andnonneutropenic mice in infections due to Klebsiella pneumoniae [37]


63 Animal Models

To study the pharmacodynamics of an antimicrobial agent the pharmacokineticsof the agent in question should be determined in the animal species and underthe same conditions that will be used in the infection model (eg neutropenicmice in the case of the neutropenic thigh model) The MICs of the organisms tobe tested should be determined by an NCCLS reference method

631 Thigh Infection Model

The thigh infection model has been used extensively to describe the pharmacody-namics of several classes of drugs particularly fluoroquinolones macrolidesβ-lactams and aminoglycosides Most investigators have used Swiss albino micefor this model although any mouse strain can be used Although occasional stud-ies are done in normal (nonneutropenic) animals most of the experience withthis model has been obtained in neutropenic mice [38] Most investigators induceneutropenia by the administration of 150 mgkg and 100 mgkg of cyclophospha-mide on days 0 and 3 respectively This results in severe neutropenia by day 4which is sustained over at least a 24ndash48 h period [38] Inocula should be preparedin a suitable medium and allowed to grow into log phase After dilution 01 mLof bacterial suspension (105ndash106 CFU) is injected into each thigh while the ani-mals are under light anesthesia (eg isoflurane sodium pentobarbital ether) Forsome experimental designs a different organism may be injected in the contralat-eral thigh (eg when studying resistance mechanisms in isogenic strains) ormixtures of organisms can be used The organisms are usually allowed to growfor 2 h prior to the start of treatment Treatment is given in multiple dosingregimens over 24 h based on the pharmacokinetics of the drug and PK-PD param-eters being tested After 24 h of treatment the animals are euthanized and theirthighs are removed homogenized in sterile saline and serially diluted and cul-tured on a suitable medium to perform colony counts In the event of agents withlong half-lives (eg azithromycin) thigh homogenates can be assayed for thedrug to exclude the possibility of drug carryover interfering with the results ora substance to inactivate drug (eg β-lactamase) may be added The CFUthighdetermined for each dosing regimen can be quantified and relationships to drugexposure fit to a suitable pharmacodynamic model

632 Pneumonia Models

The pneumonia model described below is a mouse model however there are anumber of different models in rats [21 39] hamsters [40] or rabbits [41] Organ-isms used in pneumonia models include but are not limited to S pneumoniaeK pneumoniae P aeruginosa S aureus P mirabilis E coli L pneumophilaand A fumigatus Adjuvants are often used to increase virulence

In the mouse model most Swiss albino mice of either sex can be used


Depending upon the organism the mice may or may not need to be renderedneutropenic Inocula should be prepared in a suitable medium and allowed togrow into log phase After dilution the animals are infected by intranasal instilla-tion of 005 mL of bacterial suspension (106ndash107 CFU) while the animals areunder anesthesia (isoflourane sodium pentobarbital ether etc) Treatment isgiven in multiple dosing regimens for up to 2 days starting 24 h after infectionAfter 24ndash48 h of treatment the animals killed and their lungs are removed ho-mogenized serially tenfold diluted and then cultured on a suitable medium toperform colony counts The CFUlungs determined for each dosing regimen canthen be fit to a suitable pharmacodynamic model The advantages of this modelare that the prolonged endpoint (24ndash48 h) allows for several cycles of bacterialgrowth

Using this model Woodnutt and Berry [21] found that the efficacy of amox-icillin-clavulanate (a combination of the two drugs) was best described by per-centage time above the MIC with the maximum bactericidal effect beingachieved at a T MIC of 35ndash40

634 PyelonephritisKidney Infection Models

Kidney infection models for study of antifungal activity have been described inboth mice [24] and rats [42] Organisms used in kidney infection models includebut are not limited to E faecalis E coli S aureus C albicans and A fumiga-tus

For the mouse model most Swiss albino Balbc and DBA2 mice can beused Swiss albino mice can be rendered neutropenic to support growth of theorganism Inocula should be prepared in a suitable medium and allowed to growinto log phase After dilution the animals are infected by intravenous injectionTreatment is started 2 h after infection in the case of C albicans but can bestarted as late as 24 h after infection in the case of E faecalis Treatment is givenin multiple dosing regimens for 24 h or longer After 24ndash120 h of treatment theanimals are killed and their kidneys are removed homogenized serially tenfolddiluted and then cultured on a suitable medium to perform colony counts TheCFUkidneys determined for each dosing regimen can then be fit to a suitablepharmacodynamic model The advantages of this model are endpoints that canrange anywhere from 24 to 120 h it generally uses an inoculum high enough tostudy resistance and it allows for several growth and regrowth cycles Using thismodel it has been shown that the AUCMIC best predicts the effects of flucona-zole against strains with a wide range of susceptibilities to this drug [244344]

635 PeritonitisSepticemia Models

The septicemia model described below is a mouse model [2645] however thereis a similar model in neutropenic rats [25] Organisms used in peritonitissepsis


models include but are not limited to E coli S aureus S epidermidis P aerugi-nosa K pneumoniae S pneumoniae E faecalis S marcescens P mirabilisgroup B Streptococcus C albicans and A fumigatus

For this model almost any mouse species can be used Mice may or maynot need to be rendered neutropenic Inocula should be prepared in a suitablemedium and allowed to grow into log phase After dilution the animals are in-fected by intraperitoneal injection of 01ndash05 mL of bacterial suspension (1ndash109 CFU mouse) Treatment is given in multiple dosing regimens for up to 72h After treatment animals still alive are considered long-term survivors Deathsrecorded throughout the experiment can be compared by KaplanndashMeier or probitanalysis Percent survival [(number alivenumber treated) 100] determined foreach dosing regimen can then be fit to a suitable pharmacodynamic model

Using this model Knudsen et al [45] found that the peakMIC ratio andtime above MIC were associated with survival for vancomycin and teicoplaninagainst S pneumoniae In a study using peritoneal washings and survival in micefor Emax modeling der Hollander et al [26] found that the peakMIC ratio wasassociated with maximum bactericidal effects for azithromycin against S pneu-moniae and was achieved at a ratio of 4 In a similar model in rats Drusano etal [25] found that for lomefloxacin a peakMIC ratio that was 201 or 101was associated with survival against P aeruginosa At peakMIC ratios of 101they found that the AUCMIC ratio was associated with survival

636 Meningitis Models

The meningitis model described below is a rabbit model [910] There are alsomodels in the rat guinea pig cat dog goat and monkey but studies in the higherspecies are discouraged Organisms used in meningitis include N meningitidisS pneumoniae H influenzae S aureus S pyogenes S agalactiae E aerogenesE coli K pneumoniae P mirabilis P aeruginosa and L monocytogenes

For this model male or female rabbits can be used A dental acrylic helmetis attached to the skull of each rabbit by screws while it is under anesthesia Thehelmet allows the animal to be secured to a stereotactic frame made for the punc-ture of the cisterna magna Meningitis is induced by injection of a bacterial sus-pension directly into the cisterna magna Treatment is started 18 h after infectionand is given in multiple dosing regimens for up to 72 h The CFUmL CSF fluidor cure rates (sterile CSF cultures) determined for each dosing regimen can thenbe fit to a suitable pharmacodynamic model

In this model Tauber et al [27] found that the peak CSF drug levelMBCratio emerged as an important factor contributing to the efficacy of ampicillinagainst S pneumoniae In earlier studies these investigators also found that therewas a linear correlation between β-lactam CSF peak concentrations and bacteri-cidal activity in rabbits with S pneumoniae meningitis [9] Antibiotic concentra-tions in the CSF in the range of the MBC produced static effects and concentra-tions of 10ndash30 times the MBC produced a maximal bactericidal effect


In the same model of pneumococcal meningitis Lutsar et al [31] foundthat the bactericidal activity of gatifloxacin in the CSF was closely related tothe AUCMBC ratio but that maximal activity was achieved only when drugconcentrations exceeded the MBC for the entire dosing interval In another studyof pneumococcal meningitis Lutsar et al [10] found that for ceftriaxone thetime above MBC predicted the bacterial killing rate and that there was a linearcorrelation between time above MBC and bacterial killing rate during the first24 h of therapy Sterilization of the CSF was achieved only with T MBC of95ndash100 Ahmed et al [46] found that concentrations of vancomycin neededto be at least four- to eightfold the MBC of S pneumoniae in order to obtainadequate bacterial clearance The suggestion was also that time above the MBCin the CSF was required for efficacy

637 Osteomyelitis Models

The osteomyelitis model described below is both a rat model [32] and a rabbitmodel [47] Organisms used in osteomyelitis models include S aureus and Paeruginosa For this model male or female rats or rabbits can be used Animalsare anesthetized then the tibia is exposed and a 1 mm hole is bored with a dentaldrill into the medullary cavity of the proximal tibia Bones are infected by firstinjecting 5 sodium morrhuate followed by an injection of bacterial suspensionThe hole is plugged with dental gypsum and the wound is closed Treatment isinitiated 10 days after surgery and is given in multiple dosing regimens for upto 21 days for each compound

In this model OrsquoReilly et al [32] chose single daily doses of 50 mgkgfor azithromycin single daily doses of 20 mgkg for rifampin and three 90 mgkg doses of clindamycin daily for treatment regimens against S aureus Despitehaving large bone peakMIC and troughMIC ratios azithromycin failed to steril-ize a single bone Clindamycin sterilized 20 of the animals treated and rifampinsterilized 53 of the animals treated These results underscore the difficulties inpredicting the efficacies of antibiotics in osteomyelitis based on in vitro studiesand antibiotic levels in bone

638 Endocarditis Models

The endocarditis model has been used extensively to evaluate antimicrobial regi-mens Its advantages are that it produces an infection comparable to that observedin humans The endocarditis model described below is a rabbit model [48] how-ever there is also a rat model [49] Organisms used in osteomyelitis modelsinclude but are not limited to S aureus P aeruginosa S epidermidis E aero-genes E faecalis E faecium S sanguis S mitis C albicans and A fumigatus

For this model New Zealand rabbits are usually used Rabbits are anesthe-tized then a catheter is placed across the heart valve and left in place for theduration of the study Animals are infected after 24 h by an intravenous injection


of bacterial inoculum Treatment begins 24 h after infection and is given as multi-ple dosing regimens for 3ndash10 days Animals are killed then heart valve vegeta-tions are collected homogenized serially tenfold diluted and then cultured ona suitable medium to perform colony counts Terminal blood samples are takento check for sepsis The CFUg vegetation determined for each dosing regimencan be fit to a suitable pharmacodynamic model

In the rabbit model Powell et al [50] found that single daily doses orcontinuous infusion of tobramycin provided equally efficacious results sug-gesting that the AUCMIC ratio may be the variable that best describes the effi-cacy of this compound In another rabbit model of endocarditis Fantin et al [51]found that RP 59500 was efficacious against S aureus despite the fact that itwas above the MIC for only 33 of the time However it was found that RP59500 penetrated vegetations well with a vegetationblood ratio of 41 and aprolonged in vitro postantibiotic effect

In a rat model of endocarditis Entenza et al [49] found that despite havingconcentrations of the quinolone Y-688 that were the same as human concentra-tions the compound failed to be efficacious against quinolone-resistant S aureusThis failure may be due to insufficient vegetation penetration [52] In separatein vitro time-kill studies with S aureus low concentrations of Y-688 selectedfor drug resistance Poor drug penetration into vegetations could have providedideal conditions for selection of resistance in vivo and hence the failure of Y-688 in this model

In an analysis of 19 publications on the treatment of experimentally inducedendocarditis caused by S aureus S epidermidis viridians streptococci E aero-genes and P aeruginosa in rabbit or rat models Andes and Craig [35] foundthat for fluoroquinolones a 24 h AUCMIC of 100 a peakMIC of 8 anda time above the MIC of 100 were all associated with significant bactericidalactivity after 3ndash6 days of therapy However they determined that the 24 h AUCMIC ratio exhibited the best linear correlation A conclusion was that the resultsfound in endocarditis with quinolones were similar to those found in the neutro-penic thigh model

7 EXAMPLES OF USE OF ANIMAL MODELS TO STUDY

PHARMACOKINETIC-PHARMACODYNAMIC ISSUES

71 In Vivo Postantibiotic Effects

Postantibiotic effects have been recognized since the very early studies of penicil-lin Although in vitro studies can describe the persistent effects of a drug follow-ing its complete removal from a growing culture they often have little relevanceto the in vivo setting when drug concentrations fall more slowly over time Incontrast demonstration of persistent antibiotic effects following a single dose can


be helpful in determining the optimal dosage interval Single dose experiments inanimal models can be used to define the in vivo postantibiotic effect (in vivoPAE) [5354] In these experiments a single dose of drug is given and the serumconcentration and tissue burden of the test organisms are measured over timeThe in vivo PAE is calculated by the equation

PAE T C M

where M is the time for which plasma levels exceeded the MIC T is the timerequired for the bacterial counts of treated mice to increase by 1 log10 CFUthighabove the count at time M and C is the time necessary for the counts in controlanimals to increase by 1 log10 CFUthigh An example is shown in Fig 6

There are some important differences between the in vitro and in vivoPAEs Most notably the in vivo PAE tends to be longer than the in vitro PAEA possible explanation for the differences between the in vitro and in vivo PAEsis the effect of subinhibitory drug concentrations on bacterial growth and re-

FIGURE 6 Example of an in vivo PAE for cefazolin Growth curves of control() and antibiotic-exposed () S aureus ATCC 25923 in mouse thighs aftera single dose of cefazolin 125 mgkg Serum levels of cefazolin after a singledose at 125 mgkg(∆) (From Ref 38)


growth Sub-MIC concentrations can increase the length of the PAE [55] asreflected in the in vitro measurement PAE-SME Measurements of the in vivoPAE from single-dose experiments can then be used to better define the durationof antimicrobial effects in vivo with declining concentrations

72 Serum Protein Binding

Unfortunately the effect of serum protein binding on antimicrobial activity stillattracts considerable controversy and confusion among many clinicians and re-searchers The proportional reduction of antimicrobial activity in the presence ofserum or binding proteins has been thoroughly demonstrated for several anti-infectives in susceptibility testing

Although of high interest there are too few examples of well-controlled ex-periments that demonstrate the importance of serum protein binding on efficacyin vivo The difficulty in showing the importance of protein binding in animalmodels largely lies in the fact that the class of anti-infectives with the greatestvariability in serum protein binding are the β-lactam antibiotics Since the in vivoefficacy of these agents is dependent upon T MIC and they have relatively shorthalf-lives in small animals large differences in serum protein binding are requiredto producesignificant differences in free-drug T MIC Merrikenet al [56] demon-strated the importance of serum protein binding on the efficacy of several structur-ally related analogs of penicillin in a mouse model of sepsis due to Staphylococcusaureus All of the agents had similar in vitro potency against the test organism (MICbetween 025 and 05 mgL) and pharmacokinetic properties but the percent boundto serum proteins ranged between 36 and 98 Although the differences in phar-macokinetic properties of total drug were small (25-fold range) there was a 70-fold difference among agents in dose required for survival in 50 of animals (ED50

ranged from 07 to 497) In a neutropenic mouse thigh model we also comparedthe efficacy of three cephalosporin analogs with varying MICs to 2 strains of methi-cillin-resistant Staphylococcus aureus pharmacokinetics and protein binding Asshown in Fig 7 bacterial killing was best described by the number of hours thatfree-drug concentrations exceeded the MIC

73 Drug Resistance

The increasing problem of resistance to anti-infective drugs has required criticalanalysis of the significance of novel resistance mechanisms The study of drugpharmacodynamics in animal models of infection either using isogenic strainswith or without a resistance factor or using relevant clinical isolates can assessthe importance of resistance under in vivo conditions This information can beimportant in establishing resistance breakpoints for in vitro MIC testing (see be-low) as well as for formulating optimal dosage regimens to prevent or overcomeestablished resistance Generally if resistance in vivo is less than that observed


in vitro this will be reflected by the disruption of expected relationships betweendrug exposure (eg AUC) and MIC

The clinical relevance of resistance to extended spectrum cephalosporinsby novel plasmid-mediated β-lactamases was studied by Craig and colleagues inthe neutropenic mouse thigh model against several isogenic strains producingextended-spectrum β-lactamases Mice were pretreated with uranyl nitrate to sim-ulate human exposures to the drug The results showed that although MICs wereelevated at a high inoculum in vivo results were best correlated with MICs ob-tained at the lower inoculum (W Craig personal communication)

Reduced susceptibility to vancomycin in enterococci and more recentlyStaphylococcus aureus is of increasing clinical concern because of the absenceof alternative therapies In experiments in the neutropenic mouse thigh modelwith S aureus strains that were vancomycin-susceptible or intermediate (VISA)the efficacy was best described by the CmaxMIC or AUCMIC ratio When resultsfor the vancomycin susceptible strains were compared with those for VISA onlyslightly higher vancomycin exposures (Cmax or AUC) were required for the samelevel of efficacy despite the higher MICs Although further studies are requiredthis suggests that these strains have a reduced level of susceptibility in vivo tovancomycin that is less than that predicted by the in vitro MIC [57] Optimizationof vancomycin dosage regimens could be a successful strategy for the clinicalmanagement of strains with reduced susceptibility to vancomycin

Drug efflux is increasingly recognized as an important mechanism of resis-tance in bacteria and fungi However little is known concerning the efficiencyof these pumps to produce resistance under in vivo conditions Using isogenicstrains of Pseudomonas aeruginosa with varying levels of expression of themulticomponent mexAB-oprM efflux pump a reduced response to levofloxacinand ciprofloxacin in the neutropenic mouse thigh model and mouse sepsis modelwas observed the reduction in efficacy due to efflux was proportional to thechange in AUCMIC [58] In contrast Andes and Craig [59] reported that AUCMIC ratios associated with response in the neutropenic mouse thigh model forNOR-A efflux-related resistance to fluoroquinolones in Streptococcus pneumon-iae were lower than that in susceptible strains or strains with reduced susceptibil-ity due to non-efflux (eg gyrA) mechanisms These data suggest that effluxmechanisms of resistance may be significant in some bacteria but not in others

Resistance to fluconazole due to target modifications andor efflux has alsobeen shown to be significant in vivo using pharmacodynamic modeling Sorensenet al studied several strains of C albicans with over a 2000-fold range in MICin a mouse model of disseminated candidiasis The reduction in counts in kidneysat 24 h following fluconazole was found to be described by the AUCMIC ratiofor all doses and strains tested [44] suggesting that elevated fluconzole MICsdue to target or efflux-based mechanisms correspond to similar levels of reducedactivity in vivo


74 Establishing Susceptibility Breakpoints

Given the usefulness of pharmacokinetic-pharmacodynamic relationships for pre-dicting efficacy in vivo animal models using these analyses have been used forestablishing breakpoints for in vitro susceptibility testing The Antimicrobial Sus-ceptibility Testing Subcommittee of the National Committee for Clinical Labora-tory Standards (NCCLS) has used such analyses for consideration of breakpointsfor certain classes of drugs where clinical data are scant as well as for new agentsA combined approach where data from human clinical trials involving treatmentwith organisms with varying MICs along with animal model PK-PD data pro-vides a rational basis for establishing susceptibility breakpoints

Pharmacokinetic-pharmacodynamic data derived in animal models of in-fection are used to establish susceptibilityresistance breakpoints for MIC testingin several ways Studies in a relevant model of infection can be used to character-ize the parameter that best describes efficacy in the model The lsquolsquotargetrsquorsquo PK-PD parameter (eg 24 h AUCMIC ratio) for producing a bacteristatic drug50 or even 90 of the maximum response can be derived from the experimentsBased on the pharmacokinetic properties in humans at safe doses PK-PD parame-ters for various dosage regimens can be calculated for various MIC values The

FIGURE 7 Relationship between exposure in vivo to free drug concentrationsand bacterial killing in a neutropenic mouse thigh infected model due to 2strains of methicillin-resistant Staphylococcus aureus and three cephalospo-rin analogs ( for each compound) with varying MICs and serum pro-tein binding


highest MIC value that still achieves the target PK-PD parameter for the drugwould correspond to a suitable MIC breakpoint for susceptibility testing Forexample a drug whose efficacy in an animal model of infection is maximal whenthe 24 h free-drug AUCMIC ratio exceeds 30 and safe regimens in humansproduce a 24 h AUC of 60 mg sdot hL then a susceptibility breakpoint of 2 mgLcould be recommended Of note is that although the exact serum concentration vstime curve dosing frequency and protein binding may differ between humans andsmall animals these differences are considered in reducing the exposure relativeto the MIC by using pharmacokinetic measures (eg free-drug AUC Cmax)

The rigor of the selected breakpoint can be further evaluated using simula-tion The availability of population pharmacokinetic parameters and their vari-ability in target patient groups can be used to determine the probability of individ-ual patients attaining a PK-PD target parameter using a selected breakpoint Thesimulation can be even further developed by incorporating the distribution ofMICs in organisms of interest and serum concentration data for individual pa-

FIGURE 8 Effect of simulated human dosage regimens of amoxicillin on re-covery of bacteria from the thighs of neutropenic mice according to amoxicil-lin MIC Mice were pretreated with uranyl nitrate to enable simulation ofamoxicillin levels corresponding to that observed in humans with usualdoses as shown in Fig 2 Organisms with an MIC 2 mgL all showed a reduc-tion in CFUthigh thus supporting a susceptibility breakpoint of this value(From Ref 8)


tients for the population means and variances [60] This corresponds to the equiv-alent of a simulated clinical trial

An alternative approach employs direct simulation of human pharmacoki-netics in the animal and testing of strains with varying MICs to the test agentOne would expect to see a graded response according to MIC and ultimately alsquolsquono effectrsquorsquo at a threshold MIC value This approach is shown in Fig 8 Humandosage regimens of amoxicillin were simulated in neutropenic mice whose thighswere then infected with several strains of S pneumoniae with varying levels ofsusceptibility to the drug For strains with MICs exceeding 2 mgL little or noeffect was seen on bacterial counts recovered from mice at 24 h thus supportinga susceptibility breakpoint of 2 mgL or less

8 CONCLUSIONS

Animal models of infection are a pivotal tool in the study of PK-PD propertiesof anti-infectives Consideration of PK-PD issues in the design and interpretationof experiments in animals have strengthened the usefulness of these models forthe study of human infection Animal experimentation has been greatly improvedbecause of recognition of the importance of these issues In addition many ofthe recognized limitations of animal models for application to treatment of humaninfections have been overcome by recognition of the importance of pharmacoki-netics in the outcome of infection These principles are routinely applied in thestudy of new drugs in all phases of drug discovery and development as well asin the optimization of dosage in the pre- and postmarketing evaluation of agents

ACKNOWLEDGMENTS

We acknowledge the excellent assistance of scientists and animal care personnelin Discovery and Preclinical Pharmacology at Microcide Pharmaceuticals whocontributed to generating data for some of the examples provided

REFERENCES

1 H Eagle R Fleishman M Levy lsquolsquoContinuousrsquorsquo vs lsquolsquodiscontinuousrsquorsquo therapy withpenicillin N Engl J Med 1953 248481ndash488

2 H Eagle R Fleishman M Levy On the duration of penicillin action in relation toits concentration in the serum J Lab Clin Med 1953 40122ndash132

3 H Eagle R Fleishman A Musselman Effect of schedule of administration on thetherapeutic efficacy of penicillin Am J Med 1950 9280ndash299

4 H Collins A Cross A Dobek S Opal J McClain J Sadoff Oral ciprofloxacin anda monoclonal antibody to lipopolysaccharide protect leukopenic rats from lethal in-fection with Pseudomonas aeruginosa J Infect Dis 1989 1591073ndash1082


5 J Tisdale M Pasko J Mylotte Antipseudomonal activity of simulated infusion ofgentamycin alone or with piperacillin assessed by serum bactericidal rate and areaunder the killing curve Antimicrob Agents Chemother 1989 19899

6 A Firsov S Vostrov A Shevchenko G Cornaglia Parameters of bacterial killingand regrowth kinetics and antimicrobial effect examined in terms of area under theconcentration-time curve relationships Action of ciprofloxacin against Escherichiacoli in an in vitro model Antimicrob Agents Chemother 1997 19976

7 M Dudley D Gilbert T Longest S Zinner Dose ranging of the pharmacodynamicsof cefoperazone plus sulbactam in an in vitro model of infection Comparison usinga new parametermdasharea under the survival fraction versus time curve Pharmacother-apy 1989 9189

8 D Andes W Craig In vivo activities of amoxicillin and amoxicillin-clavulanateagainst Streptococcus pneumoniae Application to breakpoint determinations Anti-microb Agents Chemother 1998 422375ndash2379

9 M Tauber C Doroshow C Hackbarth M Rusnak T Drake M Sande Antibacterialactivity of β-lactam antibiotics in experimental meningitis due to Streptococcuspneumoniae J Infect Dis 1984 149568ndash574

10 I Lutsar A Ahmed I Friedland M Trujillo L Wubbel K Olsen GH McCracken JrPharmacodynamics and bactericidal activity of ceftriaxone therapy in experimentalcephalosporin-resistant pneumococcal-meningitis Antimicrob Agents Chemother1997 412414ndash2417

11 K Madaras-Kelly A Larsson J Rotschafer A pharmacodynamic evaluation of ci-profloxacin and ofloxacin against two strains of Pseudomonas aeruginosa J Antimi-crob Agents Chemother 1996 37703ndash710

12 H Hanberger Pharmacodynamic effects of antibiotics Studies on bacterial morphol-ogy initial killing postantibiotic effect and effective regrowth time Scand J InfectDis Suppl 1992 811ndash52

13 H Mattie A predictive parameter of antibacterial efficacy in vivo based on efficacyin vitro and pharmacokinetics Scand J Infect Dis Suppl 1990 74133ndash136

14 J Zhi C Nightingale R Quintiliani A pharmacodynamic model for the activity ofantibiotics against microorganisms under measurable conditions J Pharm Sci 1986751063ndash1067

15 M Dudley S Zinner Simultaneous mathematical modeling of the pharmacokineticand pharmacodynamic properties of cefoperazone 89th Meeting of the AmericanSociety of Clinical Pharmacology and Therapeutics San Diego CA 1988

16 D DrsquoArgenio A Schumitsky ADAPT II A program for simulation identificationand optimal experimental design User manual Biomedical Simulations ResourceUniv Southern California Los Angeles 1992

17 M Dudley W Shyu C Nightingale R Quintiliani Effects of saturable protein bind-ing on the pharmacokinetics of unbound cefonicid Antimicrob Agents Chemother1986 30565ndash569

18 J Mordenti Pharmacokinetic scale-up Accurate prediction of human pharmacoki-netic profiles from animal data J Pharm Sci 1985 741097ndash1099

19 M Tsuji Y Ishii A Ohno S Miyazaki K Yamaguchi In vitro and in vivo antibacte-rial activities of S-4661 a new carbapenem Antimicrob Agents Chemother 19984294ndash99


20 A Gerber W Craig Aminoglycoside-selected subpopulations of Pseudomonas aeru-ginosa Characterization and virulence in normal and leukopenic mice J Lab ClinMed 1982 100671ndash681

21 G Woodnutt V Berry Two pharmacodynamic models for assessing the efficacy ofamoxicillin-clavulanate against experimental respiratory tract infections causedby strains of Streptococcus pneumoniae Antimicrob Agents Chemother 1999 4329ndash34

22 R Roosendaal I Bakker-Woudenber J v d Bergh M Michel Therapeutic efficacyof continuous versus intermittent administration of ceftazidime in an experimentalKlebsiella pneumoniae pneumonia in rats J Infect Dis 1985 152373ndash378

23 E Azoulay-Dupuis E Vallee J Bedos M Muffat-Joly J Pocidalo Prophylactic andtherapeutic efficacies of azithromycin in a mouse model of pneumococcal pneumo-nia Antimicrob Agents Chemother 1991 351024ndash1028

24 D Andes M v Ogtrop Characterization and quantitation of the pharmacodynamicsof fluconazole in a neutropenic murine disseminated candidiasis infection modelAntimicrob Agents Chemother 1999 432116ndash2120

25 G Drusano D Johnson M Rosen M Standiford Pharmacodynamics of a fluoroqui-nolone antimicrobial agent in a neutropenic rat model of Pseudomonas sepsis Anti-microb Agents Chemother 1993 37483ndash490

26 J der Hollander J Knudsen J Mouton K Fuurstad N Frimodt-Moller H VerbrughF Espersen Comparison of pharmacodynamics of azithromycin and erythromycinin vitro and in vivo Antimicrob Agents Chemother 1998 42377ndash382

27 M Tauber S Kunz O Zak M Sande Influence of antibiotic dose dosing intervaland duration of therapy on outcome in experimental pneumococcal meningitis inrabbits Antimicrob Agents Chemother 1989 33418ndash423

28 A Gerber H Brugger C Feller T Stritzko B Stalder Antibiotic therapy of infectionsdue to Pseudomonas aeruginosa in normal and granulocytopenic mice Comparisonof murine and human pharmacokinetics J Infect Dis 1986 15390ndash97

29 L Mizen Methods for obtaining human-like pharmacokinetic patterns in experimen-tal animals In O Zak M Sande eds Handbook of Animal Models of InfectionAcademic Press New York 1999 pp 93ndash103

30 J Lin T Lin Renal handling of drugs in renal failure I Differential effects of uranylnitrate- and glycerol-induced acute renal failure on renal excretion of TEAB andPAH in rats J Pharmacol Exp Ther 1988 246896ndash901

31 I Lutsar I Friedland L Wubbel C McCoig J Jafri W Ng F Ghaffar GH McCrackenJr Pharmacodynamics of gatifloxacin in cerebrospinal fluid in experimental cephalo-sporin-resistant Pneumococcal meningitis Antimicrob Agents Chemother 1998 422650ndash2655

32 T OrsquoReilly S Kunz E Sande O Zak M Sande M Tauber Relationship betweenantibiotic concentration in bone and efficacy of treatment of staphylococcal osteomy-elitis in rats Azithromycin compared with clindamycin and rifampin AntimicrobAgents Chemother 1992 362693ndash2697

33 F Genko T Mannion C Nightingale J Schentag Integration of pharmacokineticsand pharmacodynamics of methicillin in curative treatment of experimental endocar-ditis J Antimicrob Chemother 1984 14619ndash631

34 T Carpenter C Hackbarth H Chembers M Sande Efficacy of ciprofloxacin for


experimental endocarditis caused by methicillin-susceptible or -resistant strains ofStaphylococcus aureus Antimicrob Agents Chemother 1986 30382ndash384

35 D Andes W Craig Pharmacodynamics of fluoroquinolones in experimental modelsof endocarditis Clin Infect Dis 1998 2747ndash50

36 A Gerber W Craig H-P Brugger C Feller A Vastola J Brandel Impact of dosingintervals on activity of gentamicin and ticarcillin against Pseudomonas aeruginosain granulocytopenic mice J Infect Dis 1983 145296ndash329

37 D Andes M V Ogtrop W Craig Impact of neutrophils on the in-vivo activity offluoroquinolones 37th Annual Meeting of the Infectious Disease Society ofAmerica Infect Dis Soc Am Philadelphia PA 1999

38 W Craig S Gudmundsson The postantibiotic effect In V Lorian ed Anti-biotics in Laboratory Medicine Williams and Wilkins Baltimore MD 1996pp 296ndash329

39 G Smith K Abbott Development of experimental respiratory infections in neutro-penic rats with either penicillin-resistant Streptococcus pneumoniae or β-lactamase-producing Haemophilus influenzae Antimicrob Agents Chemother 1994 38608ndash610

40 S Arai Y Gohara A Akashi K Kuwano N Nishimoto T Yano K Oizumi KTakeda T Yamaguchi Effects of new quinolones on Mycoplasma pneumoniae-in-fected hamsters Antimicrob Agents Chemother 1993 37287ndash292

41 L Piroth L Martin A Coulon C Lequeu M Duong M Buisson H Portier P Chava-net Development of an experimental model of penicillin-resistant Streptococcuspneumoniae pneumonia and amoxicillin treatment by reproducing human pharmaco-kinetics Antimicrob Agents Chemother 1999 432484ndash2492

42 T Rogers J Galgiani Activity of fluconazole (UK 49858) and ketoconazole againstCandida albicans in vitro and in vivo Antimicrob Agents Chemother 1986 30418ndash422

43 A Louie G Drusano P Banerjee Q-F Liu W Liu P Kaw M Shayegani H TaberH Miller Pharmacodynamics of fluconazole in a murine model of systemic candidia-sis Antimicrob Agents Chemother 1998 421105ndash1109

44 K Sorensen E Corcoran S Chen D Clark V Tembe O Lomovskaya M DudleyPharmacodynamic assessment of efflux- and target-based resistance to fluconazole(FLU) on efficacy against C albicans in a mouse kidney infection model 39th Inter-science Conference on Antimicrobial Agents and Chemotherapy San Francisco CA1999

45 J Knudsen K Fuursted F Esperson N Frimodt-Moller Activities of vancomycinand teicoplanin against penicillin-resistant pneumococci in vitro and in vivo andcorrelations to pharmacokinetic parameters in the mouse peritonitis model Antimi-crob Agents Chemother 1997 411910ndash1915

46 A Ahmed H Jafri I Lutsar C McCoig M Trujillo L Wubbel S Shelton GHMcCracken Jr Pharmacodynamics of vancomycin for the treatment of experimentalpenicillin- and cephalosporin-resistant pneumococcal meningitis AntimicrobAgents Chemother 1999 43876ndash881

47 M Smeltzer J Thomas S Hickmon R Skinner C Nelson D Griffith TR Parr JrR Evans Characterization of a rabbit model of staphylococcal osteomyelitis J Or-thop Res 1997 15414ndash421


48 M Sande M Johnson Antimicrobial therapy of experimental endocarditis causedby Staphylococcus aureus J Infect Dis 1975 131367ndash375

49 J Entenza O Marchetti M Glauser P Moreillon Y-688 a new quinolone activeagainst quinolone-resistent Staphylococcus aureus Lack of in vivo efficacy in exper-imental endocarditis Antimicrob Agents Chemother 1998 421889ndash1894

50 S Powell W Thompson M Luthe R Stern D Grossniklaus D Bloxham D GrodenM Jacobs A Discenna H Cash J Klinger Once-daily vs continuous aminoglycosidedosing Efficacy and toxicity in animal and clinical studies of gentamicin netilmicinand tobramicin J Infect Dis 1983 147918ndash932

51 B Fantin R Leclercq M Ottaviani J Valois B Maziere J Duval J Pocidalo CCarbon In vivo activities and penetration of the two components of the strepto-gramin RP 59500 in cardiac vegetations of experimental endocarditis AntimicrobAgents Chemother 1994 38432ndash437

52 A Cremieux B Maziere J Vallois M Ottaviani A Azancot H Raffoul A BouvetJ Pocidalo C Carbon Evaluation of antibiotic diffusion into cardiac vegetations byquantitative autoradiography J Infect Dis 1989 159938ndash944

53 W Craig Post-antibiotic effects in experimental infection models Relationship toin-vitro phenomena and to treatment of infections in man J Antimicrob Chemother1993 31149ndash158

54 B Vogelman S Gudmundsson J Turnidge J Leggett W Craig In vivo postantibioticeffect in a thigh infection in neutropenic mice J Infect Dis 1988 157287ndash298

55 O Cars I Odenholt-Tornqvist The post-antibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 1993 31159ndash166

56 D Merrikin J Briant G Rolinson Effect of protein binding on antibiotic activity invivo J Antimicrob Chemother 1983 11233ndash238

57 M Dudley D Griffith E Corcoran C Liu K Sorensen V Tembe S ChamberlandD Cotter S Chen Pharmacokinetic-pharmacodynamic (PK-PD) indices for vanco-mycin (V) treatment of susceptible (VSSA) and intermediate (VISA) S aureus inthe neutropenic mouse thigh model 39th Interscience Conference on AntimicrobialAgents and Chemotherapy San Francisco CA 1999

58 D Griffith O Lomovskaya L Harford A Lee MN Dudley V Lee Effect of effluxpumps (EPs) on the activity of fluoroquinolones against Pseudomonas aeruginosain murine models of infection 37th Interscience Conference on AntimicrobialAgents and Chemotherapy Toronto Canada 1997

59 D Andes W Craig Pharmacodynamics of gemifloxacin (GEM) against quinolone-resistant strains of S pneumoniae (SP) with known resistance mechanisms 39thInterscience Conference on Antimicrobial Agents and Chemotherapy San Fran-cisco CA 1999

60 M Dudley PG Ambrose Pharmacodynamics in the study of drug resistance andestablishing in vitro susceptibility breakpoints ready for prime time Curr Opin Mi-crobiol 2000 3515ndash521

5

β-Lactam Pharmacodynamics

JoCarol J McNabb

University of Nebraska Medical Center Omaha Nebraska

Khanh Q Bui

Bristol-Myers Squibb Company Chicago Illinois

1 INTRODUCTION

11 Historical Overview

Penicillin G the original β-lactam antibiotic was discovered by Fleming in 1928and used for the first time in 1941 to treat a staphylococcal infection in a Britishpoliceman By the end of World War II penicillin was commercially availablein the United States [1] Although initially given as a continuous infusion due tothe abundance of penicillin and the difficulties with the intravenous drip deliverysystem long-acting repository forms of penicillin G became popular with clini-cians The pharmacodynamic concepts that apply to β-lactams were actually pion-eered in the late 1940s by Harry Eagle an immunologist at the National Institutesof Health Calling upon both in vitro and in vivo animal studies Eagle was thefirst investigator to propose the concept of time-dependent bactericidal killingfor β-lactams Eagle demonstrated in vivo that a penicillin-free interval prolongsthe duration of treatment necessary for cure and that less total daily drug givenin frequent multiple doses is more effective than larger infrequent doses Eagle

99

100 McNabb and Bui

also demonstrated in vivo with nonneutropenic mice and rabbits that the durationof the penicillin concentration above the MIC of a specific pathogen correlatesto drug efficacy In Eaglersquos experiments a serum concentration 2ndash5 times theMIC of the bacteria correlated with penicillinrsquos bactericidal activity [2] The con-cept of dividing antibacterials into groups based on the pattern of bactericidalactivity was not formally proposed by investigators until the late 1970s As earlyas 1953 however Eagle clearly discussed not only the time-dependent bacteri-cidal activity of penicillin but also the concentration dependence of streptomycinthe first aminoglycoside [3]

In like manner Eagle also elucidated the effect of antibiotic concentrationon the rate at which a pathogen is killed both in vitro and in vivo Marked differ-ences in the effective concentration of penicillin and the maximal rate of killexist between organisms depending on the generation time and the MIC of theorganism For gram-positive bacteria however 3ndash10 times the MIC of the patho-gen kills at a maximal rate that does not continue to increase as the concentrationof penicillin increases [4ndash7] Excessively high concentrations of penicillin donot increase its bactericidal effect In fact in relation to S pneumoniae Eagleobserved a paradoxical decrease in the rate of killing in vitro at very high concen-trations of penicillin [8] Although two case reports of streptococcal endocarditiscured by reducing the dose of penicillin appear in the literature the clinical sig-nificance of this paradoxical phenomenon now termed the lsquolsquoEagle effectrsquorsquo hasnever been studied in a clinical trial [9ndash10]

As experience with penicillin increased clinicians soon realized that inpractice the concentration did not have to be continuously maintained above theMIC of a given pathogen to effect a cure in humans [11] Several investigatorsmost notably Bigger and Parker demonstrated in vitro that once gram-positivebacteria were exposed to a lethal dose of penicillin a period of delayed organismregrowth ensued during which surviving organisms did not immediately beginto multiply [12ndash14] Schmidt and Eagle were the first investigators to report thisphenomenon in animal studies of S pneumoniae infection [15ndash17] Eagle pro-posed an elegant hypothesis explaining what is now known as the post antibioticeffect Calling this phenomenon the lsquolsquoslow recovery of bacteria from the toxiceffects of penicillinrsquorsquo he proposed that bacteria that are damaged but not killedby exposure to penicillin remain susceptible to host defenses for several hoursafter the penicillin is removed [1819] Eaglersquos in vitro and in vivo experimentsare the foundation for β-lactam pharmacodynamics His experiments howeverwere not designed to answer one of the most crucial pharmacodynamic questionsWhat is the optimal time above the MIC for the β-lactams Although not specifi-cally proven by his own experiments Eagle consistently asserted throughout hiswork that the best dosing strategy for penicillin is to maintain antibiotic concen-trations above the pathogenrsquos MIC for the entire dosing interval This conceptguided the dosing of β-lactams for the next 50 years


12 Pharmacokinetics Versus Pharmacodynamics

The differentiation between pharmacokinetics and pharmacodynamics has beendiscussed earlier in this book To review these concepts the reader is encouragedto refer back to Chapter 2 Because of the large number of β-lactam antibioticsavailable a complete review of each product will not be undertaken Where possi-ble generalizations will be made to simplify the discussion as it pertains to thisclass β-Lactams are best represented by a two-compartment model in which theyundergo rapid distribution followed by an elimination or terminal phase Thepharmacokinetic parameters of the β-lactams show many similarities in relationto the steady-state volume of distribution In a review of eight β-lactams (includ-ing members of the semisynthetic penicillin cephalosporin and carbapenemgroup) Drusano [20] showed that the volume of distribution following the post-distributive phase for these agents is much less than 1 Lkg (range 015ndash024 Lkg) indicating that they remain in the extracellular water as opposed to movinginto mammalian cells β-Lactams can be found in most body sites and secretionsmaking them an option for many infections

Although the volumes of distribution are similar for the compounds in thisclass other pharmacokinetic parameters such as clearance route of eliminationand half-life show more variability The clearance can vary by threefold from7 to 24 Lh [20] Because the volume of distribution falls within a narrow rangethis change in clearance is the primary determinant of half-life variation for thisclass The terminal half-life for penicillin and its analogs tends to be shorter (08ndash12 h) than those of the cephalosporins (14ndash8 h) [20ndash23] This is the rationalefor different dosing regimens as some require administration every 4 h whereasothers produce reliable outcomes with more extended dosing intervals of every12 or 24 h

The renal route of elimination is the primary pathway for the clearance ofβ-lactams The notable exceptions for this would be ceftriaxone cefoperazoneand nafcillin which undergo extensive nonrenal elimination as well [22]

2 FACTORS AFFECTING PHARMACODYNAMICS


The postantibiotic effect (PAE) is the suppression of bacterial growth that persistswhen drug is removed after a short exposure of a microorganism to an antimicro-bial All antimicrobials appear to have a PAE to gram positive bacteria in vitroThe mechanism of the PAE is not well understood It may relate to nonlethaldamage of the bacteria or limited persistence of the drug at a cellular site ofaction Factors that have been shown to influence the PAE include the type oforganism the dose and concentration of antibiotic the duration of exposure andthe size of the bacterial inoculum

102 McNabb and Bui

In vitro one determines the PAE by observing bacterial growth kineticsafter antibiotic is removed The PAE retards the growth of antibiotic-exposedculture in relation to a control and is usually defined as the difference betweenthe time for each culture (exposed and control) to increase in concentration by1 log10 (tenfold) after removal of the drug [24] There is no standard protocol formeasurement of the PAE in vitro however Some investigators measure the PAEindirectly by viable colony counts Others use direct methods including biolum-inescense or optical density [25] The methodology will affect the results A PAEobtained from a bioluminescense assay is typically longer than the PAE deter-mined by viable counts [26] This knowledge is important in evaluating resultsfrom different studies The magnitude of the PAE depends upon drug exposuretime and antibiotic concentration up to some maximal response [27 28] Byviable counts the PAE of penicillins and cephalosporins to gram-positive bacteriais consistently 1ndash3 h [29] Imipenem and meropenem are the only β-lactams thatdemonstrate a PAE to gram-negative bacteria primarily P aeruginosa [30ndash32]The PAE of these carbapenems to P aeruginosa appears to be strain-dependentand varies up to 2 h in length depending on the particular strain [33]

In general the PAE observed in vivo is of greater duration than the PAEobserved in vitro for the same bacteria Several theories have been proposedto explain this phenomenon The sub-MIC effect is one possible explanationInvestigators evaluating the effects of residual drug on bacterial growth haveshown that in vitro a post antibiotic sub-MIC effect will prolong the durationof the PAE For example the PAE of benzylpenicillin is 24 h to S pyogenesWhen exposed to sub-MIC concentrations of penicillin (03 MIC) the PAEincreases to 22 h Continued exposure to antibiotics at sub-MIC concentrationsis actually closer to the reality of declining in vivo drug levels than the in vitromodels where antibiotic is completely removed from the system when PAE istested [34] Another possible explanation for the longer in vivo PAE is the postan-tibiotic leukocyte enhancement (PALE) effect As long as there is otherwise aPAE leukocytes have been shown in vitro to enhance the killing of antibiotic-damaged bacteria and prolong the PAE [35]

The neutropenic mouse thigh model and the neutropenic mouse pneumoniamodel are the two most widely accepted animal models used by investigators tostudy the PAE in vivo By eliminating the effects of white blood cells one candetermine the PAE attributable solely to the antibiotic In a neutropenic mousethigh model of infection all β-lactams induce a PAE of 12ndash45 h to S aureusno PAE to gram-negative bacteria and no PAE to S pneumoniae [36] Theseresults are consistent with in vitro results with the exception of penicillin in rela-tion to S pneumoniae [37] Even though penicillin does exert an in vitro PAEto S pneumoniae investigators have not been able to reproduce the effect in vivoUsing the mouse thigh model to evaluate the effect of combination antibiotics


investigators have also shown that when both drugs (an aminoglycoside plus aβ-lactam to treat S aureus) induce a PAE alone then the combination prolongsthe PAE beyond the longer of the individual PAEs [38]

The most important clinical application of the PAE as a pharmacodynamicparameter is in defining optimal dosing schedules for β-lactams The PAE iscrucial in determining the optimal time above the MIC for any given drugndashpatho-gen combination The existence of a PAE implies that the time above the MICcan be less than 100 of the dosing interval and in fact the PAE is the theoreticalrationale for the intermittent dosing of β-lactams

22 Protein Binding

The effect of protein binding continues to be a topic of debate even as more islearned about the pharmacodynamics of β-lactams It is recognized that proteinbinding is a rapid process that produces a reversible interaction between antibioticand protein principally albumin [39] A constant equilibrium exist between thetotal (T) drug concentration and freeunbound (F) and protein bound (DB) frac-tions T harr F DB It is accepted that only the free drug is able to diffuse fromthe bloodstream to the site of infection and subsequently into the bacteria Thisconcept is important for the time-dependent antibiotics that rely on maintainingunbound serum concentrations in excess of the MIC for a prolonged period oftime For highly protein bound drugs failures may be predicted as free-drugconcentrations drop below the MIC

In vivo models with mice and rabbits have demonstrated that the degreeof β-lactam protein binding correlates with the penetration into implanted tissuecages When table tennis balls were implanted into rabbits it was found that thepenetration of cephalothin (65 protein bound) was 27 versus 12 that ofcefazolin (86 protein bound) [40] This was further substantiated with work ina mouse peritonitis infection model with S aureus showing that the degree ofprotein binding was inversely proportional to the dose of antibiotic required toproduce a protective dose [41] Merrikin et al [41] used a set of β-lactams thathad the same intrinsic activity to the test strain while maintaining the pharmacoki-netics relatively constant to derive their data They also noted that the more highlyprotein bound drugs had a longer half-life but that this did not correlate with theprotective dose because the MICs of all agents were identical

Studies evaluating the effect of protein binding and clinical outcome inhumans are difficult to perform Instead the skin blister fluid model has been usedin healthy subjects to estimate the relationship between serum protein binding andtissue penetration This is interpreted as a movement of drug from the intravascu-lar to the extravascular space that is necessary during the treatment of an activeinfection When flucloxacillin (95 protein-bound) was compared to amoxicillin

104 McNabb and Bui

(17 protein-bound) penetration into blister fluid was markedly less for thehighly protein bound antibiotic [42] This suggests that amoxicillin is a betteragent for treatment but many other factors (intrinsic antimicrobial activity solu-bility) must be considered when making a determination for the appropriatenessof use The authors also pointed out that the effect of protein binding on penetra-tion becomes more pronounced only when the proportion of bound drug is highBecause most β-lactams exhibit less than 70 protein binding their penetrationwill be minimally affected [42]

The in vitro and in vivo reduction in cephalosporin activity is less predict-able when cephalosporins are tested against gram-negative organisms Leggettand Craig [43] found that the ceftriaxone cefoperazone moxalactam and cefti-zoxime MICs for E coli and K pneumoniae in human serum ultrafiltrate wereless than predicted by simply examining the protein binding alone The sameeffect was not observed with S aureus or P aeruginosa A disproportionate risein the MIC was observed when the antibiotics were placed into 25 50 and95 serum The hypothesis for this observation was that the serum containedproducts that enhanced the killing of gram-negative bacilli In essence a proteinfound in the serum effectively lowered the MIC when these cephalosporins wereexposed to Enterobacteriaceae This finding may explain why ceftriaxone (95protein bound) provides activity at or slightly below its MIC even though failurewould be predicted

Clinical data in humans to show that protein binding can alter the outcomeof an infection were presented in a case report by Chambers et al [44] Theseworkers reported three therapeutic failures with cefonicid used once daily for thetreatment of S aureus endocarditis This is a highly bound (95) cephalosporinand it was suggested that free drug concentrations were not sustained for a longenough period of time The measured total drug concentrations (free and bound)exceeded 150 microgmL but the serum bactericidal titer (SBT) was 18 Success-ful outcomes are predicted when the SBTs are 18 Another consequence ofthe high protein binding was the MIC difference of cefonicid in broth versusserum When the organisms were originally tested in broth the geometric meanfor all patients was 46 microgmL whereas use of 50 serum as the diluent resultedin a six fold increase (279 microgmL) As noted previously protein binding particu-larly in situations with highly bound cephalosporins against gram-positive bacte-ria will result in an increase of the MIC This latter observation is often over-looked when susceptibility tests are performed From these data it appears thatthis regimen allowed free-drug concentrations to remain above the MIC for onlya very short period of time The role of protein binding may affect the pharmaco-dynamics of specific antibioticndashbacterium combinations especially in situationswhere the free drug concentrations do not exceed the time above the MIC for β-lactams


3 RESEARCH STUDIES

31 In Vitro Studies

The in vitro evidence supporting the role of β-lactams as concentration-indepen-dent (time-dependent) agents was reported by Craig and Andes [45] who useda standard time-kill method with ticarcillin ciprofloxacin and tobramycin Fol-lowing the addition of antibiotic to the experimental strain of Pseudomonas aeru-ginosa they found that only ticarcillin failed to produce a faster rate of bacterialkilling after concentrations of 4 MIC were achieved The rate and extent ofbacterial killing between 4 MIC as compared to 16 MIC and 64 MIC weresimilar leading them to suggest that exceeding the MIC by more than fourfold isnot necessary

32 In Vivo Animal Studies

The in vivo data with animals correlate with observations from in vitro experi-ments An undertaking to describe and highlight all of the previous work in thisarea will not be attempted here Instead studies describing important results thatcontribute to the understanding of β-lactam pharmacodynamics will be used Tominimize the confusion related to host factors most of the experimental datawere derived using a neutropenic infection model Eliminating the activity ofwhite blood cells from these models has a distinct advantage over using normalanimals only antibioticndashorganism interactions are studied without any additionalinterference from neutrophils

Using a mouse model Gerber et al [46] correlated bacterial regrowth tothe time above the MIC (T MIC) When ticarcillin was administered as eithera single bolus or a fractionated dose (more closely simulating human pharmacoki-netics resulting in similar AUCs) neutropenic animals were found to have re-duced bacterial growth with more frequent dosing extending the T MIC Theextremely large peakMIC ratio produced from a bolus dose (201) did not reducethe bacteria count more than the fractionated doses (51) This same study evalu-ated the effect of these two dosing schemes in normal mice Unlike previousresults the bacterial regrowth with the bolus dose was not demonstrated sug-gesting that the effect of the combination with antibiotic and white blood cellswas significant in suppressing P aeruginosa No attempt was made to determinethe optimal T MIC required to produce a good outcome but an examinationof these data show that concentrations during bolus dosing were above the MICfor only 25 of the dosing interval

The required T MIC for obtaining the best outcome may vary for individ-ual antibioticndashpathogen combinations A univariate analysis of ticarcillin cefa-zolin and penicillin all demonstrated that the T MIC was the most important

106 McNabb and Bui

parameter in determining outcome as opposed to the AUC or peak concentrations[47] To produce a bactericidal effect E coli required a longer exposure to cefa-zolin (60 versus 20) compared to S aureus This large difference is theresult of a significant PAE of cefazolin for S aureus This evidence supportsearlier work with cephalosporins and S pneumoniae that found a relationshipbetween T MIC and the effective dose required for protection of 50 of theanimals [48]

Studies comparing normal and neutropenic animals can show the pharma-codynamic influence of host defenses As described previously the work ofGerber et al [46] demonstrated that normal mice could suppress bacterial growthregardless of the optimal dosing scheme Roosendaal et al [49] also discovereda striking difference between normal and leukopenic rats For normal rats theintermittent (every 6 h) and continuous administration of ceftazidime requiredequivalent doses (035 and 036 mgkg respectively) to reach the protective dosefor 50 (PD50) of the animals For leukopenic rats the continuous infusion re-quired 375 mgkg to produce the PD50 whereas 30 mgkg of ceftazidime wasneeded with the intermittent dosing This study highlights two important differ-ences between normal and neutropenic animals (1) Host defenses can overcomethe deficiencies when the pharmacodynamics are not maximized and (2) neutro-penic hosts require larger doses than normal hosts to eradicate organisms

33 Clinical Studies

Human studies to determine the optimal pharmacodynamics of β-lactams havefocused on infections in acute otitis media (AOM) or continuous infusion (CI)settings AOM is an attractive model because of the relative ease of fluid collec-tion that can be used for determining antibiotic concentation or MIC over timeA more in-depth discussion of AOM is presented in Section 7 Clinical data withcontinuous infusion provide important information because the pharmacodynam-ics are maximized with a prolonged time above the MIC The following sectionpresents a summary of relevant continuous infusion studies

4 CONTINUOUS INFUSION

The goal of antibiotic therapy is to achieve the best possible clinical outcomeswhile consuming the least amount of hospital resources Health care systems areunder intense pressure to increase quality of care and at the same time reducecosts Pressure to reduce the cost of antimicrobial therapy is especially intensebecause these drugs may account for up to 50 of a hospital pharmacy budgetAlthough β-lactam antibiotics have traditionally been given by intermittent infu-sion administration by continuous infusion is gaining popularity because it takesfull advantage of the known pharmacodynamics of the β-lactams and potentially


consumes the least amount of hospital resources Other options that will alsomaintain the time above MIC for the entire dosing interval are the use of antibiot-ics with longer half-lives or more frequent larger doses An advantage of continu-ous infusion however is that it can be given at a lower total daily dose thanstandard dosing schedules Although the IV drip method used in the past wascumbersome and inaccurate today because of lightweight portable and accurateinfusion pumps a continuous infusion is relatively easy and cost-effective toadminister Giving a loading dose prior to starting the continuous infusion willbring the antibiotic concentration into the therapeutic range immediately and min-imize any lag time in drug tissue equilibration Constant infusion of β-lactamantibiotics has the potential for appreciable cost reductions because it may repre-sent the best method to maintain levels above the MIC during the entire dosinginterval using the least amounts of drug labor and supplies

A number of in vitro and animal models substantiate the equivalent orsuperior efficacy of continuous infusion of β-lactams compared to standard dos-ing These models have been used extensively to elucidate the pharmacodynamicsof β-lactams because they hold a significant advantage over studies in humansThe doses and dosing intervals of antibiotic are easily varied reducing the inter-dependence of the pharmacodynamic parameters Using an in vitro model inves-tigators have demonstrated that a continuous infusion (CI) at 5 MIC of Paeruginosa is as efficacious as intermittent dosing (II) using less total daily drug[5051] Experiments in animal models have confirmed these in vitro results anddemonstrated further that a continuous infusion may be the better dosing strategyif the same total daily dose is used [5253]

The mouse thigh model has been used to evaluate the difference betweenneutropenic and nonneutropenic host response [54] The real difference in effi-cacy between CI and II shows up in the neutropenic host Even with a lowertotal daily dose CI provides much better efficacy than bolus dosing [5556] Inneutropenic rats with gram-negative infection a ceftazidime bolus dose that pro-tects 50 of the animals from death (PD50) had to be 65-fold higher than thePD50 dose for a continuous infusion of the same drug Once again this experimentconfirms that time above MIC correlates to efficacy for the β-lactams and whenhost defenses are not present the β-lactam concentration should exceed the MICof the pathogen for the entire dosing interval [57]

Continuous infusion is a practical way to maintain 100 time above MICwith less total daily drug ( eg 3 g24 h CI of ceftazidime versus 1ndash2 g every8 h) Although clinical efficacy data are sparse the pharmacodynamics of contin-uous infusion are well-characterized in both normal volunteers and critically illpatients and several small clinical trials have shown the equivalence of continu-ous infusion and standard dosing [58ndash62] In one of the first clinical trials ofcontinuous infusion Bodey et al [63] compared the efficacy of cefamandoledosed as either a continuous infusion or an intermittent dose given in combination

108 McNabb and Bui

with carbenicillin There was no significant difference in clinical cure betweenthe two regimens By subgroup analysis patients with persisting severe neutro-penia had a better clinical outcome (65 versus 21 p 003) with continuousinfusion [63] In a study of CI benzylpenicillin versus daily IM procaine penicillinG in 123 patients with pneumococcal pneumonia there was also no differencein clinical cure rates [64] A nonrandomized trial of continuous versus intermit-tent dosing of cefuroxime showed that the CI results in a lower total antibioticdose The CI results in equivalent efficacy shorter length of hospital stay andoverall cost savings to the institution [65]

At Hartford Hospital there have been two continuous infusion β-lactamclinical studies The investigators compared cefuroxime given either as a continu-ous infusion of 1500 mg or intermittently as 750 mg every 8 h (n 25 in eachgroup) to treat hospitalized community-acquired pneumonia (CAP) [66] Steadystate cefuroxime serum concentrations were 1325 629 microgmL more than 2ndash4 times the MIC90 of typical CAP pathogens There was no difference in clinicalcure rates but the CI regimen was associated with a shorter length of treatmentdecreased length of stay lower total cefuroxime dose and overall cost savingsThe average amount of intravenous cefuroxime per patient decreased significantly(p 004) from 80 34 g for intermittent dosing to 59 32 g for thecontinuous infusion The average daily costs (including antibiotics labor andsupplies) decreased significantly (p 004) $6364 3095 for the continuousinfusion compared with $8385 3482 for intermittent dosing

The second Hartford Hospital study was a prospective randomized trial ofthe efficacy and economic impact of ceftazidime given as either a continuousinfusion (3 gday) or intermittent infusion (2 g q8h) plus once-daily tobramycinfor the treatment of nosocomial pneumonia in the ICU [67] The investigatorsevaluated 35 patients 17 in the CI group and 18 in the II group Clinical efficacydid not vary significantly between groups (94 success in CI vs 83 in II)Number of adverse events duration of treatment and total length of hospital stayalso did not vary significantly The continuous infusion regimen used half of theintermittent dose and maintained concentrations above the MIC of the pathogenfor 100 of the dosing interval The intermittent dosing regimen maintainedconcentrations above the MIC for 76 of the dosing interval The costs (includ-ing drug acquisition antibiotic preparation and administration adverse eventsand treatment failures) associated with the CI of ceftazidime $62569 38784were significantly lower (p 0001) than with the II $100464 42995

There are some potential disadvantages to giving antibiotics by continuousinfusion For patients with limited IV access the continuous infusion may requiretheir only IV line Drug compatibility will be an issue if two drugs must beinfused simultaneously through one line Although the cost of infusion pumpsshould be considered in any cost-effectiveness analysis of drug delivery by con-tinuous infusion since most manufacturers will provide the use of the infusion


pumps with a supply contract the cost to the hospital is usually limited to theprice of the administration sets Overall the advantages of the continuous infu-sion both pharmacodynamically and economically far outweigh the disadvan-tages The continuous infusion of β-lactams in place of frequent intermittentdosing is a good example of how the knowledge and application of pharmacody-namic concepts can lead to cost-effective antibiotic therapy

5 TISSUE PENETRATION

Because of the difficulties associated with measuring tissue concentrations ofdrugs we generally use serum concentration as a surrogate marker in pharmaco-dynamic models Although most common bacterial pathogens are extracellularinfections occur in the tissues and it is the pharmacodynamic profile of an antibi-otic at the site of infection that ultimately determines its clinical efficacy Antibi-otic concentrations can vary significantly depending on the type of tissue Drugpenetration into tissues depends on the properties of the specific tissue type theproperties of the antibiotic and the interactions that occur at the tissuedrug inter-face Protein binding tissue permeability tissue metabolic processes and drugphysiochemical properties such as lipid solubility pKa and molecular weight allinfluence an antibioticrsquos movement into human tissues Only the free fraction(non-protein-bound) of an antibiotic is available to exert a pharmacological ef-fect For β-lactams the percent tissue penetration is inversely proportional to theprotein binding of the drug Animal studies show a direct correlation betweenserum protein binding and penetration into peripheral lymph The penetrationinto rabbit lymph of ceftriaxone a drug that is highly protein bound is 673 Incomparison the penetration of amoxicillin a drug with much less serum proteinbinding is 976 [68]

Methodological problems in obtaining tissue samples are the limiting factorin characterizing antibiotic tissue concentrations A complete pharmacodynamicpicture would include a concentrationndashtime curve for the various tissue compart-ments Most studies done in humans to date however simply characterize drugpenetration into various tissue compartments In spite of the limitations thesestudies have provided important information about the extracellular and intracel-lular disposition of these drugs Tissue penetration studies consistently show thatpenicillins and cephalosporins have rapid and good penetration into interstitialfluid [69ndash72] The opposite occurs in relation to intracellular space There isessentially no uptake of β-lactams into peripheral blood mononuclear cells poly-morphonuclear cells or alveolar macrophages thus explaining the ineffec-tiveness of β-lactams against intracellular pathogens such as Mycoplasma orChlamydia [7374]

Standard methods of characterizing tissue concentrations by using wholetissue homogenates tend to underestimate the interstitial concentration of β-

110 McNabb and Bui

lactams A promising new technique currently being developed for studying thepharmacodynamics of drug tissue penetration in both animals and humans hasbeen applied to the study of β-lactams with some success Microdialysis is anin vivo sampling technique for the continuous monitoring of drugs or other ana-lytes in the extravascular space The advantage of microdialysis is that it can beused in a variety of tissue compartments with minimal invasiveness and can there-fore be used in subjects that are awake and freely moving Because it overcomesthe limitations of other methods microdialysis offers a unique opportunity tocharacterize the pharmacokinetic profile of drugs in tissue compartments such asthe interstitial fluid adipose tissue muscle or dermis

Two microdialysis studies in animals have evaluated the tissue pharmacoki-netic profile of various β-lactams In the rat thigh model investigators found anexcellent correlation between piperacillin and ceftriaxone concentrations inplasma and predicted free levels of drug in the tissue [7576] In a separate studyusing awake and freely moving rats the investigators measured ceftriaxone andceftazidime concentrations in two regions of the animalrsquos brain Interestinglynot only are the half-lives of the antibiotics in the brain different from their re-spective half-lives in serum but drug distribution within the brain differs byregion and is not homogenous Based on this study antibiotic distribution appearsto differ by specific tissue compartment and region [77] Similar differences bytissue compartment in equilibration rates and pharmacokinetic profiles have beenobserved in human subjects [78] Microdialysis promises to be a useful tool inpharmacodynamic studies of the future

The central nervous system presents special problems in relation to study-ing the pharmacodynamics of drugs The brain is a unique tissue compartmentTight junctions of endothelial cells and a low rate of transcellular drug transportboth act to exclude antibiotics from the CNS Impaired host defenses and theslow rate of bacterial growth in the CNS decrease the effectiveness of antibioticsThe primary determinants of antibiotic blood-brain barrier penetration are adrugrsquos lipid solubility degree of protein binding ionization and active transportinto or out of the CSF [79] Although highly lipophilic compounds pass readilyinto the CSF β-lactams are hydrophilic weak organic acids and their penetrationis normally less than 10 This penetration significantly increases during theinflammation associated with meningitis [80ndash83] An additional issue in relationto β-lactams is the active transport mechanism that pumps these drugs out of theCSF Benzylpenicillin has the highest affinity for the transport pump but otherβ-lactams are also subject to elimination by this route

Antibiotic concentrations are especially difficult to measure in the braindue to the invasiveness in obtaining either spinal fluid (CSF) or tissue samplesand relatively few pharmacodynamic studies have examined the activity of β-lactams in the CNS Although there is limited evidence that antibiotic pharmaco-dynamics in the brain differ from the dynamics in other tissue compartmentsbecause we cannot easily characterize this in human subjects antibiotic dosing


in CNS infections is still largely empirical The data we do have have beenobtained from in vitro models or animal studies

In vitro for cefotaxime investigators have shown a limited PAE to E coliof about 05 h in pooled human CSF The same studies do not demonstrate aPAE for cefotaxime to E coli in Mueller-Hinton broth (MHB) indicating thatthere may be a mechanism for PAE that is unique to the CSF [8485] There areconflicting results from animal studies measuring the PAE of β-lactams in theCSF [86] In one study adding β-lactamase to the CSF reversed the PAE indicat-ing that small amounts of residual drug in the CSF may actually be responsiblefor the in vivo PAE [87]

Although studies in animals have suggested that the β-lactam pharmacody-namic parameter in CSF that correlates to efficacy is actually the peak concentra-tion rather than the time above MIC the results from these studies are inconclu-sive [88ndash90] Using a rabbit pneumococcal meningitis model to investigate therelationship between CSF penicillin concentration and bactericidal rate of killingthe investigators demonstrated that maximal killing occurred at 10ndash30 times theminimal bactericidal concentration (MBC) of the pathogen [91] Unfortunatelythey did not also determine the duration of time above MBC As the concentrationof drug increases the time above MBC in the CSF will also increase and willmost likely approach 100 of the dosing interval When the dose of the antibioticis high enough to provide 100 time above MIC the pharmacodynamic parame-ters of time and concentration cannot be separated to determine which parameterreally correlates to efficacy

In contrast the one study to examine the relationship between the pharma-codynamic parameters of time above MBC peakMBC and AUCMBC demon-strated that T MIC continues to be the important pharmacodynamic parameterfor β-lactams even in the CSF By varying the doses and dosing intervals ofceftriaxone in a rabbit meningitis model of cephalosporin-resistant S pneumoniae(MIC and MBC 40 microgmL) the investigators determined that T MBC isthe only pharmacodynamic parameter that independently correlated with ceftriax-onersquos bactericidal activity During the first 24 h the highest rate of bactericidalkilling occurred when T MBC exceeded 95 of the dosing interval Also inthe first 24 h twice-daily administration of the same total ceftriaxone dose re-sulted in longer time MBC and higher killing rates [92] On the basis of thisstudy it appears that the same pharmacodynamic model time-dependent killingthat defines β-lactam activity in serum and other tissue compartments also definesβ-lactam efficacy in the CSF [93]

6 -LACTAM AND -LACTAMASE INHIBITOR

COMBINATIONS

The incidence of gram-negative bacterial resistance has been on the rise in recentyears Escherichia coli the leading cause of gram-negative bacteremia in both

112 McNabb and Bui

the community and hospital setting develops resistance to β-lactams by the pro-duction of TEM-1 and TEM-2 plasmid-mediated β-lactamases β-Lactamases area large family of enzymes ubiquitous to bacteria that hydrolyze the β-lactamring of penicillins and cephalosporins The incidence of gram-negative bacterialresistance as well as the increasing incidence of nosocomial gram-positive infec-tions (due to bacteria such as S aureus that are intrinsically resistant to β-lactams)have led to the search for new antibiotics One approach is to combine a β-lactamwith a potent β-lactamase inhibitor Currently in the United States there are threeβ-lactamase inhibitors on the market (clavulanate sulbactam and tazobactam)that are used in combination with a β-lactam antibiotic The goal of β-lactamndashβ-lactamase inhibitor combinations is to expand the coverage of the β-lactam togram-positive bacteria and overcome drug resistance to gram-negative bacteria[9495]

Strategies for the optimal dosing of β-lactamndashβ-lactamase inhibitors basedon pharmacodynamic principles have not been established or even extensivelystudied In addition to the pharmacodynamic issues that relate to the individualβ-lactam several additional pharmacodynamic questions arise in relation to com-bination drugs These questions include Would the sequential dosing of inhibitorand β-lactam affect the bactericidal activity of the combination Second is it theratio of the combination of drugs that matters or is it the time above the MICof the component drugs that determines efficacy Finally if it is the time abovethe MIC of each drug that correlates to overall efficacy what is the optimal timeabove the MIC for the inhibitor and how does that affect the time above theMIC required for the β-lactam

Two pharmacodynamic studies have addressed the issue of sequential dos-ing In vitro the sequential dosing of tazobactam followed by piperacillin doesnot enhance the bactericidal activity of piperacillin [96] Similarly in vivo asstudied in E coli bacteremia in mice the pharmacodynamics of ampicillin-sul-bactam does not depend on whether sulbactam is dosed sequentially or simulta-neously with ampicillin [97]

The question of optimal T MIC is complicated for β-lactamndashβ-lactamaseinhibitor combinations because the turnover rate of both the enzyme and theinhibitor will affect the inactivation of the β-lactamase [98] The amount andtype of enzyme produced by the bacteria have a marked effect on the dynamicsof the inhibitor [99] Subsequently the amount of inhibitor present determinesrestoration of susceptibility to the partner drug [100] Current dosing regimensprovide concentrations of inhibitor that exceed the in vitro susceptibilitybreakpoint for only 2ndash3 h not the entire dosing interval Inasmuch as these drugshave been shown to work clinically the unique pharmacodynamics of the β-lactamndashβ-lactamase inhibitor combination is clearly the determining factor [101]

One explanation for the clinical efficacy of these drugs may relate to apost-β-lactamase inhibition effect In an adaptation of the model used to deter-mine post-antibiotic effect the effect of tazobactam was evaluated in β-lacta-


mase-producing strains of E coli Preincubation of bacteria with tazobactam andpiperacillin resulted in piperacillin-induced killing during a second exposure topiperacillin alone Bacteria not initially exposed to tazobactam were not killedby piperacillin during the second exposure [102] Similarly other investigatorshave reported a post-β-lactamase inhibition effect in which regrowth of amoxicil-lin-resistant bacteria is prevented by amoxicillin alone following exposure to andremoval of amoxicillin and clavulanate [103]

In an in vitro study of piperacillin versus piperacillintazobactam as ex-pected the addition of tazobactam to piperacillin did not alter the killing of anE coli piperacillin-susceptible strain By adding tazobactam to piperacillin thekilling of an isogenic TEM-3 E coli resistant to piperacillin became equivalentto the killing of the susceptible strain even though the concentration of tazobac-tam fell below 4 mgL (the concentration used in susceptibility testing) at 3 hAs an explanation the investigators suggest that several hours of an essentiallyβ-lactamase-negative state during which bacteria must reaccumulate sufficientquantities of β-lactamase to inactivate the drug follow exposure to high levelsof inhibitor Therefore the dosing interval of β-lactamndashβ-lactamase inhibitorcombinations can be extended for some finite period of time limited by the reaccumulation of β-lactamase by persisting bacteria [104] On the basis of thissmall number of in vitro studies it appears that dosing piperacillin-tazobactamevery 8 h instead of every 6 h is a reasonable dosing regimen

7 CORRELATION TO IN VITRO RESISTANCE

Clinical studies evaluating resistance rates and patient outcomes have been pri-marily conducted in respiratory tract infections In comparison to other infectionsites respiratory tract infections are more prevalent and recovery of bacteria isprimarily noninvasive allowing more data to be compiled A limitation to inter-preting these data is that only a handful of the most commonly isolated bacteriaare studied often excluding serious pathogens such as vancomycin-resistant En-terococcus faecium (VRE) or methicillin resistant Staphylococcus aureus(MRSA) which are not associated with these body sites Although limited thesestudies provide clues for the treatment of other infections that require aggressiveantibiotic therapy The reporting of resistance continues to rise but the impacton clinical outcomes may not always correlate with microbiological reports

Using a retrospective review of pneumonia caused by Streptococcus pneu-moniae Pallares and colleagues compared cases involving penicillin-nonsensi-tive strains (MIC 012ndash8 microgmL) to a matched control group with penicillin-sensitive strains (MIC 01 microgmL) [105] Of the 24 cases 14 presented withintermediate-resistant pneumococci (012 MIC 1 microgmL) and the remaining10 were resistant (MIC 2 microgmL) A higher mortality rate (54 vs 25 P 00298) was observed when these cases were compared to the control groupThis difference may have been multifactorial because cases also had statistically

114 McNabb and Bui

significant more episodes of pneumonia in the past year (P 001) exposureto β-lactam therapy in the past 3 months (P 00008) recent hospitalizations(P 00038) and nosocomial pneumonia (P 00032) and were initially criti-cally ill (P 00030) These factors provide evidence that cases with penicillin-nonsensitive strains had more comorbidities When the authors examined theirdata they reported that of the 19 cases that received a β-lactam as treatment 11recovered Furthermore all eight deaths occurred in patients who were initiallycritically ill suggesting that this parameter not the choice of drug therapy ac-counted for mortality differences Two strains with a higher MIC (4 and 8 microgmL) resulted in therapeutic failures providing support that current breakpointsneed to be revised upward to correlate with clinical outcomes The authors con-cluded that penicillin-resistant strains with MIC 2 microgmL responded well toβ-lactam therapy if patients were not critically ill at the initial presentation

As a followup the same group performed a 10-year prospective study[106] It was shown that mortality was higher (38 vs 24 P 0001) whena penicillin-resistant S pneumoniae was isolated As described previously whenall other factors relating to severity were included no statistical significance wasfound (P 032) The mortality resulting from the administration of penicillinG ampicillin ceftriaxone or cefotaxime was in the range of 19ndash25 regardlessof the susceptibility patterns or agent used These data suggest that the use ofcephalosporin or high dose intravenous penicillin may be effective for treatmenteven when the MIC of penicillin predicts resistance (2 microgmL)

These studies have since been substantiated by the work of other investiga-tors [107108] Gress et al [107] compared the outcomes of intermediate-resistantand susceptible S pneumoniae and found no difference in length of stay (P 096) fever (P 074) or mortality (P 015) The treatment in this study waspenicillin or ampicillin and the conclusion was that both agents provided ade-quate serum and tissue concentrations to effectively treat organisms that had anMIC of 1 microgmL Cabellos et al [108] confirmed the usefulness of procainepenicillin in the treatment of pneumococcal pneumonia Fifteen of 16 patientswere cured and the only failure occurred in a patient who had a S pneumoniaewith penicillin MIC of 4 microgmL The findings were supported with pharmacoki-netic and serum bactericidal activity data

Friedland [109] prospectively studied children with pneumococcal pneu-monia and found that after 3 days of therapy clinical improvement was notedin 70 (1217) of patients with penicillin-resistant strains compared to 62 (2845) of patients with penicillin-susceptible strains [109] At the day 7 evaluation88 (1517) of patients with a penicillin-resistant strain had improved along with93 (4245) in whom a penicillin-susceptible S pneumoniae was isolated Twodeaths occurred in each group and all were treated with intravenous therapyEvery patient treated with oral β-lactam therapy improved regardless of the peni-cillin susceptibility profile These data show poor correlation between penicillin


resistance (2 microgmL) and clinical outcome for non-CNS infections caused byS pneumoniae

Although resistance data for pneumococcal pneumonia do not correlatewell with clinical outcomes studies in acute otitis media (AOM) have producedmore predictable outcomes In a comparative study utilizing either cefuroximeaxetil or cefaclor for the treatment of AOM Dagan et al [110] found that cefuro-xime axetil consistently produced better outcomes These data showed that in-creasing penicillin resistance correlated with statistically significant (P 0001)bacteriological failures despite cephalosporin therapy The penicillin MIC rangesof 01 0125ndash025 and 038ndash1 microgmL resulted in overall failure rates of 621 and 64 respectively When further stratified to the drug regimens thefailure rate of cefuroxime axetil was 9 8 and 50 respectively In thesesame ranges the cefaclor failure rate was 4 43 and 80 The escalatingfailure rate in these patients probably resulted from poor antibiotic penetrationinto the middle ear fluid because the pharmacodynamics was not optimized withthese dosage regimens

Gehanno et al [111] also reported the diminished activity of cefuroximeaxetil as the penicillin MIC increased When 84 children with S pneumoniae weretreated with 30 mgkg of cefuroxime suspension for 8 days a clinical success rateof 86 (7284) was observed for the penicillin MIC range of 0015 to 4 microgmL The clinical success rate was 92 (3942) for penicillin-susceptible strains90 (910) for penicillin-intermediate strains and 75 (2432) for penicillin-resistant strains The conclusion was that cefuroxime axetil was clinically effec-tive for AOM but may not be the drug of choice when resistant strains emergedue to the higher failure rate

Craig and Andes [112] had predicted this scenario when they evaluatedthe pharmacokinetics and antimicrobial effects of these agents in AOM Againstpenicillin-intermediate and -resistant S pneumoniae failure was expected withcefaclor because the usual dosage regimen did not produce any appreciable timeabove the minimal inhibitory concentration (T MIC) that is required of β-lactams for clinical success Cefuroxime was more likely to produce a successfuloutcome because the T MIC against penicillin-intermediate S pneumoniaeranged from 33 to 53 whereas the T MIC for penicillin-resistant strainswas 0ndash23 The T MIC can be thought of as a surrogate marker for resistancebecause as the MIC increases the T MIC is reduced predicting failure In theanalysis of AOM an inverse correlation was observed between resistant strainsand clinical outcomes

The previous examples do not provide a strong correlation between micro-biological resistance and clinical outcomes The primary reason for these discrep-ancies is that microbiological tests are performed with only bacteria and antibiot-ics whereas clinical outcomes include an additional component of host-specificfactors When the host has a functional immune system or is not critically ill

116 McNabb and Bui

outcomes may be much better than predicted by pharmacodynamic relationshipsalone Conversely a host with comorbidities or situations that compromise thepharmacodynamics can unmask the true effectiveness of an antibiotic Lindenet al [113] reported that the poor outcomes between vancomycin-resistant and-susceptible Enterococcus faecium (VREF vs VSEF) were multifactorial withthe antibiotic accounting for a partial effect The effect of reduced antibioticsusceptibility also increased the number of adverse events for patients infectedwith Pseudomonas aeruginosandashresistant isolates [114] These patients simulta-neously presented with additional problems that may have added to their pooroutcomes The in vitro reporting of bacterial resistance should be used as a guidebut cannot serve as the sole determinant of therapeutic decision making becauseit is the triad of antibiotic host and bacteria that determines outcomes

8 CONCLUSION

β-Lactam antibiotics are time-dependent agents that display bactericidal activitywithin the therapeutically achievable dosages Because most agents in this drugclass have short half-lives it is often necessary to administer frequent doses or usenovel dosing strategies to optimize their pharmacodynamic and pharmacokineticproperties The data gathered over the past half century using in vitro animaland human studies clearly show their usefulness in the treatment of numerousinfections when factors such as tissue penetration postantibiotic effect and pro-tein binding are considered

REFERENCES

1 A Fleming On the antibacterial action of cultures of a penicillium with specialreference to their use in the isolation of B influenzae Br J Exp Pathol 192910226

2 H Eagle R Fleischman AD Musselman Effect of schedule of administration ontherapuetic efficacy of penicillin Importance of aggregate time penicillin remainsat effectively bactericidal levels Am J Med 19509280ndash299

3 PM Shah W Junghanns W Stille Dosis-Wirkuns-Beziehung der Bakterizidie beiE coli K pneumoniae und Staphylococcus aureus Dent Med Wochenschr 1976101325ndash328

4 H Eagle R Fleischman AD Musselman Effective concentrations of penicillin invitro and in vivo for streptococci pneumococci and Treponema pallidum J Bacte-riol 195059625ndash43

5 H Eagle R Fleischman M Levy On duration of penicillin action in relation to itsconcentration in serum J Lab Clin Med 195341122ndash132

6 H Eagle HJ Magnuson R Fleischman Effect of method of administration on thera-


peutic efficacy of sodium penicillin in experimental syphilis Bull Johns HopkinsHosp 194679168ndash89

7 H Eagle AD Musselman The rate of bactericidal action of penicillin in vitro asfunction of its concentration and its paradoxically reduced activity at high concen-trations against certain organisms J Exp Med 19488899ndash131

8 WMM Kirby Bacteriostatic and lytic actions of penicillin on sensitive and resistantstaphylococci J Clin Invest 194524165ndash169

9 LR Griffiths HT Green Paradoxical effect of penicillin in-vivo (letter) J Antimi-crob Chemother 198515507ndash508

10 RH George A Dyas Paradoxical effect of penicillin in vivo (letter) J AntimicrobChemother 198717684ndash685

11 H Eagle R Fleischman M Levy lsquolsquoContinuousrsquorsquo vs lsquolsquodiscontinuousrsquorsquo therapy withpenicillin N Engl J Med 1953248481ndash488

12 RF Parker S Luse Action of penicillin on Staphylococcus Further observationsof effect of short exposure J Bacteriol 19485675ndash81

13 RF Parker HC Marsh Action of penicillin on staphylococcus J Bacteriol 194651181ndash186

14 JW Bigger Bactericidal action of penicillin on Staphylococcus pyogenes Irish JMed Sci 1944228585ndash595

15 E Jawetz Dynamics of the action of penicillin in experimental animals Arch InternMed 1946771

16 LH Schmidt A Walley RD Larson The influence of the dosage regimen on thetherapeutic activity of penicillin G J Pharmacol Exp Ther 194996258ndash268

17 LH Schmidt A Walley The influence of the dosage regimen on the therapeuticeffectiveness of penicillin G in experimental lobar pneumonia J Pharmacol ExpTher 1951103479ndash488

18 H Eagle R Fleischman AD Musselman Bactericidal action of penicillin in vivoParticipation of host and slow recovery of surviving organisms Ann Intern Med195033544ndash571

19 H Eagle AD Musselman The slow recovery of bacteria from the toxic effects ofpenicillin J Bacteriol 194958475ndash490

20 GL Drusano Role of pharmacokinetics in the outcome of infections AntimicrobAgents Chemother 198832289ndash297



23 M Fassbender H Lode T Schaberg K Borner P Koeppe Pharmacokinetics ofnew oral cephalosporins including a new carbacephem Clin Infect Dis 199316646ndash653

24 A Kumar MB Hay GA Maier JW Dyke Post-antibiotic effect of ceftazidimeciprofloxacin imipenem piperacillin and tobramycin for Pseudomonas cepacia JAntimicrob Chemother 199230597ndash602

25 H Hanberger LE NilssonM Nilsson R Maller Post-antibiotic effect of β-lactamantibiotics on gram-negative bacteria in relation to morphology initial killing andMIC Eur J Clin Microbiol Infect Dis 199110927ndash934

118 McNabb and Bui

26 H Hanberger E Svensson LE Nilsson M Nilsson Pharmacodynamic effects ofmeropenem on gram-negative bacteria Eur J Clin Microbiol Infect Dis 199514383ndash390


28 RW Bundtzen AU Gerber DL Cohn WA Craig Postantibiotic suppression ofbacterial growth Rev Infect Dis 1981328ndash37

29 F Baquero E Culebras C Patron JC Perez-Diaz JC Medrano MF Vicente Postan-tibiotic effect of imipenem of gram-positive and gram-negative micro-organismsJ Antimicrob Chemother 198618(suppl E)47ndash59

30 H Erlendsdottir S Gudmundsson The post-antibiotic effect of imipenem and peni-cillin-binding protein 2 J Antimicrob Chemother 199230231ndash232

31 HL Nadler DH Pitkin W Sheikh The postantibiotic effect of meropenem andimipenem on selected bacteria J Antimicrob Chemother 198924(suppl A)225ndash231

32 CI Bustamante GL Drusano BA Tatem HC Standiford Postantibiotic effect ofimipenem on Pseudomonas aeruginosa Antimicrob Agents Chemother 198426678ndash682

33 S Gudmundsson B Vogelman WA Craig The in-vivo postantibiotic effect of imi-penem and other new antimicrobials J Antimicrob Chemother 198618(suppl E)67ndash73

34 O Cars I Odenholt-Tornqvist The post-antibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 199331(suppl D)159ndash166

35 PJ McDonald BL Wetherall H Pruul Postantibiotic leukocyte enhancement in-creased susceptibility of bacteria pretreated with antibiotics to activity of leuko-cytes Rev Infect Dis 1981338ndash44

36 B Vogelman S Gudmundsson J Turnidge J Leggett WA Craig In vivo postantibi-otic effect in a thigh infection in neutropenic mice J Infect Dis 1988157287ndash298

37 WA Craig Post-antibiotic effects in experimental infection models Relationshipto in vitro phenomena and to treatment of infections in man J Antimicrob Chemo-ther 199331(suppl D)149ndash158

38 S Gudmundsson S Einarsson H Erlendsdottir J Moffat W Bayer WA Craig Thepost-antibiotic effect of antimicrobial combinations in a neutropenic murine thighinfection model J Antimicrob Chemother 199331(suppl D)177ndash191

39 R Wise Protein binding of β-lactams The effects on activity and pharmacologyparticularly tissue penetration I J Antimicrob Chemother 1983121ndash18

40 DN Gerding WH Hall The penetration of antibiotics into peritoneal fluid BullNY Acad Med 1975511016ndash1019

41 DJ Merrikin J Briant GN Rolinson Effect of protein binding on antibiotic activityin vivo J Antimicrob Chemother 198311233ndash238

42 R Wise AP Gillett B Cadge SR Durham S Baker The influence of protein bindingupon tissue fluid levels of six β-lactam antibiotics J Infect Dis 198014277ndash82

43 JE Leggett WA Craig Enhancing effect of serum ultrafiltrate on the activity of


cephalosporins against gram-negative bacilli Antimicrob Agents Chemother 19893335ndash40

44 HF Chambers J Mills TA Drake MA Sande Failure of a once-daily regimen ofcefonicid for treatment of endocarditis due to Staphylococcus aureus Rev InfectDis 19846S870ndash874

45 WA Craig SC Ebert Killing and regrowth of bacteria in vivo A review ScandJ Infect Dis Suppl 19916463ndash70

46 AU Gerber H-P Brugger C Fell T Stritzko B Stadler Antibiotic therapy of infec-tions due to Pseudomonas aeruginosa in normal and granulocytopenic mice Com-parison of murine and human pharmacokinetics J Infect Dis 198615390ndash97

47 B Vogelman S Gudmundsson J Leggett J Turnidge S Ebert WA Craig Correla-tion of antimicrobial pharmacokinetic parameters with therapeutic efficacy in ananimal model J Infect Dis 1988158831ndash847

48 N Frimodt-Moslashller MW Bentzon VF Thomsen Experimental infection with Strep-tococcus pneumoniae in mice Correlation of in vitro activity and pharmacokineticparameters with in vivo effect for 14 cephalosporins J Infect Dis 1986154511ndash517

49 R Roosendaal IAJM Bakker-Woudenbert MVDBV Raffe MF Michel Continu-ous versus intermittent administration of ceftazidime in experimental Klebsiellapneumoniae pneumonia in normal and leukopenic rats Antimicrob Agents Chemo-ther 198630403ndash408

50 JW Mouton JG den Hollander Killing of Pseudomonas aeruginosa during continu-ous and intermittent infusion of ceftazidime in an in vitro pharmaocokinetic modelAntimicrob Agents Chemother 199438931ndash936

51 DM Cappelletty SL Kang SM Palmer MJ Rybak Pharmacodynamics of ceftazi-dime administered as continuous infusion or intermittent bolus alone and in combi-nation with single daily-dose amikacin against Pseudomonas aeruginosa in an invitro infection model Antimicrob Agents Chemother 1995331797ndash1801

52 JJ Mordenti R Quintiliani CH Nightingale Combination antibiotic therapy Com-parison of constant infusion and intermittent bolus dosing in an experimental animalmodel J Antimicrob Chemother 198515313ndash321

53 DH Livingston MT Wang Continuous infusion of cefazolin is superior to intermit-tent dosing in decreasing infection after hemorrhagic shock Am J Surg 1993165203ndash207

54 AU Gerber Impact of the antibiotic dosage schedule on efficacy in experimentalsoft tissue infections Scand J Infect Dis 1991suppl 74147ndash154

55 IAJM Bakker-Woudenberg JC van den Berg P Fontijne MF Michel Efficacyof continuous versus intermittent administration of penicillin G in Streptococcuspneumoniae pneumonia in normal and immunodeficient rats Eur J Clin Microbiol19843131ndash135

56 K Totsuka K Shimizu J Leggett B Vogelman WA Craig Correlation betweenthe pharmacokinetic parameters and the efficacy of combination therapy with anaminoglycoside and a β-lactam In Y Ueda ed Proceedings of the InternationalSymposium on Netilmicin Professional Postgraduate Services Japan 198541ndash48

57 R Roosendaal IAJM Bakker-Woudenberg M van den Berghe-van Raffe MF Mi-

120 McNabb and Bui

chel Continuous versus intermittent administration of ceftazidime in experimentalKlebsiella pneumoniae pneumonia in normal and leukopenic rats AntimicrobAgents Chemother 198630403ndash408

58 DP Nicolau JC McNabb MK Lacy J Li R Quintiliani CH Nightingale Pharma-cokinetics and pharmacodynamics of continuous and intermittent ceftazidime dur-ing the treatment of nosocomial pneumonia (in press)

59 DP Nicolau CH Nightingale MA Banevicius Q Fu R Quintiliani Serum bacteri-cidal activity of ceftazidime Continuous infusion versus intermittent injectionsAntimicrob Agents Chemother 19964061ndash64

60 S Daenen Z Erjavec DRA Uges HG de Vries-Hospers P de Jonge MR HalieContinuous infusion of ceftazidime in febrile neutropenic patients with acute my-eloid leukemia Eur J Clin Microbiol Infect Dis 199514188ndash192

61 AS Benko DM Cappelletty JA Kruse MJ Rybak Continuous infusion versus in-termittent administration of ceftazidime in critically ill patients with suspectedgram-negative infections Antimicrob Agents Chemother 199640691ndash695

62 H Lagast F Meunier-Carpenter J Klastersky Treatment of gram-negative bacillarysepticemia with cefoperazone Eur J Clin Microbiol 19832554ndash558

63 GP Bodey SJ Ketchel V Rodriguez A randomized study of carbenicillin pluscefamandole or tobramycin in the treatment of febrile episodes in cancer patientsAm J Med 197967608ndash611

64 A Brewin L Arango WK Hadley JF Murray High-dose penicillin therapy andpneumococcal pneumonia JAMA 1974230409ndash413

65 JA Zeisler JD McCarthy WA Richelieu MB Nichol Cefuroxime by continuousinfusion A new standard of care Infect Med 19921154ndash60

66 PG Ambrose R Quintiliani CH Nightingale DP Nicolau Continuous vs intermit-tent infusion of cefuroxime for the treatment of community-acquired pneumoniaInfect Dis Clin Prac 19987463ndash470

67 DP Nicolau J McNabb MK Lacy J Li R Quintiliani CH Nightingale Pharmaco-kinetics and pharmacodynamics of continuous and intermittent ceftazidime duringthe treatment of nosocomial pneumonia Clin Drug Invest 1999in press

68 G Woodnutt V Berry L Mizen Effect of protein binding on penetration of β-lactams into rabbit peripheral lymph Antimicrob Agents Chemother 1995392678ndash2683

69 DM Ryan B Hodges GR Spencer SM Harding Simultaneous comparison of threemethods for assessing ceftazidime penetration into extravascular fluid AntimicrobAgents Chemother 198222995ndash998

70 T Kalager A Digranes T Bergan CO Solberg The pharmacokinetics of ceftriax-one in serum skin blister and thread fluid J Antimicrob Chemother 198413479ndash485

71 R Wise IA Donovan MR Lockley J Drumm JM Andrews The pharmacokineticsand tissue penetration of imipenem J Antimicrob Chemother 198618(suppl E)93ndash101

72 JW Mouton MF Michel Pharmacokinetics of meropenem in serum and suctionblister fluid during continuous and intermittent infusion J Antimicrob Chemother199128911ndash918

73 H Koga High-performance liquid chromatography measurement of antimicrobial


concentrations in polymorphonuclear leukocytes Antimicrob Agents Chemother1987311904ndash1908

74 WL Hand N King-Thompson JW Holman Entry of roxithromycin (RU 965) imi-penem cefotaxime trimethoprim and metronidazole into human polymorphonu-clear leukocytes Antimicrob Agents Chemother 1987311553 -1557

75 A Nolting TD Costa R Vistelle KH Rand H Derendorf Determination of freeextracellular concentrations of piperacillin by microdialysis J Pharm Sci 199685369-372

76 A Kovar TD Costa H Derendorf Comparison of plasma and free tissue levels ofceftriaxone in rats by microdialysis J Pharm Sci 19978652ndash56

77 L Granero M Santiago J Cano A Machado JE Peris Analysis of ceftriaxone andceftazidime distribution in cerebrospinal fluid of and cerebral extracellular spacein awake rats by in vivo microdialysis Antimicrob Agents Chemother 1995392728ndash2731

78 M Muller O Haag T Burgdorff A Georgopoulos W Weninger B Jansen G Sta-nek H Pehamberger E Agneter HG Eichler Characterization of peripheral-compartment kinetics of antibiotics by in vivo microdialysis in humans AntimicrobAgents Chemother 1996402703ndash2709

79 RA Fishman Blood-brain and CSF barriers to penicillin and related organic acidsArch Neurol 196615113ndash124

80 LJ Strausbaugh CR Bodem PR Laun Penetration of aztreonam into cerebrospinalfluid and brain of noninfected rabbits and rabbits with experimental meningitiscaused by Pseudomonas aeruginosa Antimicrob Agents Chemother 198630701ndash704

81 E Marmo L Coppola R Pempinello G di Nicuolo E Lampa Levels of amoxicillinin the liquor during continuous intravenous administration Chemotherapy 198228171ndash175

82 R Yogev WE Schultz SB Rosenman Pentrance of nafcillin into human ventricularfluid Correlation with ventricular pleocytosis and glucose levels AntimicrobAgents Chemother 198119545ndash548

83 DT Mullaney JF John Cefotaxime therapy Evaluation of its effect on bacterialmeningitis CSF drug levels and bactericidal activity Arch Intern Med 19831431705ndash1708

84 GG Zhanel JA Karlowsky RJ Davidson DJ Hoban Effect of pooled human cere-brospinal fluid on the postantibiotic effects of cefotaxime ciprofloxacin and genta-micin against Escherichia coli Antimicrob Agents Chemother 1992361136ndash1139

85 JA Karlowsky GG Zhanel RJ Davidson SR Zieroth DJ Hoban In vitro postantibi-otic effects following multiple exposures of cefotaxime ciprofloxacin and genta-micin against Escherichia coli in pooled human cerebrospinal fluid and Mueller-Hinton broth Antimicrob Agents Chemother 1993371154ndash1157

86 MA Sande OM Korzeniowski GM Allegro RO Brennan O Zak WM ScheldIntermittent or continuous therapy of experimental meningitis due to Streptococcuspneumoniae in rabbits Preliminary observations on the postantibiotic effect in vivoRev Infect Dis 1981398ndash109

87 MG Tauber O Zak WM Scheld B Hengstler MA Sande The postantibiotic effect

122 McNabb and Bui

in the treatment of experimental meningitis caused by Streptococcus pneumoniaein rabbits J Infect Dis 1984149575ndash583

88 MG Tauber S Kunz O Zak MA Sande Influence of antibiotic dose dosing inter-val and duration of therapy on outcome in experimental pneumococcal meningitisin rabbits Antimicrob Agents Chemother 198933418ndash423

89 JM Decazes JD Ernst MA Sande Correlation of in vitro time-kill curves andkinetics of bacterial killing in cerebrospinal fluid during ceftriaxone therapy of ex-perimental Escherichia coli meningitis Antimicrob Agents Chemother 198324463ndash467

90 I Lustar GH McCracken Jr IR Friedland Antibiotic pharmacodynamics in cere-brospinal fluid Clin Infect Dis 1998271117ndash1129

91 MG Tauber CA Doroshow CJ Hackbarth MG Rusnak TA Drake MA SandeAntibacterial activity of β-lactam antibiotics in experimental meningitis due toStreptococcus pneumoniae J Infect Dis 1984149568ndash574

92 I Lutsar A Ahmed IR Friedland M Trujillo L Wubbel K Olsen GH McCrackenJr Pharmacodynamics and bactericidal activity of ceftriaxone therapy in experi-mental cephalosporin-resistant pneumococcal meningitis Antimicrob Agents Che-mother 1997412414ndash2417

93 I Lustar A Ahmed IR Friedland M Trujillo L Wubbel K Olsen GH McCrackenJr Pharmacodynamics and bactericidal activity of ceftriaxone therapy in experi-mental cephalosporin-resistant pneumococcal meningitis Antimicrob Agents Che-mother 1997412414ndash2417

94 MR Jacobs SC Aronoff S Johenning DM Shlaes S Yamabe Comparative activi-ties of the β-lactamase inhibitors YTR 830 clavulanate and sulbactam combinedwith ampicillin and broad-spectrum penicillins against defined β-lactamase-produc-ing aerobic gram-negative bacilli Antimicrob Agents Chemother 198629980ndash985

95 EE Stobberingh In vitro effect of YTR 830 (tazobactam) in plasmid and chromo-somally mediated β-lactamases Chemotherapy 199036209ndash214

96 PD Lister AM Prevan CC Sanders Importance of β-lactamase inhibitor pharmaco-kinetics in the pharmacodynamics of inhibitor-drug combinations Studies with pip-eracillin-tazobactam and piperacillin-sulbactam Antimicrob Agents Chemother199741721ndash727

97 M Alexov PD Lister CC Sanders Efficacy of ampicillin-sulbactam is not depen-dent upon maintenance of a critical ratio between components Sulbactam pharma-cokinetics in pharmacodynamic interactions Antimicrob Agents Chemother 1996402468ndash2477

98 K Bush C Macalintal BA Rasmussen VJ Lee Y Yang Kinetic interactions oftazobactam with β-lactamases from all major structural classes Antimicrob AgentsChemother 199337851ndash858

99 KS Thomson DA Weber CC Sanders WE Sanders Jr β-lactamase productionin members of the family Enterobacteriaceae and resistance to β-lactam-enzymeinhibitor combinations Antimicrob Agents Chemother 199034622ndash627

100 DM Livermore Determinants of the activity of β-lactamase inhibitor concentra-tions J Antimicrob Chemother 199331(suppl A)9ndash21

101 RN Jones MN Dudley Microbiologic and pharmacodynamic principles applied


to the antimicrobial susceptibility testing of ampicillinsulbactam Analysis of thecorrelations between in vitro test results and clinical response Diagn MicrobiolInfect Dis 19971595ndash18

102 AB Maderazo DH Gilbert MN Dudley Post-exposure inhibition of TEM-3 β-lactamase by high concentrations of tazobactam In Program and Abstracts of the36th Interscience Conference on Antimicrobial Agents and Chemotherapy SanFrancisco 1996 Am Soc Microbiol Washington DC Abstract A95 p 19

103 CE Thorburn J Molesworth The post β-lactamase inhibitor effect A novel aspectof the activity of clavulanic acid in antibacterial tests In Program Abstracts of32nd Interscience Conference on Antimicrobial Agents and ChemotherapeuticsAbstract 540

104 AH Strayer DH Gilbert P Pivarnik AA Medeiros SH Zinner MN Dudley Phar-macodynamics of piperacillin alone and in combination with tazobactam againstpiperacillin-resistant and -susceptible organisms in an in vitro model of infectionAntimicrob Agents Chemother 1994382351ndash2356

105 R Pallares F Gudiol J Linares J Ariza G Rufi L Murgui J Dorga PF ViladrichRisk factors and response to antibiotic therapy in adults with bacteremic pneumoniacaused by penicillin-resistant pneumococci N Engl J Med 198731718ndash22

106 R Pallares J Linares M Vadillo C Cabellos F Manresa PF Viladrich R MartinF Gudiol Resistance to penicillin and cephalosporin and mortality from severepneumococcal pneumonia in Barcelona Spain N Engl J Med 1995333474ndash480

107 TW Gress KW Yingling RJ Stanek MA Mufson Infection with Streptococcuspneumoniae moderately resistant to penicillin does not alter clinical outcome InfectDis Clin Pract 19965435ndash439

108 C Cabellos J Ariza B Barreiro F Tubau J Linares R Pallares F Manresa FGudiol Current usefulness of procaine penicillin in the treatment of pneumococcalpneumonia Eur J Clin Microbiol Infect Dis 199817265ndash268

109 IR Friedland Comparison of the response to antimicrobial therapy to penicillin-resistant and penicillin-susceptible pneumococcal disease Ped Infect Dis J 199514885ndash890

110 R Dagan O Abramson E Leibovitz R Lang S Goshen D Greenberg P YagupskyA Leiberman DM Fliss Impaired bacteriologic response to oral cephalosporins inacute otitis media caused by pneumococci with intermediate resistance to penicillinPed Infect Dis J 199615980ndash986

111 P Gehanno G Lenoir P Berche In vivo correlates for Streptococcus pneumoniaepenicillin resistance in acute otitis media Antimicrob Agents Chemother 199539271ndash272

112 WA Craig D Andes Pharmacokinetics and pharmacodynamics of antibiotics inotitis media Pediat Infect Dis J 199615255ndash259

113 PK Linden AW Pasculle R Manez DJ Dramer JJ Fung AD Pinna S KusneDifferences in outcomes for patients with bacteremia due to vancomycin-resistantEnterococcus faecium or vancomycin-susceptible E faecium Clin Infect Dis 199622663ndash670

114 Y Carmeli N Troillet AW Karchmer MH Samore Health and economic outcomesof antibiotic resistance in Pseudomonas aeruginosa Arch Intern Med 19991591127ndash1132

6

Aminoglycoside Pharmacodynamics

Myo-Kyoung Kim and David P Nicolau


1 INTRODUCTION

Aminoglycosides are highly potent broad-spectrum antibiotics that have re-mained an important therapeutic option for the treatment of life-threatening in-fections Since the introduction of this class of agents into clinical practice somefive decades ago the major obstacle to their use is the potential for drug-relatedtoxicity However over the last decade new information concerning the phar-macodynamic profile of these agents has been revealed that leads not only tothe potential for improved antibacterial effectiveness but also to the minimiza-tion of their toxicodynamic profile As a result of our contemporary understand-ing of these principles parenteral dosing techniques for the aminoglycosideshave been modified from the administration of frequent small intermittent dos-ages to once-daily regimens that not only optimize the pharmacodynamic andtoxicodynamic profiles but also substantially reduce the expenditure associatedwith this therapeutic option Application of these new principles together withthe aminoglycosidesrsquo in vitro activity proven clinical effectiveness and syner-gistic potential form the rationale for their continued use in the management ofserious infections

125

126 Kim and Nicolau

2 HISTORY AND MECHANISM OF ACTION

OF AMINOGLYCOSIDES

The aminoglycosides include an important group of natural and semisyntheticcompounds The first parenterally administered aminoglycoside streptomycinwas introduced in 1944 and was followed by a number of other naturally oc-curring compounds including neomycin kanamycin tobramycin gentamicinsisomicin and paromomycin Amikacin and netilmicin are semisynthetic deriva-tives of kanamycin and sisomicin respectively whereas isepamicin is a semisyn-thetic derivative of gentamicin

Like that of many antibiotics (ie macrolides tetracyclines streptogram-ins) the bactericidal activity of the aminoglycosides is thought to be ribosomallymediated Existing data suggest that their antibacterial activity results from inhi-bition of protein biosynthesis by irreversible binding of the aminoglycoside tothe bacterial ribosome The intact bacterial ribosome is a 70S particle that consistsof two subunits (50S and 30S) that are assembled from three species of rRNA(5S 16S and 23S) and 52 ribosomal proteins The smaller 30S ribosomal submitwhich contains the 16S rRNA has been identified as a primary target for amino-glycoside that ultimately induces mistranslation on prokaryotic ribosomes [12]

To reach their cytoplasmic ribosomal target aminoglycosides must initiallycross the outer membrane (in gram-negative organisms) and the cytoplasmicmembrane (in gram-negative and gram-positive bacteria) In gram-negative bac-teria the initial step involves ionic binding of the highly positively charged amino-glycosides to negatively charged phosphates mainly in lipopolysaccharides onthe outer membrane surface uptake across this membrane is likely due a lsquolsquoself-promoted uptakersquorsquo mechanism [34] The cationic aminoglycosides may act bycompetitively displacing the divalent cations in the membrane resulting in theentry of the antibiotic [3] The rapid initial binding of the aminoglycosides tothe cell accounts for the rapid bactericidal activity which appears to increase withincreasing aminoglycoside concentration This characteristic of concentration- ordose-dependent killing explains in part the recent attention given to administeringthe entire dose of the aminoglycoside on a once-daily basis in order to maximizebacterial killing

Aminoglycoside uptake across the cytoplasmic membrane is the result ofits electrostatic binding to the polar heads of phospholipids whereas the drivingforce for aminoglycoside entry is provided by a cellular transmembrane electricalpotential The combination of these effects is characterized by rapid binding to theribosome and an acceleration of aminoglycoside uptake across the cytoplasmicmembrane

Aminoglycosides of the gentamicin kanamycin and neomycin familiesinduce misreading of mRNA codons during translation and also inhibit transloca-tion [5] Streptomycin induces misreading of the genetic code in addition to inhib-

Aminoglycosides 127

iting translational initiation By contrast spectinomycin an agent with only bacte-riostatic activity does not cause translation errors but inhibits translocationThese findings support the notion that translational misreading is at least partlyresponsible for the bactericidal activity characteristic of aminoglycosides [5ndash8]

Although the ribosome has been identified as a primary target for theseagents the precise mechanism by which aminoglycosides exert their bactericidalactivity has remained elusive because these drugs manifest pleiotropic effectson bacterial cells These effects include but are not limited to disruption of theouter membrane irreversible uptake of the antibiotic and blockade of initiationof DNA replication [9]

3 MICROBIOLOGIC SPECTRUM

Although the aminoglycosides are highly potent broad-spectrum antibiotics theirin vitro activity is considered to be most notable against a variety of gram-negative pathogens These pathogens include common clinical isolates of Acinet-obacter spp Citrobacter spp Enterobacter spp Escherichia coli Klebsiellaspp Serratia spp Proteus spp Morganella spp and Pseudomonas aeruginosaHowever although these agents are generally considered active against thesemicrobes substantial differences in antimicrobial potency exist among the vari-ous aminoglycosides For example even though the antimicrobial spectra of gen-tamicin and tobramycin are quite similar tobramycin is generally more active invitro against P aeruginosa whereas gentamicin is more active against Serratia

Although streptomycin has been used extensively for many years theemergence of resistance against Mycobacterium tuberculosis and aerobic gram-negative bacilli and the relatively frequent occurrence of vestibular toxicity com-bined with the availability of less toxic antibiotics have greatly diminished itsclinical utility The introduction of kanamycin provided a broader spectrum ofactivity against gram-negative bacilli including streptomycin-resistant strainsbut it was not active against P aeruginosa As with streptomycin extensive useof kanamycin quickly led to the emergence and widespread dissemination ofkanamycin resistance among Enterobacteriaceae The development of otheragents within the class (ie gentamicin tobramycin netilmicin and amikacin)further expanded the spectrum of antimicrobial activity of this class to covermany kanamycin-resistant strains including P aeruginosa

Although the aminoglycosides are also active against Salmonella spp Shi-gella spp N gonorrhea and Haemophilus influenzae they are not recommendedfor infections caused by these species because of the wide availability of effectiveand less toxic drugs Also although they are generally active against Staphylo-cocci aminoglycosides are not advocated as single agents for infections due tothis genera However an aminoglycoside usually gentamicin is frequently ad-ministered in combination with a cell-wall-active agent to provide synergy in the

128 Kim and Nicolau

treatment of serious infections due to Staphylococci Enterococci and Viridansstreptococci

4 PHARMACOKINETIC CHARACTERIZATION

Although the focus of this chapter is related to the pharmacodynamics of amino-glycosides and their application in the management of systemic infection itshould be noted that although these agents are poorly absorbed after oral adminis-tration the prolonged use of large oral doses in patients with altered renal functionmay result in detectable aminoglycoside serum concentrations and the develop-ment of toxicity In addition although they penetrate poorly through intact skintheir use as a topical antibacterial for large areas of denuded skin (ie thermalinjury) may lead to substantial systemic absorption Similarly the use of amino-glycosides for local irrigation of closed body cavities may result in considerablesystemic accumulation and potential toxicity

As a consequence of poor oral bioavailability the aminoglycosides mustbe given parenterally in order to achieve a consistent serum concentration profileThe intramuscular route is well tolerated and results in essentially complete ab-sorption but intravenous administration is generally preferred because of therapid and predictable serum profile The importance of the rapid and reliableattainment of sufficient peak concentrations will be further discussed in subse-quent sections of this chapter

The aminoglycosides are weakly bound to serum proteins and thereforefreely distribute into the interstitial or extracellular fluid The apparent volumeof distribution of this class of agents is approximately 25 of the total bodyweight which corresponds to the estimated extracellular fluid volume Althoughthe volume of distribution is generally approximated at 025ndash03 Lkg patientswho are malnourished obese or pregnant are in the intensive care unit or haveascites may have substantial alterations in this parameter that require dosage andor schedule modifications to maintain the desired serum profile In general theconcentrations of the aminoglycosides attained in tissue and body fluids are lessthan that obtained in serum with the notable exceptions of the kidney perilymphof the inner ear and urine Approximately 20ndash50 of the serum concentrationcan be achieved in bronchial sputum pleural or synovial fluid and unobstructedbile Although low aminoglycoside bronchial fluid concentrations have been re-ported the administration of large single daily doses versus conventional dosingsubstantially improves drug penetration into this fluid while reducing drug accu-mulation in the renal tissue [10ndash12] Compared with other body sites the penetra-tion into prostate tissue and bone is poor Penetration of aminoglycosides intocerebral spinal fluid in the presence of inflammation or into the fluid of the eye isinadequate and variable therefore direct instillation is often required to providesufficient concentrations at these sites Additionally these agents cross the pla-

Aminoglycosides 129

centa therefore the potential risk to the fetus and mother must be consideredprior to use

The kidneys via glomerular filtration are responsible for essentially allaminoglycoside elimination from the body As a result there is a proportionalrelationship between drug clearance and glomerular filtration rate which is rou-tinely used to assist with aminoglycoside dosage modification [13] In adults andchildren older than 6 months with normal renal function the elimination half-life is approximately 2ndash3 h In premature or low birth weight infants less than1 week old the half-life is 8ndash12 h whereas the half-life decreases to 5 h forneonates whose birth weight exceeds 2 kg As a result of the primary renal elimi-nation it should be expected that substantial increases in the half-life would beobserved in patients with renal dysfunction

5 PHARMACODYNAMIC OVERVIEW

Over the last several decades new data emerged that extended our understandingof the complex interactions that take place among the pathogen drug and hostduring the infection process Much of the focus has been on the influence of drugconcentration on bacterial cell death The pharmacodynamic properties or thecorrelation of drug concentration and the clinical effect (eg bacterial killing)of a specific antibiotic class are therefore an integration of two related areas onebeing microbiological activity and the other pharmacokinetics (refer to Chapter2 for a more complete review of this topic) Distinct pharmacodynamic profilesexist for all antimicrobials because the influence of drug concentration on therate and extent of bactericidal activity differs among the various classes of drugs

The pharmacodynamic profile of the aminoglycosides has been character-ized both in vitro and in vivo Utilization of both static (ie time-kill studies)and dynamic (ie pharmacokinetic modeling) in vitro techniques has providedfundamental information concerning the pharmacodynamic profile of the amino-glycosides These data indicate that a general pharmacodynamic division amongantimicrobials occurs between agents whose rate and extent of bactericidal activ-ity is dependent upon drug concentration (aminoglycosides and fluoroquinolones)and agents such as the β-lactams whose bactericidal activity is independent ofdrug concentration when their concentration exceeds four times the minimuminhibitory concentration (MIC) [14ndash17]

Figure 1 depicts these principles as illustrated with ciprofloxacin tobra-mycin and ticarcillin In this experiment bacteria are exposed to various multiplesof the MIC in vitro and as shown with ticarcillin little difference in the rate ofbactericidal activity is noted when its concentration exceeds 4 times the MICTherefore this type of killing which is characteristic of β-lactams is termedconcentration- or dose-independent bactericidal activity (refer to Chapter 5 fora more complete discussion of β-lactam pharmacodynamics)

130 Kim and Nicolau

FIGURE 1 Influence of drug concentration on the bactericidal activity of tobra-mycin ciprofloxacin and ticarcillin against Pseudomonas aeruginosa (FromRef 15)

By contrast when the same multiples of the MIC are studied with tobra-mycin and ciprofloxacin the number of organisms is seen to decrease more rap-idly with each rising MIC interval Because these agents eliminate bacteria morerapidly when their concentrations are appreciably above the MIC of the organismtheir killing activity is referred to as concentration- or dose-dependent bacteri-cidal activity [15] These in vitro data indicate that optimum bactericidal activityfor the aminoglycosides is achieved when the exposure concentration is approxi-mately 8ndash10 times the MIC [14151819] In addition to maximal bactericidalactivity Blaser et al [20] demonstrated that the peakMIC ratio of 81 is corre-lated with a decrease in the selection and regrowth of resistant subpopulationsoccurring during treatment with netilmicin

Antimicrobial activity in vivo is a complex and multifactorial process Asdescribed elsewhere in this text to be effective an antimicrobial must reach andmaintain adequate concentrations at the target site and interact with the targetsite for a period of time so as to interrupt the normal functions of the cell Invivo this description of the interaction between the pathogen and the drug (iemicrobiological activity) is influenced by the drug disposition or pharmacokinetic

Aminoglycosides 131

profile of the host species As a result of the complex interactions occurringamong the pathogenndashdrugndashhost triad in vivo pharmacodynamic characterizationof compounds is required

Because we are not yet able to measure drug concentrations at the site ofaction (ie ribosome for the aminoglycosides) we commonly employ a microbio-logical parameter (ie the MIC) as the critical value in the interpretation of thesein vivo pharmacodynamic relationships When integrating the microbiologicalactivity and pharmacokinetics several parameters appear to be significant constit-uents of drug efficacy The pharmacokinetic parameters AUC (area under theconcentrationndashtime curve) maximum observed concentration (Cmax or peak) andhalf-life are often integrated with the MIC of the pathogen to produce pharmaco-dynamic parameters such as the AUCMIC peakMIC and the time for whichthe drug concentration remains above the MIC (T MIC) For the aminoglyco-sides the AUCMIC peakMIC and T MIC have all been shown to be pharma-codynamic correlates of efficacy [142021] However it is not surprising thatseveral pharmacodynamic parameters have been related to efficacy with theseagents because all of these parameters are related (Fig 2) Therefore since theamount of drug delivered against the pathogen is proportional to the amount ofdrug delivered to the host (AUC) the AUC is the primary pharmacokinetic

FIGURE 2 Antimicrobial pharmacodynamics integration of microbiologicalpotency and selected pharmacokinetic parameters

132 Kim and Nicolau

parameter associated with efficacy However because the AUC is a product ofconcentration and time under certain conditions the influence of concentrationwill appear to be a predominant factor whereas under a different set of conditionsthe exposure to the drug or the time above the MIC may assume a larger rolein bacterial eradication In the case of the aminoglycosides which display concen-tration-dependent killing and a relatively long PAE the influence of the timeabove the MIC is small compared to the influence of peak concentration As aresult the pharmacodynamic parameter that is believed to best characterize theprofile of the aminoglycosides in vivo is the peakMIC ratio

In support of the concentration-dependent bactericidal activity of theaminoglycosides displayed in in vitro studies and animal models of infectionseveral studies in humans have also demonstrated the importance of achievingsufficient peakMIC ratios as related to defined treatment success Aminoglyco-side concentrations have been associated with treatment success in a three studiesreported by Moore and colleagues [22ndash24] In the first report by this group higherCmax concentrations were associated with improved outcomes in gram-negativepneumonia whereas in the second report higher concentrations were associatedwith improved survival in gram-negative bacteremia [2223] Results of thesestudies suggest the importance of achieving adequate and early aminoglycosideconcentrations in severely ill patients with gram-negative infection Their laststudy published in 1987 evaluated the relationship between the ratio of the peakconcentration to MIC and clinical outcome through data collection from fourrandomized double-blind controlled clinical trials that used gentamicin tobra-mycin or amikacin for the treatment of gram-negative bacterial infections [22]For the purposes of the study the maximal peak concentration (Cmax) was definedas the highest concentration determined during therapy and the mean peak con-centration was calculated as the average of all Cmax values during the course oftreatment The investigators demonstrated that ratios of high maximal and meanpeak aminoglycoside concentrations (85 50 microgmL and 66 39 microgmLrespectively) to MIC were significantly (p 000001 and p 00001 respec-tively) correlated with clinical response Of the 188 patients who had a clinicalresponse to therapy the CmaxMIC average value was 85 50 microgmL whereasthe 48 nonresponders had a ratio of 55 46 microgmL (p 000001) Althoughthese studies used fixed dosing intervals and were not designed to assess the invivo pharmacodynamic parameters and their relationship to outcome the dataprovide the backbone for our commitment to the value of the peakMIC ratio inclinical practice Deziel-Evans et al [25] demonstrated that a 91 cure rate wasobserved in patients with peakMIC ratios greater than 8 whereas only a 125cure rate was observed for patients with ratios of 4 in a retrospective studywith 45 patients In a study by Keating et al [26] response rates of 57 67and 85 were observed in neutropenic patients with mean serum aminoglycosideconcentrationMIC ratios of 1ndash4 4ndash10 and 10 respectively Williams et al

Aminoglycosides 133

[27] have also reported that CmaxMIC ratios correlated significantly with cure in42 patients undergoing amikacin treatment who could be evaluated for clinicaloutcome [27] Additionally Fiala and Chatterjee [28] noted that infection wascured more frequently in patients with severe gram-negative infections whoachieved higher peakMIC ratios The association of high peakMIC ratios withimproved outcomes has also been noted in orthopedic patients receiving genta-micin and recently a similar relationship was noted for 61 febrile neutropenicpatients with hematological malignancies [2930] Other investigators have alsoobserved beneficial correlations between serum concentrations or pharmacody-namic parameters and therapeutic outcomes in patients treated with aminoglyco-sides [31ndash33]

More recently Kashuba et al [3435] reported that achieving an aminogly-coside peakMIC of 10 within 48 h of initiation of therapy for gram-negativepneumonia resulted in a 90 probability of therapeutic response by day 7 oftherapy They also note that aggressive aminoglycoside dosing (initial dose of 7mgkg) followed by individualized pharmacokinetic monitoring should maximizethe rate and extent of response in this patient population

Additionally many once-daily aminoglycoside trials and their meta-analy-sis studies (which will be described in a later section in this chapter) also provedthe importance of the correlation between the peakMIC ratio and clinical out-come

6 POSTANTIBIOTIC EFFECT

The postantibiotic effect (PAE) is defined as the persisting suppressive activityagainst bacterial growth after limited exposure of bacteria to an antibiotic Inorder to measure the in vitro PAE of aminoglycosides after a 1ndash2 h exposureof an antibiotic or a combination antibiotics to bacteria the drug is removed rap-idly by dilution drug inactivation [eg cellulose phosphate powder or tobra-mycin-acetylating enzyme AAC (3)-II and acetyl coenzyme A] or filtration(045 microm pore size filters) After this drug elimination process viable counts(CFUmL) at each time point are required to develop viability curves The invitro PAE is calculated with the equation PAE T C where T is the timerequired for the count of CFUs in the test culture to increase by 1 log10 abovethe count observed immediately after drug removal and C is the time neededfor the count of CFUs in an untreated control culture to increase by 1 log10

above the count observed immediately after the same procedure used on the testculture for drug removal [36]

Like quinolones aminoglycosides represent a antibiotic class that has aclinically meaningful PAE however several factors may affect the presence andduration of the PAE A range of 05ndash75 h has been reported for the PAE ofaminoglycosides [3637] Major factors influencing PAE include organism con-

134 Kim and Nicolau

centration of antibiotic duration of antimicrobial exposure and antimicrobialcombinations Minor factors include size of the inoculum growth phase of theorganism at the time of exposure mechanical shaking of the culture type ofmedium pH and temperature of the medium and the effect of re-exposure [36]

The examples below illustrate how the above factors affect the presenceand duration of the PAE Unlike β-lactam antibiotics with PAEs against onlygram-positive organisms aminoglycosides exhibit a PAE on both gram-positiveand gram-negative organisms [3738] However the PAE duration depends onthe type of bacterium For example the duration of the PAE following exposureof Pseudomonas aeruginosa to gentamicin and tobramycin is 22 h and 21 hrespectively whereas that of Escherichia coli is 18 h and 12 h respectively[36] Additionally it has been shown that there is a positive correlation betweensubsequent dose or concentration of aminoglycosides and duration of the PAE[39ndash42] The maximum concentration to exert the maximal PAE effect of amino-glycosides is difficult to determine because most bacteria are completely andrapidly killed at high drug concentrations In contrast the PAE of penicillin Ggradually increases up to a point of maximal effect at a concentration 8ndash16 timesthe MIC [42ndash44] The same theory also applies to duration of exposure

The duration of PAE also varies depending on concurrently applied antibi-otics when they are tested as a combination therapy The combined effect ofaminoglycosides and cell wall inhibitors on the duration of the PAE was studiedby several researchers [45ndash48] In general these combinations produced additiveeffects (ie similar to the sum of PAEs for individual drugs) or synergistic effects(ie at least 1 h longer than the sum of PAEs for individual drugs) in Staphylococ-cus aureus and various streptococci The effects of antibiotic combinationsagainst gram-negative bacilli were mainly additive or indifferent (ie no differentfrom the longest of the individual PAEs) As an exception the addition of tobra-mycin to rifampin which can achieve prolonged PAEs in gram-negative bacillishowed synergism of the PAE in P aeruginosa E coli and K pneumonia

However there are unavoidable limitations in this in vitro determinationof the PAE duration One major drawback is that bacteria undergo a single expo-sure for a short period of time to a fixed concentration of a testing antimicrobialagent However in a clinical setting the antimicrobial agent should be used multi-ple times In addition it should maintain the concentration above the MIC for arelatively longer time period than that which occurs during PAE testing andthe concentration should decline continuously throughout the dosing intervalKarlowsky et al [4950] demonstrated that multiple exposures of E coli and Paeruginosa to aminoglycosides significantly decreased the duration of PAE alongwith attenuating bacterial killing activity McGrath et al [51] suggested that thereasons for this phenomenon may be adaptive resistance or the selection of drug-resistant variants Li et al [52] demonstrated that P aeruginosa organism ex-posed to constant tobramycin concentrations have a longer PAE than those ex-posed to exponentially decreasing tobramycin concentrations at similar AUCs

Aminoglycosides 135

above the MIC These studies suggest that conventional testing yields an overesti-mate of the PAE in comparison to the PAE presented in a clinical situation withcontinuously changing concentrations

Furthermore the duration of PAE significantly varies depending on thetesting environment important factors influencing the testing environment in-clude inoculum concentration temperature pH oxygen tension and free cation(Ca2 Mg2) content [53ndash57] The other obstacle to applying this in vitro PAEduration to clinical practice is that the PAE does not consider host immunityHowever some effort has been undertaken to include host immunity by usingother terminology such as postantibiotic leukocyte enhancement (PALE) andpostantibiotic sub-MIC effect PALE describes the phenomenon that pathogensin the PAE phase are more susceptible to the antimicrobial effect of human leuko-cytes than non-PAE controls The postantibiotic sub-MIC effect illustrates theadditive effects of PAE and the bactericidal effects when the drug is still presentonly in sub-MIC levels

Despite some of the limitations involved in predicting the exact durationof PAE the general consensus is that PAE is an important factor to be consideredin the development of a drug regimen The precise mechanisms of the PAE arelargely unknown However several hypotheses have been suggested They in-clude limited persistence of antibiotic at the site of action recovery from nonle-thal damage to cell structures and the time required for synthesis of new proteinsor enzymes before growth Drug-induced nonlethal damage due to the irreversiblebinding to bacterial ribosomes represents a feasible mechanism of the PAE ofaminoglycoside [4259] In a study measuring the rate of [3H]-adenosine incorpo-ration Gottfredsson et al [60] showed that DNA synthesis by P aeruginosa afterexposure to tobramycin was markedly affected during the PAE phase Howeverin a study that used cumulative radiolabeled nucleoside precursor uptake in aclinical strain of E coli Barmada et al [61] showed that DNA and RNA synthesisresumed almost immediately after exposure to tobramycin whereas protein syn-thesis did not recover until 4 h later Therefore the duration of PAE producedby aminoglycosides against E coli seems to be better correlated with inhibitionof protein synthesis than with inhibition of DNA or RNA synthesis [61] Evenif the rationale for this difference is unknown it may be due to a discrepancy inthe mechanisms of action of two species Theoretically provided that a certainthreshold of growth suppression to restrain DNA synthesis is attained greateraccumulation or entrapment of intracellular tobramycin in P aeruginosa mayaccount for this disagreement [6061]

Although numerous data are available on the in vitro PAE there is less invivo information Six animal models have been developed to evaluate the in vivoPAE thigh infection in mice pneumonia in mice infected subcutaneous threadsin mice meningitis in rabbits infected tissue cages in rabbits and endocarditisin rats [36] Among these models the mice thigh infection model is commonlyused to evaluate the PAE of aminoglycosides although the pneumonia model is

136 Kim and Nicolau

also adopted for them [62ndash65] The endocarditis rat model has been used toevaluate the in vivo PAEs of aminoglycosides when they are added to penicillinor imipenem [4066] In these models antibiotic is administered to achieve aconcentration that exceeds the MIC during the first 1ndash2 h Next bacterial loadsfrom tissue are counted at various time points and drug concentrations of plasmaare measured simultaneously After graphing the bacterial growth curve in vivoPAE can be calculated by the equation

PAE T C M

where M is the length of time serum concentration exceeds the MIC T is thetime required for the counts of CFU in tissue to increase by 1 log10 above thecount at the time closest to but not less than time M and C is the time requiredfor the counts of CFU in tissue of untreated control to increase by 1 log10 abovethe count at time zero [36]

Major factors affecting the in vivo PAE include the infection site typeof organism type of antimicrobial agent the drug dose simulation of humanpharmacokinetics and the presence of leukocytes [3637] For example the invivo PAEs for 15 clinical isolates of Enterobacteriaceae following administrationof gentamicin (8 mgkg) ranged from 14 to 73 h [67] Like the in vitro PAEhigher doses of drugs are correlated with longer in vivo PAEs [36] The PAEsof single doses of 4 12 and 20 mgkg tobramycin in the thighs of neutropenicmice infected with P aeruginosa were 22 h 48 h and 73 h respectively Gener-ally the combinations of aminoglycoside and β-lactam lengthened the PAE forS aureus and P aeruginosa by 10ndash33 h compared to the longest PAE of theindividual drugs However no difference was observed against E coli and Kpneumoniae [65] The adoption of a different infection model also may influencethe duration of the in vivo PAE PAEs with amikacin against Klebsiella pneumon-iae in the mouse pneumonia model was roughly 15ndash25 times as long as thatobserved in the mouse thigh model at the corresponding dose [65] Furthermoreother environmental conditions also influence the in vivo PAE [67]

The in vivo PAE can be used to incorporate the effect of host immunityin conjunction with the PAE The duration of the in vivo PAE of aminoglycosideswas prolonged 19ndash27-fold by the presence of leukocytes [67] In addition neu-trophils are also proven to prolong the in vivo PAEs for aminoglycosides againsta standard strain of K pneumoniae [68] The other benefit of the in vivo PAEis that the half-life of some antimicrobials can be prolonged to simulate humanpharmacokinetics by inducing transient renal impairment in mice with uranylnitrite The in vivo PAE in the renally impaired mice was approximately 7 hlonger than that observed in normal mice with large doses inducing a similareffect This difference is likely due to sub-MIC levels that persist for a longertime with renal impairment than with normal renal function [36]

Aminoglycosides 137

In spite of the lack of an ideal method to apply the PAE to clinical practicethe PAE has a major impact on antimicrobial dosing regimens For antibioticswith longer PAEs dosing may be less frequent than that of antibiotics with shorterPAEs Therefore PAE may be one of the rationales for the implementation ofonce-daily aminoglycoside dosing

7 RESISTANCE AND SYNERGY

Although this chapter primarily concerns the pharmacodynamic profile of theaminoglycosides and its implications for clinical practice it is important to realizethat the development of antimicrobial resistance is often the rate-limiting stepfor a compoundrsquos clinical utility Not unlike other antimicrobials the aminogly-cosides face similar issues regarding resistance Although this topic is beyondthe scope of this chapter it should be noted that at least three mechanisms conferresistance to the aminoglycosides impaired drug uptake mutations of the ribo-some and enzymatic modification of the drug Intrinsic resistance is often dueto impaired uptake whereas acquired resistance usually results from acquisitionof transposon- and plasmid-encoded modifying enzymes [69] To this end thepharmacodynamic implications regarding resistance are that one should select aregimen that maximizes the rate and extent of killing If this approach is univer-sally endorsed it will likely minimize the development of resistance in vivobecause the pharmacodynamic optimization of aminoglycosides has been shownto have this effect in vitro [2070] Additionally adaptive resistance and refracto-riness to aminoglycosides has been demonstrated in vitro and in a neutropenicmurine model by exposing Pseudomonas aeruginosa to concentrations below orat the MIC of the organism [71ndash73] Exposure to an aminoglycoside without adrug-free period leads to decreased bacterial killing Therefore longer dosingintervals which can be achieved with the pharmacodynamically based once-dailyaminoglycoside dosing approach allow for a drug-free period in which the bacte-ria are not exposed to an aminoglycoside yet they still preserve the antibacterialactivity of these agents after multiple doses

Aminoglycosides exhibit synergistic bactericidal activity when given incombination with cell-wall-active agents such as β-lactams and vancomycin[7475] For example enterococcal endocarditis should be treated with a combi-nation of an aminoglycoside plus a penicillin or vancomycin because by itselfneither agent is sufficiently bactericidal However when combination therapy isadvocated to achieve synergy for gram-negative organisms maximally effectivedoses of both agents should be maintained because synergy does not occur uni-versally for all pathogens to all β-lactamndashaminoglycoside combinations [7476]It should also be noted that combination exposure may also prolong the in vitroand in vivo PAE observed with the aminoglycosides (see Sect 6) although theclinical relevance of this effect is not fully understood

138 Kim and Nicolau

8 TOXICODYNAMICS

Since the introduction of aminoglycosides into clinical practice a variety of ad-verse events have been reported during aminoglycoside therapy however most(eg gastrointestinal) are mild and resolve when the drug is discontinued Theaminoglycosides rarely produce hypersensitivity reactions and despite direct in-jection into the central nervous system and the eye local adverse events (ieseizures hypersensitivity reactions) are generally not observed Although infre-quent in contemporary clinical practice the aminoglycosides have the potentialto cause or exacerbate neuromuscular blockade Despite the concern for increasedrisk with the administration of the high doses routinely used in once-daily dosingprotocols this adverse event has not been observed [7778] However althoughthey are generally well tolerated the major obstacle that has curtailed the use ofaminoglycosides is the potential for ototoxicity and nephrotoxicity

Although ototoxicity has long been recognized as a potential complicationof aminoglycoside therapy questions still remain regarding the full delineationof risk factors and a universally accepted definition As a result of discrepanciesin both the definition of ototoxicity and the sensitivity of testing the reportedincidence of ototoxicity has spanned a wide range (2ndash25) Two distinct formsof ototoxicitymdashauditory and vestibularmdashhave been reported and may occuralone or simultaneously The precise mechanism of injury remains elusive how-ever ototoxicity is believed to result from the destruction of the sensory haircells in the cochlea and the vestibular labyrinth [79]

Auditory toxicity often occurs at frequencies that are higher than that re-quired for conversation and thus patient complaints that usually manifest as tinni-tus or a feeling of fullness in the ear are usually voiced after considerable auditorydamage has already been done [80] Like the progression of auditory loss theinitial symptoms of vestibular toxicity often go unrecognized due to the nonspe-cific nature of its initial presentation (ie nausea vomiting cold sweats nystag-mus vertigo and dizziness) [81] Although considered to be less frequent thanauditory toxicity these vestibular effects are by and large irreversible and there-fore may have a profound impact on the daily function status Owing to the lackof well controlled comparative trials with sufficient power to detect differences inototoxicity among aminoglycosides and the generally poor risk factors analysis itis difficult if not impossible to substantiate that a particular agent may preferen-tially result in one form of ototoxicity rather than another

Although serum concentration data may be useful to ensure an adequatepharmacodynamic profile these data cannot accurately predict the developmentof ototoxicity Recently acquired data suggest that toxicity is related to drug accu-mulation within the ear not peak concentrations which supports the concept ofsaturable transport and reinforces the belief that higher peak concentrationsshould not result in increased ototoxicity [82] For these reasons the once-daily

Aminoglycosides 139

administration techniques may minimize drug accumulation and therefore drug-related toxicity [8384]

Although nephrotoxicity has been reported in more than half of patientsreceiving aminoglycoside therapy the broad range of definitions and the poor riskfactor assessment of the affected patient population often make the true incidencedifficult if not impossible to determine Considered by many to be a noteworthyevent toxicity is nevertheless generally mild and reversible and few patientshave progressive toxicity severe enough to warrant dialysis [85] At present it isthought that this toxicity is due to aminoglycoside accumulation in the lysosomesof the renal proximal tubule cells which results in necrosis of the tubular cellsand the clinical presentation of acute tubular necrosis manifested by nonoliguricrenal failure within a week [86]

Several investigators have reported that advanced age pre-existing renaldysfunction hypovolemia shock liver dysfunction obesity duration of therapyuse of concurrent nephrotoxic agents and elevated peaktrough aminoglycosideconcentrations are risk factors for development of nephrotoxicity [87ndash90] Addi-tionally in the last-cited study [90] multiple logistic regression analysis alsorevealed that trough concentration duration of therapy advanced age leukemiamale gender decreased albumin ascites and concurrent clindamycin vancomy-cin piperacillin or cephalosporins were independent risk factors for nephrotoxic-ity Similar risk factors were identified in patients receiving once-daily aminogly-cosides [91]

Similar to that previously described in the inner ear a saturable aminogly-coside transport system has been used to describe the uptake of drug in the kidneyTherefore less frequent single daily dose administration may minimize accumu-lation and nephrotoxicity [1092] In this regard once-daily regimens have beenreported to lessen the incidence of nephrotoxicity [788493]

Although the risk of these toxicities cannot be completely eliminated rec-ognition of risk factors and the implementation of regimens that minimize drugaccumulation will lead to optimal therapeutic outcomes and minimize toxicity

9 CLINICAL USE AND APPLICATION

OF PHARMACODYNAMICS

The parenteral aminoglycosides particularly gentamicin tobramycin and ami-kacin have long been used empirically for treatment of the febrile neutropenicpatient or of patients with serious nosocomial infection Although aminoglyco-side utilization has generally been declining owing to the introduction of paren-teral fluoroquinolones emergence of fluoroquinolone-resistant P aeruginosa willlikely result in resurgence in clinical use of the aminoglycosides To this pointit is also apparent that the antipseudomonal β-lactams should not be given aloneto treat systemic pseudomonal infections because this organism often develops

140 Kim and Nicolau

resistance under therapy Thus the aminoglycosides play an important role incombination therapy for gram-negative infections

As discussed earlier the aminoglycosides are also commonly used with acell-wall-active agent for synergistic purposes for gram-positive infections Inthis situation gentamicin is frequently administered to provide synergy in thetreatment of serious infections due to Staphylococci Enterococci and Viridansstreptococci

In the current era of aminoglycoside utilization two predominant intrave-nous administration techniques are employed in clinical practice The older ofthe two approaches is the administration of multiple doses usually 17ndash2 mgkg every 8 h for gentamicin and tobramycin whereas amikacin was frequentlydosed using regimens of 5 mgkg every 8 h or 75 mgkg every 12 h (Fig 3)Using this technique maintenance of concentrations within the therapeutic rangefor patients with alterations in elimination or volume of distribution was achievedwith the use of a nomogram or by individualized pharmacokinetic dosing meth-ods based on the patient-specific aminoglycoside disposition Of the nomogram-based methods the scheme of Sarubbi and Hull [94] appears to have gained thewidest acceptance By this method a loading dose of 1ndash2 mgkg gentamicin ortobramycin and of 5ndash75 mgkg amikacin based on ideal body weight was givento adults with renal impairment After the loading dose subsequent doses were

FIGURE 3 Concentrationndashtime profile comparison of () conventional q8h in-termittent dosing versus () the once-daily daily administration technique

Aminoglycosides 141

selected as a percentage of the chosen loading dose according to the desireddosing interval and the estimated creatinine clearance of the patient (Table 1)Alternatively one-half the loading dose may be given at intervals equal to thatof the estimated half-life Although the nomogram approach has been utilizedfrequently the preferred method of dosage adjustment is to individualize theregimen by using the standard pharmacokinetic dosing principles when aminogly-coside concentrations are available [9596]

The second method has been referred to as the once-daily single-daily orextended interval dosing method (Fig 3) Although the potential benefits of thissecond method were not well described until the early 1990s this administrationtechnique has now become the standard of practice in the United States threeof every four hospitals surveyed in 1998 used it [9798] For this reason and thewide availability of tertiary text references concerning the dosing of aminoglyco-sides using the more frequent intermittent approach the remainder of this sectionwill focus on the once-daily dosing methodology

When considering the pharmacodynamic profile of aminoglycosides as de-scribed earlier in this chapter four distinct advantages of using extended dosing

TABLE 1 Selection of Aminoglycoside MaintenanceDosing Using the Method of Sarubbi and Hull

Dose intervalCreatinine clearance Half-life(mLmin) (hs) 8 h 12 h 24 h

90 31 84 mdash mdash80 34 80 91 mdash70 39 76 88 mdash60 45 71 84 mdash50 53 65 79 mdash40 65 57 72 9230 84 48 63 8625 99 43 57 8120 119 37 50 7517 136 33 46 7015 151 31 42 6712 179 27 37 6110 204 24 34 56

7 259 19 28 475 315 16 23 412 468 11 16 300 693 8 11 21

Source Ref 94

142 Kim and Nicolau

intervals are readily apparent [99] As stated previously giving aminoglycosidesas a single daily dose as opposed to conventional strategies provides the opportu-nity to maximize the peak concentrationMIC ratio and the resultant bactericidalactivity (Fig 3) Second this administration technique should minimize drugaccumulation within the inner ear and kidney and therefore minimize the potentialfor toxic effects to these organs Third the PAE may also allow for longer periodsof bacterial suppression during the dosing interval Finally this aminoglycosidedosing approach may prevent the development of bacterial resistance

Once-daily aminoglycoside therapy has been evaluated in several largeclinical studies with a total study population of 100 or more patients [100ndash109]Compared with multidose aminoglycoside regimens the once-daily regimen wasshown to be as efficacious as or superior to traditional dosing for the treatmentof a wide variety of infections Toxicity evaluations showed that there were nodifferences between the two dosing methods for either nephrotoxicity or ototoxi-city These toxicity data have been supported by other recent observations frominvestigators in Detroit [8493] In addition clinical experience at our own institu-tion in a large patient population who received 7 mgkg of either gentamicin ortobramycin indicates a reduced potential for nephrotoxicity [78]

Studies have also been conducted in pediatrics [110111] and pregnant pop-ulations [112] for determination of serum concentrations as well as clinical effi-cacy Several recently published meta-analyses evaluating once-daily dosing withstandard dosing regimens also demonstrate that increased bacterial killing andtrends for decreased toxicity are actually borne out in clinical practice when theextended interval dosing is used [113ndash121]

At present the strategy for once-daily dosing has not been consistent inthe literature as doses for gentamicin tobramycin and netilmicin have rangedfrom 3 to 7 mgkg whereas the usual amikacin dose is 15ndash20 mgkg Dosingregimens that use doses of less than 6 mgkg for gentamicin tobramycin andnetilmicin have arrived at the dose by converting the conventional mgkg doseto a dose that is then administered once daily At present there appear to be fourcommonly advocated methods for the once-daily administration of aminoglyco-sides Although these approaches differ somewhat with regard to dose andorinterval all reflect the need for dosage modification in the patient with renaldisease As of yet no method has been shown to be superior to any of the othersConcerns about the extended intervals and possible risk of increased toxicity inpatients with reduced drug clearance should be mentioned but they should beno greater than those encountered with conventional dosing based on our currentunderstanding of aminoglycoside-induced toxicity

The first method of once-daily dosage determination was proposed andimplemented on the basis of the pharmacokinetic and pharmacodynamic profilesof these agents This method which was developed at our institution is intendedto optimize the peakMIC ratio in the majority of clinical situations by adminis-

Aminoglycosides 143

tering a dose of 7 mgkg of either gentamicin or tobramycin [78] Like conven-tional regimens once-daily protocols require modification for patients with renaldysfunction in order to minimize drug accumulation In the Hartford Hospitalprogram this is accomplished by administering a fixed dose with dosing intervaladjustments for patients with impaired renal function [78] Due to the high peakconcentrations obtained and the drug-free period at the end of the dosing intervalit is no longer necessary to draw standard peak and trough samples rather asingle random blood sample is obtained between 6 and 14 h after the start of theaminoglycoside infusion This serum concentration is used to determine the dos-ing interval based on a nomogram for once-daily dosing (Fig 4) Although Dem-czar et al [122] suggested that the nomogram may be inappropriate for the moni-toring of therapy based on their assessment of aminoglycoside distribution in11 healthy subjects a subsequent population pharmacokinetic analysis using dataderived from more than 300 patients receiving 7 mgkg of tobramycin furthersupports the clinical utility of the original nomogram [123]

As a result of low toxicity the short duration of therapy and the excellentrenal function of most patients criteria have been developed to withhold theinitial random concentration (which is obtained after the first or second dose) inpatients (1) receiving 24 h dosing (2) without concurrently administered nephro-toxic agents (eg amphotericin cyclosporine vancomycin) (3) without exposure

FIGURE 4 Once-daily aminoglycoside nomogram for the assessment of dos-ing interval using a 7 mgkg dose of gentamicin or tobramycin (From Ref 78)

144 Kim and Nicolau

to contrast media (4) neither quadriplegic nor amputee (5) not in the intensivecare unit and (6) less than 60 years of age [78] Even though the initial randomconcentration may be withheld in eligible patients monitoring of the serum creat-inine should continue to occur at 2ndash3 day intervals throughout the course oftherapy For patients who continue on the once-daily regimen for 5 or more daysa random concentration is obtained on the fifth day and weekly thereafter Eventhough an initial random concentration may no longer be necessary in many pa-tients for those experiencing rapidly changing creatinine clearances or those inwhom the creatinine clearance is significantly reduced (ie 30 mLmin) it maybe necessary to obtain several samples to adequately structure the administrationschedule to maximize efficacy and minimize toxicity The 7 mgkg dosage regi-men has also been advocated by other investigators to rapidly obtain sufficientaminoglycoside exposures [34124]

The second method proposed by Gilbert uses a 5 mgkg gentamicin ortobramycin dose in patients without renal dysfunction [99125] If dosage adjust-ment is required to compensate for diminished renal function the dose andordosing interval may be modified to optimize therapy and minimize drug accumu-lation (Table 2) A similar scheme for dosage modification was advocated by

TABLE 2 Suggested Once-Daily Dosage Requirements for Patients withAltered Renal Function

Creatinine clearance Dosage DoseAminoglycoside (mlmin) interval (h) (mgkg)

Gentamicintobramycin 80 24 5070 24 4060 24 4050 24 3540 24 2530 24 2520 48 4010 48 30

Hemodialysisa 48 20Amikacin 80 24 15

70 24 1250 24 7530 24 4020 48 7510 48 40

Hemodialysisa 48 50

a Administered post-hemodialysisSource Adapted from Ref 125

Aminoglycosides 145

Prin et al [126] for patients with renal dysfunction Finally Begg et al [127]suggested two methods to optimize once-daily dosing The first suggested forpatients with normal renal function uses a graphical approach with target AUCvalues The second method for patients with renal dysfunction uses two amino-glycoside serum concentrations and a target AUC value based on the 24 h AUCthat would result with multiple-dose regimens for dosage modifications

Although all the above-noted once-daily methodologies have used fixeddoses subsequent dosage adjustments may be guided by individualized pharma-cokinetic methods similar to that used for the conventional multiple-dose ap-proach On the other hand although individualization of therapy can be accom-plished no data are available to support the assumption that these manipulationswill improve outcomes or minimize toxicity further than the fixed dose methodol-ogies

At the time of once-daily implementation the methodology was introducedinto clinical practice to further optimize the clinical outcomes of patients receiv-ing these agents for serious infections However in addition to meeting this goaland reducing the incidence of drug-induced adverse events this approach hasalso substantially reduced expenditures associated with the initiation of aminogly-coside therapy compared to traditional dosing techniques [128ndash130]

10 SUMMARY

The pharmacodynamic profile of aminoglycosides is maximized when high doseextended interval aminoglycoside therapy is employed The use of this aminogly-coside administration technique has considerable in vitro and in vivo scientificsupport which justifies its wide-scale use within this country The implementa-tion of such programs should maximize the probability of clinical cure and mini-mize toxicity and may help to avoid the development of resistance Althoughsuch dosing is not appropriate for all patients this strategy appears to be useful inthe majority of patients requiring aminoglycoside therapy and can be successfullyemployed as a hospital-wide program

REFERENCES

1 Cundliffe E Antibiotics and prokaryotic ribosomes Action interaction and resis-tance In WE Hill A Dahlberg RA Garrett PB Moore D Schlessinger JR Warnereds Ribosomes Am Soc Microbiol Washington DC 1990479ndash490

2 Davies JE Resistance to aminoglycosides Mechanisms and frequency Rev InfectDis 19835S261ndashS266

3 Hancock REW Aminoglycoside uptake and mode of action With special referenceto streptomycin and gentamicin J Antimicrob Chemother 19818249ndash276

4 Hancock REW Farmer SW Li Z Poole K Interaction of aminoglycosides with

146 Kim and Nicolau

the outer membranes and purified lipopolysaccharide and OmpF porin of Esche-richia coli Antimicrob Agents Chemother 1991351309ndash1314

5 Gale EF Cundliffe E Reynolds PE Richmond MH Waring MJ The MolecularBasis of Antibiotic Action Wiley London 1981

6 Davis BD Tai PC Wallace BJ Complex interactions of antibiotics with the ribo-some In M Nomura A Tissieres P Lengyel eds Ribosomes Cold Spring HarborLaboratory Cold Spring Harbor NY 1974771ndash789

7 Gorini L Streptomycin and misreading of the genetic code In M Nomura A Tis-sieres P Lengyel eds Ribosomes Cold Spring Harbor Laboratory Cold SpringHarbor NY 1974791ndash803

8 Ruusala T Kurland CG Streptomycin preferentially perturbs ribosomal proofread-ing Mol Gen 1984198100ndash104

9 Matsunaga K Yamaki H Nishimura T Tanaka N Inhibition of DNA replicationinitiation by aminoglycoside antibiotics Antimicrob Agents Chemother 198630468ndash474

10 De Broe ME Verbist L Verpooten GA Influence of dosage schedule on renalcortical accumulation of amikacin and tobramycin in man J Antimicrob Chemother199127(suppl C)41ndash47

11 Santre C Georges H Jacquier JM Leroy O Beuscart C Buguin D Beaucaire GAmikacin levels in bronchial secretions of 10 pneumonia patients with respiratorysupport treated once daily versus twice daily Antimicrob Agents Chemother 199539264ndash267

12 Valcke YJ Vogelaers DP Colardyn FA Pauwels RA Penetration of netilmicinin the lower respiratory tract after once-daily dosing Chest 19921011028ndash1032

13 Zarowitz BJ Robert S Peterson EL Prediction of glomerular filtration rate usingaminoglycoside clearance in critically ill medical patients Ann Pharmacother 1992261205ndash1210

14 Begg EJ Peddie BA Chambers ST Boswell DR Comparison of gentamicin dosingregimens using an in-vitro model J Antimicrob Chemother 199229427ndash433

15 Craig WA Ebert SC Killing and regrowth of bacteria in vitro A review ScandJ Infect Dis 1991suppl 7463ndash70

16 Drusano GL Johnson DE Rosen M Standiford HC Pharmacodynamics of a flu-oroquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sep-sis Antimicrob Agents Chemother 199337483ndash490

17 Dudley MN Pharmacodynamics and pharmacokinetics of antibiotics with specialreference to the fluoroquinolones Am J Med 199191(suppl 6A)45Sndash50S

18 Davis BD Mechanism of the bactericidal action of the aminoglycosides MicrobiolRev 198751341ndash350

19 Ebert SC Craig WA Pharmacodynamic properties of antibiotic Application todrug monitoring and dosage regimen design Infect Control Hosp Epidemiol 199011319ndash326

20 Blaser J Stone B Groner M et al Comparative study with enoxacin and netilmicinin a pharmacodynamic model to determine importance of ratio of antibiotic peakconcentration to MIC for bactericidal activity and emergence of resistance Antimi-crob Agents Chemother 1987311054ndash1060

21 Leggett JE Ebert S Fantin B Craig WA Comparative dose-effect relationships

Aminoglycosides 147

at several dosing intervals for β-lactam aminoglycoside and quinolone antibioticsagainst gram-negative bacilli in murine thigh-infection and pneumonitis modelsScan J Infect Dis 199174179ndash184

22 Moore RD Smith CR Lietman PS Association of aminoglycoside plasma levelswith therapeutic outcome in gram-negative pneumonia Am J Med 198477657ndash662

23 Moore RD Smith CR Lietman PS The association of aminoglycoside plasma lev-els with mortality in patients with gram-negative bacteremia J Infect Dis 1984149443ndash448

24 Moore RD Lietman PS Smith CR Clinical response to aminoglycoside therapyImportance of the ratio of peak concentration to minimal inhibitory concentrationJ Infect Dis 198715593ndash99

25 Deziel-Evans L Murphy J Job M Correlation of pharmacokinetic indices withtherapeutic outcomes in patients receiving aminoglycoside Clin Pharm 19865319ndash324

26 Keating MF Bodey GP Valdivieso M Rodriguez V A randomized comparativetrial of three aminoglycosides Comparison of continuous infusions of gentamicinamikacin and sisomicin combined with carbenicillin in the treatment of infectionsin neutropenic patients with malignancies Medicine 197958159ndash170

27 Williams PJ Hull JH Sarubbi FA Rogers JF Wargin WA Factors associatedwith nephrotoxicity and clinical outcome in patients receiving amikacin J ClinPharmacol 19862679ndash86

28 Fiala M Chatterjee SN Antibiotic blood concentrations in patients successfullytreated with tobramycin Postgrad Med 198157548ndash551

29 Berirtzoglou E Golegou S Savvaidis I Bezirtzoglou C Beris A Xenakis T Arelationship between serum gentamicin concentrations and minimal inhibitor con-centration Drugs Exp Clin Res 19962257ndash60

30 Binder L Schiel X Binder C Menke CF Schuttrumpf S Armstrong VW UnterhaltM Erichsen N Hiddemann W Oellerich M Clinical outcome and economic impactof aminoglycoside peak concentrations in febrile immuocompromised patients withhematologic malignancies Clin Chem 199844408ndash414

31 Anderson ET Young LS Hewitt WL Simultaneous antibiotic levels in lsquolsquobreak-throughrsquorsquo gram-negative rod bacteremia Am J Med 197661493ndash497

32 Noone P Parsons TMC Pattison JR Slack RCB Garfield-Davies D Hughes KExperience in monitoring gentamicin therapy during treatment of serious gram-negative sepsis Br Med J 19741477-481

33 Reymann MT Bradac JA Cobbs CG Dismukes WE Correlation of aminoglyco-side dosage with serum concentrations during therapy of serious gram-negativebacillary disease Antimicrob Agents Chemother 197916353ndash361

34 Kashuba ADM Bertino JS Jr Nafziger AN Dosing of aminoglycosides to rapidlyattain pharmacodynamic goals and hasten therapeutic response by using individual-ized pharmacokinetic monitoring of patients with pneumonia caused by gram-negative organism Antimicrob Agents Chemother 1998421842ndash1844

35 Kashuba ADM Nafziger AN Drusano GL Bertino JS Jr Optimizing aminoglyco-side therapy for nosocomial pneumonia caused by gram-negative bacteria Antimi-crob Agents Chemother 1999431623ndash1629

148 Kim and Nicolau

36 Craig W Gunmundsson S Postantibiotic effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams amp Wilkins Baltimore 1987296ndash329

37 Zhanel G Hoban D Harding G The postantibiotic effect a review of in vitro andin vivo data Ann Pharmacother 199125153ndash163

38 Bundtzen R Gerber A Cohn D et al Postantibiotic suppression of bacterialgrowth Rev Infect Dis 1981328ndash37


40 Hessen M Pitsakis P Levison M Postantibiotic effect of penicillin plus gentamicinversus Enterococcus faecalis in vitro and in vivo Antimicrob Agents Chemother198933608ndash611

41 McGrath B Marchbanks C Gilbert D et al In vitro postantibiotic effect followingexposure to imipenem temafloxacin and tobramycin Antimicrob Agents Chemo-ther 1993371723ndash1725

42 Vogelman B Craig W Postantibiotic effects J Antimicrob Chemother 198515(suppl A)37ndash46

43 Odenholt-Tornqvist I Pharmacodynamics of beta-lactam antibiotics Studies on theparadoxical effect and postantibiotic effects in vitro and in an animal model ScandJ Infect Dis 198958(suppl)1ndash55

44 Craig W Leggett K Totsuka K et al Key pharmacokinetic parameters of antibioticefficacy in experimental animal infections J Drug Dev 19981(suppl 3)7ndash15

45 Dornbusch K Henning C Linden E In-vitro activity of the new penems FCE 22101and FCE 24362 alone or in combination with aminoglycosides against streptococciisolated from patients with endocarditis J Antimicrob Chemother 198923(supplC)109ndash117

46 Fuursted K Comparative killing activity and postantibiotic effect of streptomycincombined with ampicillin ciprofloxacin imipenem piperacillin or vancomycinagainst strains of Streptococcus faecalis and Streptococcus faecium Chemotherapy198834229ndash234

47 Winstanley T Hastings J Penicillin-aminoglycosides synergy and post-antibioticeffect for enterococci J Antimicrob Chemother 198923189ndash199

48 Gudmundsson S Erlendsdottir H Gottfredsson M et al The postantibiotic effectinduced by antimicrobial combinations Scand J Infect Dis 19917480ndash93

49 Karlowsky J Zhanel G Davidson R et al Postantibiotic effect in Pseudomonasaeruginosa following single and multiple aminoglycoside exposures in vitro J Anti-microb Chemother 199433937ndash947

50 Karlowsky J Zhanel G Davidson R et al In vitro postantibiotic effects followingmultiple exposures of cefotaxime ciprofloxacin and gentamicin against Esche-richia coli in pooled human cerebrospinal fluid and Mueller-Hinton broth Antimi-crob Agents Chemother 1993371154ndash1157

51 McGrath B Marchbanks C Gilbert D et al In vitro postantibiotic effect followingrepeated exposure to imipenem temafloxacin and tobramycin Antimicrob AgentsChemother 1993371723ndash1725

52 Li R Zhu Z Lee S et al Antibiotic exposure and its relationship to postantibioticeffect and bactericidal activity Constant versus exponentially decreasing tobra-

Aminoglycosides 149

mycin concentrations against Pseudomonas aeruginosa Antimicrob Agents Che-mother 1997411808ndash1811

53 Rescott D Nix D Holden P et al Comparison of two methods for determiningin vitro postantibiotic effects of three antibiotics on Escherichia coli AntimicrobAgents Chemother 199832450ndash453

54 Fuursted K Postexposure factors influencing the duration of postantibiotic effectSignificance of temperature pH cations and oxygen tension Antimicrob AgentsChemother 1997411693ndash1696

55 Gudmundsson A Erlendsdottir H Gottfredsson M et al The impact of pH andcationic supplementation on in vitro postantibiotic effect Antimicrob Agents Che-mother 1991352617ndash2624

56 Park MK Myers R Marzella L Hyperoxia and prolongation of aminoglycoside-induced postantibiotic effect in Pseudomonas aeruginosa Role of reactive oxygenspecies Antimicrob Agents Chemother 199337120ndash122

57 Hanberger H Nilsson L Maller R et al Pharmacodynamics of daptomycin andvancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated bystudies of initial killing and postantibiotic effect and influence of Ca2 and albuminon these drugs Antimicrob Agents Chemother 1991351710ndash1716

58 Cars O Odenholt-Tornqvist I The postantibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 199331(suppl D)159ndash166

59 Craig W Vogelman B The postantibiotic effect Ann Intern Med 1987106900ndash902

60 Gottfredsson M Erlendsdottir H Gudmundsson et al Different patterns of bacte-rial DNA synthesis during the postantibiotic effect Antimicrob Agents Chemother1995391314ndash1319

61 Barmada S Kohlhepp S Leggett J et al Correlation of tobramycin-induced inhibi-tion of protein synthesis with postantibiotic effect in Escherichia coli AntimicrobAgents Chemother 1993372678ndash2683

62 Minguez F Izquierdo J Caminero M et al In vivo postantibiotic effect of isepa-micin and other aminoglycosides in a thigh infection model in neutropenic miceChemotherapy 199238179ndash184


64 Gudmundsson S Einarsson S Erlendsdottir H The post-antibiotic effect of antimi-crobial combinations in a neutropenic murine thigh infection model J AntimicrobChemother 199331(suppl D)177ndash191

65 Craig W Redington J Ebert S Pharmacodynamics of amikacin in vitro and inmouse thigh and lung infections J Antimicrob Chemother 199127(suppl C)29ndash40

66 Hessen M Pitsakis P Levison M Absence of a postantibiotic effect in experimentalPseudomonas endocarditis treated with imipenem with or without gentamicin JInfect Dis 1998158542ndash548

67 Craig W Post-antibiotic effects in experimental infection model relationship toin-vitro phenomena and to treatment of infection in man J Antimicrob Chemother199331(suppl D)149ndash158

150 Kim and Nicolau

68 Fantin B Craig W Factors affecting duration of in vivo postantibiotic effect foraminoglycosides against gram-negative bacilli Antimicrob Agents Chemother199127829ndash836

69 Mingeot-Leclercq MP Glupczynski Y Tulkens PM Aminoglycosides Activityand resistance Antimicrob Agents Chemother 199943727ndash737

70 Karlowsky JA Zhanel GG Davidson RJ Hoban DJ Once-daily aminoglycosidedosing assessed by MIC reversion time with Pseudomonas aeruginosa AntimicrobAgents Chemother 1994381165ndash1168

71 Daikos GL Jackson GG Lolans V Livermore DM Adaptive resistance to amino-glycoside antibiotics from first-exposure down-regulation J Infect Dis 1990162414ndash420

72 Daikos GL Lolans VT Jackson GG First-exposure adaptive resistance to amino-glycoside antibiotics in vivo with meaning for optimal clinical use AntimicrobAgents Chemother 199135117ndash123

73 Gerber AU Vastola AP Brandel J Craig WA Selection of aminoglycoside-resistant variants of Pseudomonas aeruginosa in an in vivo model J Infect Dis1982146691ndash697

74 Owens RC Jr Banevicius MA Nicolau DP Nightingale CH Quintiliani R In vitrosynergistic activities of tobramycin and selected β-lactams against 75 gram-nega-tive clinical isolates Antimicrob Agents Chemother 1997412586ndash2588

75 Marangos MN Nicolau DP Quintiliani R Nightingale CH Influence of gentamicindosing interval on the efficacy of penicillin containing regimens in experimentalEnterococcus faecalis endocarditis J Antimicrob Chemother 199739519ndash522

76 Hallander HO Donrbusch K Gezelius L Jacobson K Karlsson I Synergism be-tween aminoglycosides and cephalosporins with anti-pseudomonal activity Inter-action index and killing curve method Antimicrob Agents Chemother 198222743ndash752


78 Nicolau DP Freeman CD Belliveau PP Nightingale CH Ross JW Quintiliani RExperience with a once-daily aminoglycoside program administered to 2184 adultpatients Antimicrob Agents Chemother 199539650ndash655

79 Hutchin T Cortopassi G Proposed molecular and cellular mechanism for amino-glycoside ototoxicity Antimicrob Agents Chemother 1994382517ndash2520

80 Fausti SA Henry JA Scheffer HI Olson DJ Frey RH McDonald WJ High-fre-quency audiometric monitoring for early detection of aminoglycoside ototoxicityJ Infect Dis 19921651026ndash1032

81 Federspil P Drug-induced sudden hearing loss and vestibular disturbances AdvOtorhinolaryngol 198127144ndash158

82 Beaubien AR Ormsby E Bayne A Carrier K Crossfield G Downes M Henri RHodgen M Evidence that amikacin ototoxicity is related to total perilymph areaunder the concentration-time curve regardless of concentration Antimicrob AgentsChemother 1991351070ndash1074

83 Proctor L Petty B Lietman P Thakor R Glackin R Shimizu H A study of poten-tial vestibulotoxicity effects of once daily versus thrice daily administration of to-bramycin Laryngoscope 1987971443ndash1449

Aminoglycosides 151

84 Rybak MJ Abate BJ Kang SL Ruffing MJ Lerner SA Drusano GL Prospectiveevaluation of the effect of an aminoglycoside dosing regimen on rates of observednephrotoxicity and ototoxicity Antimicrob Agents Chemother 1999431549ndash1555

85 Garrison MW Zaske DE Rotschafer JC Aminoglycosides Another perspectiveDICP Ann Pharmacother 199024267ndash272

86 Mingeot-Leclercq MP Tulkens PM Aminoglycosides Nephrotoxicity AntimicrobAgents Chemother 1999431003ndash1012

87 Moore RD Smith CR Lietman PS Risk factors for nephrotoxicity in patientstreated with aminoglycosides Ann Intern Med 1984100352ndash357

88 Sawyers CL Moore RD Lerner SA Smith CR A model for predicting nephrotox-icity with aminoglycosides J Infect Dis 19861531062ndash1068

89 Whelton A Therapeutic initiatives for avoidance of aminoglycoside toxicity J ClinPharmacol 19852567ndash81

90 Bertino JS Jr Booker LA Franck PA Jenkins PL Nafziger AN Incidence andsignificant risk factors for aminoglycoside-associated nephrotoxicity in patientsdosed by using individualized pharmacokinetic monitoring J Infect Dis 1993167173ndash179

91 Nodoushani M Nicolau DP Hitt CH Quintiliani R Nightingale CH Evaluation ofnephrotoxicity associated with once-daily aminoglycoside administration J PharmTech 199713258ndash262

92 Verpooten GA Giuliano RA Verbist L Eestermans G De Broe ME Once dailydosing decreases the accumulation of gentamicin and netilmicin Clin PharmacolTher 19894522ndash27

93 Murray KR McKinnon PS Mitrzyk B Rybak MJ Pharmacodynamic characteriza-tion of nephrotoxicity associated with once-daily aminoglycoside Pharmacother-apy 1999191252ndash1260

94 Sarubbi FA Jr Hull JH Amikacin serum concentrations Prediction of levels anddosage guidelines Ann Intern Med 197889612ndash618

95 Sawchuk RJ Zaske DE Pharmacokinetics of dosing regimens which utilize multi-ple intravenous infusions Gentamicin in burn patients J Pharmacokinet Biopharm19764183ndash195

96 Sawchuk RJ Zaske DE Cipolle RJ Wargin WA Strate RG Kinetic model forgentamicin dosing with the use of individual patient parameters Clin PharmacolTher 197721362ndash369

97 Schumock GT Raber SR Crawford SY Naderer OJ Rodvold KA National surveyof once-daily dosing of aminoglycoside antibiotics Pharmacotherapy 199515201ndash209

98 Chuck SK Raber SR Rodvold KA Areff D National survey of extended-intervalaminoglycoside dosing Clin Infect Dis 200030433ndash439


100 DeVries PJ Verkooyen RP Leguit P Verbrugh HA Prospective randomized studyof once-daily versus thrice-daily netilmicin regimens in patients with intraabdomi-nal infections Eur J Clin Microbiol Infect Dis 19909161ndash168

101 Mauracher EH Lau WY Kartowisastro H et al Comparison of once-daily and

152 Kim and Nicolau

thrice-daily netilmicin regimens in serious systemic infections A multicenter studyin six asian countries Clin Ther 198911604ndash613

102 Maller R Ahrne H Eilard T et al Efficacy and safety of amikacin in systemicinfections when given as a single daily dose or in two divided doses J AntimicrobChemother 199127(suppl C)121ndash128

103 Maller R Ahrne H Holmen C et al Once- versus twice-daily amikacin regimenefficacy and safety in systemic gram-negative infections J Antimicrob Chemother199331939ndash948

104 Prins JM Buller HR Kuijper EJ Tange RA Speelman P Once versus thrice dailygentamicin in patients with serious infections Lancet 1993341335ndash339

105 Rozdzinski E Kern WV Reichle A et al Once-daily versus thrice-daily dosing ofnetilmicin in combination with β-lactam antibiotics as empirical therapy for febrileneutropenic patients J Antimicrob Chemother 199331585ndash598

106 TerBraak EW DeVries PJ Bouter KP et al Once-daily dosing regimen for amino-glycoside plus β-lactam combination therapy of serious bacterial infections Com-parative trial with netilmicin plus ceftriaxone Am J Med 19908958ndash66

107 International Antimicrobial Therapy Cooperative Group of the EORTC Efficacyand toxicity of single daily doses of amikacin and ceftriaxone versus multiple dailydoses of amikacin and ceftazidime for infection in patients with cancer and granulo-cytopenia Ann Intern Med 1993119584ndash593

108 Beaucaire G Leroy O Beuscart C et al Clinical and bacteriological efficacy andpractical aspects of amikacin given once daily for severe infections J AntimicrobChemother 199127(suppl C)91ndash103

109 Prins JM Buller HR Kuijper EJ Tange RA Speelman P Once-daily gentamicinversus once-daily netilmicin in patients with serious infections A randomized clini-cal trial J Antimicrob Chemother 199433823ndash835

110 Marik PE Lipman J Kobilski S Scribante J A prospective randomized study com-paring once- versus twice-daily amikacin dosing in critically ill adult and paediatricpatients J Antimicrob Chemother 199128753ndash764

111 Nicolau DP Quintiliani R Nightingale CH Once-a-day aminoglycoside therapyRep Ped Infect Dis 1997728

112 Bourget P Fernandez H Delouis C Taburet AM Pharmacokinetics of tobramycinin pregnant women Safety and efficacy of a once-daily dose regimen J Clin PharmTher 199116167ndash176

113 Galoe AM Graudal N Christensen HR Kampmann JP Aminoglycosides Singleor multiple daily dosing A meta-analysis on efficacy and safety Eur J Clin Phar-macol 19954839ndash43

114 Freeman CD Strayer AH Mega-analysis of meta-analysis An examination ofmeta-analysis with an emphasis on once-daily aminoglycoside comparative trialsPharmacotherapy 1996161093ndash102

115 Hatala R Dinh T Cook D Once-daily aminoglycoside dosing in immunocompe-tent adults A meta-analysis Ann Intern Med 1996124717ndash725

116 Barza M Ioannidis JP Cappelleri JC Lau J Single or multiple doses of aminogly-cosides A meta-analysis Br Med J 1996312338ndash345

117 Munckhof WJ Grayson JL Turnidge JD A meta-analysis of studies on the safety

Aminoglycosides 153

and efficacy of aminoglycosides given with once daily or as divided doses J Anti-microb Chemother 199637645ndash663

118 Ferriols-Lisart R Alos-Alminana M Effectiveness and safety of once-daily amino-glycosides A meta-analysis Am J Health-Syst Pharm 1996531141ndash1150

119 Bailey TC Little JR Littenberg B Reichley RM Dunagan WC A meta-analysisof extended-interval dosing versus multiple daily dosing of aminoglycosides ClinInfect Dis 199724786ndash795


121 Hatala R Dinh TT Cook DJ Single daily dosing of aminoglycosides in immuno-compromised adults A systematic review Clin Infect Dis 199724810ndash815

122 Demczar DJ Nafziger AN Bertino JS Jr Pharmacokinetics of gentamicin at tradi-tional versus high doses Implications for once-daily aminoglycoside dosing Anti-microb Agents Chemother 1997411115ndash1119

123 Xuan D Lu JF Nicolau DP Nightingale CH Population pharmacokinetics studyof tobramycin after once-daily dosing in hospitalized patients Int J AntimicrobAgents 200015185ndash191

124 Konrad F Wagner R Neumeister B Rommel H Georgieff M Studies on drugmonitoring in thrice and once daily treatment with aminoglycosides Intensive CareMed 199319215ndash220

125 Gilbert DN Bennett WM Use of antimicrobial agents in renal failure Infect DisClin North Am 19893517ndash531

126 Prins JM Koopmans RP Buller HR Kuijper EJ Speelman P Easier monitoringof aminoglycoside therapy with once-daily dosing schedules Eur J Clin MicrobiolInfect Dis 199514531ndash535

127 Begg EJ Barclay ML Duffull SB A suggested approach to once-daily aminoglyco-side dosing Br J Clin Pharmacol 199539605ndash609

128 Nicolau DP Wu AHB Finocchiaro S Udeh E Chow MSS Quintiliani R Nightin-gale CH Once-daily aminoglycoside dosing Impact on requests for therapeuticdrug monitoring Thera Drug Mon 199618263ndash266

129 Hitt CM Klepser ME Nightingale CH Quintiliani R Nicolau DP Pharmacoeco-nomic impact of a once-daily aminoglycoside administration Pharmacotherapy199717810ndash17814

130 Parker SE Davey PG Once-daily aminoglycoside administration in gram-negativesepsis Economic and practical aspects PharmacoEconomics 19957393ndash402

7

Pharmacodynamics of Quinolones

Robert C Owens Jr

Maine Medical Center Portland Maine and University of Vermont Collegeof Medicine Burlington Vermont

Paul G Ambrose


1 INTRODUCTION

lsquolsquoWe know everything about antibiotics except how much to giversquorsquo MaxwellFinland once stated With the proliferation of pharmacodynamics as a sciencewe are finally addressing the question of how much to give We are watchingthe pendulum swing from an era of more-or-less arbitrary dosage selection towardthe present-day science that integrates both pharmacokinetic and microbiologicdata to determine optimal dosing strategies and to set perhaps more clinicallymeaningful breakpoints It is conceivable that body-site-specific susceptibilitybreakpoints based on the pharmacodynamic profiles of antimicrobial agents willexist in the not-too-distant future replacing the one breakpoint fits all approachthat we have historically endured Pharmacodynamics is central to the idea ofoptimizing antimicrobial therapy Figure 1 illustrates the relationship betweenpharmacodynamics and the optimal selection dosing and duration of antimicro-bial therapy A comprehensive review by Ron Polk [1] provides in great detailthe various aspects of the optimal use of antibiotics

155


FIGURE 1 Pharmacodynamics is central to the optimization of antimicrobialtherapy (Courtesy of Robert Owens PharmD)

During the three decades since nalidixic acid was first introduced thou-sands of related quinolone compounds have been synthesized and most havebeen abandoned prior to development for a variety of reasons A better under-standing of structurendashactivity and structurendashtoxicity relationships has allowedchemists to modify the adaptable basic quinolone structure to enhance or limitthe extent of antimicrobial activity and to improve various other product attri-butes such as the pharmacokinetic profile tolerability drug interaction potentialand toxicity of each new agent The quinolones have undergone a lsquolsquostructuralevolutionrsquorsquo that began in 1962 upon the inadvertent discovery of nalidixic acidand the pilgrimage continues today with the search for the perfect compound[2]

For some time now clinicians have been familiar with antibiotic classescategorized by generations (eg cephalosporins quinolones macrolides) Cer-tain schemes have been based upon pharmacodynamics [2ndash5] (Table 1) ratherthan relying on simply microbiological susceptibility data or merely the date acompound was licensed for use Classifying quinolones by generations allowsone to realize the pharmacokinetic and microbiological evolution of the quino-lones as a direct result of structural changes To this day the fluoroquinolonesremain the prototypical class of antibiotics because they are available in bothoral and parenteral dosage forms have excellent oral bioavailability are activeagainst a wide range of bacteria achieve therapeutic concentrations both intracel-lularly and extracellularly distribute widely into organ tissue and secretions andare rapidly bactericidal It is for these reasons that the fluoroquinolones haverevolutionized transitional therapy as well as other aspects of the treatment ofmodern infectious diseases


TA

BLE

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osa

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fect

ion

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ne

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ne

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ne

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ual

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hw

ays

exis

tS

ou

rce

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urt

esy

of

Ro

ber

tO

wen

sP

har

m

D


2 PHARMACODYNAMIC CONCEPTS

Since the dawn of the antibiotic era in the late 1930s controversy has existedas to the most appropriate method to administer and dose antibiotics to maximizethe killing of microorganisms while minimizing toxicity Harry Eagle a half-century ago pioneered the first pharmacodynamic studies using penicillin instreptococcal and syphylitic animal models of infection [6ndash8] It was at this timethat dosing methods (eg continuous infusion versus intermittent injection) aswell as the dose of penicillin employed in relation to the minimum inhibitoryconcentration (MIC) were evaluated for their ability to impact in vivo outcomeFollowing a long respite the science known as pharmacodynamics reemergedas Shah et al [9] classified antimicrobial agents on the basis of their patternsof bactericidal activity One of the two patterns described was a concentration-dependent killing effect where an increase in the rate and extent of bacterialkilling occurred with increasing drug concentrations in relation to the MIC of thebacteria Agents that demonstrated concentration-dependent bactericidal activityincluded the aminoglycosides (and now include the fluoroquinolones and mostlikely metronidazole) (see Fig 2)

The second pattern now known as time-dependent killing exhibited a satu-rable concentration-dependent increase in the rate and extent of bacterial killingoccurring at approximately 2ndash4 times the MIC of the pathogen In essence theseagents proceeded to kill bacteria most efficiently when the concentration of drugremained in excess of the MIC of the pathogen for a specified length of timerather than when doses were employed that provided high serum concentrations

FIGURE 2 Concentration-dependent versus time-dependent bactericidal ac-tivity (From Ref 20)


(relative to the MIC) because the latter did not result in enhanced bacterial kill-ing β-Lactams (eg penicillins cephalosporins monobactams carbapenems)lincosamides (eg clindamycin) macrolides (eg erythromycin clarithro-mycin) and oxazolidinones (eg linezolid) best fit this pattern of bactericidalactivity For the purposes of this chapter we will focus on the quinolones andconcentration-dependent bacterial killing

Historically the MIC has been used by the practicing clinician to selectdrug therapy for a particular infection The lower the MIC the better the drugmust be and the choice has been traditionally made accordingly and sometimesstill is today Unfortunately the MIC has many shortcomings and therefore can-not be used alone to predict the outcome for a given infection The MIC is ameasure of a drugrsquos potency against a particular organism It should be remem-bered that the MIC is an artificial value and is further subject to a one-tubedilution standard error if broth dilution techniques are used This can translateinto a considerable difference in actual MIC values (eg is the MIC 8 microgmLor 16 microgmL or somewhere in between) The MIC does not describe the rateand extent of bacterial killing or persistent antimicrobial effects such as the post-antibiotic effect (PAE) or the more clinically relevant sub-MIC effect (SME)The PAE and SME are used to describe the continued suppression of bacterialgrowth despite either complete removal of the antimicrobial agent from the organ-ismrsquos milieu or concentrations of the agent that are below the MIC value for theparticular organism respectively Also the MIC does not describe the impactof increasing drug concentrations or increased time of exposure of the drug onbacteriological outcome MIC testing does not account for protein binding al-though somewhat controversial this has historically misled clinicians to choosetherapies with confidence based solely on in vitro data For example the use ofoxacillin for the treatment of enterococcal endocarditis (despite MIC values inthe susceptible range) has led to clinical and microbiological failures in humanssimilarly the use of cefonicid and teicoplanin for the treatment of staphylococcalinfections has led to treatment failures some so extensive that clinical trials wereterminated sooner than planned [10ndash12] Fortunately these weaknesses can beovercome or a drug selection can be modified if the appropriate pharmacokineticaspects of the antimicrobial agent are also factored into the equation

Bacterial killing can be characterized mathematically For example theproduct of concentration and time (C t) may be reflected by the pharmacoki-netic term lsquolsquoarea under the concentrationndashtime curversquorsquo (AUC) Hence bacterialkilling is a function of a drugrsquos AUC when it is indexed to the MIC The 24 hAUCMIC ratio is the pharmacodynamic correlate that can be used to describethe time course of antimicrobial activity and to predict clinical or microbiologicaloutcome and perhaps the development of resistance

Under certain circumstances one of the terms of the product (either concen-tration or time) makes a small or negligible contribution to the killing process


and can therefore be ignored The pharmacodynamic parameter can be simplifiedto the peak concentration (peak)MIC ratio or the length of time the serum con-centration remains above the MIC (t MIC) How this simplification occursdepends on the pattern of bactericidal activity demonstrated by the antimicrobialagent in question (eg concentration-dependent killing or time-dependent kill-ing) For concentration-dependent killing agents (eg aminoglycosides quino-lones) the 24 h AUCMIC ratio and peakMIC ratio have been used to correlatein vivo outcome For the time-dependent killing agents (eg β-lactams) t MIC has best correlated with efficacy

3 CLINICAL PHARMACODYNAMIC TARGETS

For the quinolones as one might expect both the peakMIC ratio and the 24 hAUCMIC ratio have been correlated with outcome The magnitude of the peakMIC ratio that has been associated with improved outcomes is 10ndash12 [13] Inaddition the peakMIC ratio has been suggested to be the parameter of choicewhen resistant subpopulations of bacteria exist [26] Unfortunately increasingthe amount of drug given in excess of standard doses for many agents in thisclass also increases the probability of unwanted adverse events so unless thepathogen is relatively susceptible this ratio may not always be the best parameterto optimize

When a peakMIC ratio of at least 101 is not possible one can no longerignore the contribution of the time of exposure (Fig 3) Under these circum-

FIGURE 3 Pharmacodynamic relationships of (a) peakMIC ratio and (b) 24 hAUCMIC ratio (Courtesy of Robert Owens Pharm D)


stances the parameter that best correlates with efficacy defaults to the 24 h AUCMIC ratio

The optimal pharmacodynamic targets are pathogen-specific Data obtainedfrom animal models of sepsis in vitro pharmacodynamic experiments and clinicaloutcome studies indicate that the magnitude of the 24 h AUCMIC ratio can beused to predict clinical response Forrest et al [14] demonstrated that a 24 hAUCMIC ratio of 125 was associated with the best clinical cure rates in thetreatment of infections caused by gram-negative enteric pathogens and P aerugi-nosa However for gram-positive bacteria data collected in humans by Drusanoand colleagues [13] and Ambrose et al [15] suggest that the 24 h AUCMICratio can be appreciably lower For infections caused by anaerobic pathogensthe optimal pharmacodynamic correlate for efficacy remains to be determinedHowever the likely 24 h AUCMIC ratio target will again be below 125 The24 h AUCMIC ratio for trovafloxacin ranged from 50 to 100 [16] similar tothat of metronidazole and was successful within this range One must also re-member that free drug concentrations should be considered so taking into ac-count that trovafloxacin is 75 protein-bound a free drug 24 h AUCMIC ratiorequired to predict success against anaerobic pathogens may in fact be as low as15ndash25 Pharmacodynamic studies evaluating quinolones against anaerobic patho-gens are needed considering that most newer agents (eg gatifloxacin moxiflox-acin gemifloxacin sitafloxacin) have in vitro activity against these organisms

4 PHARMACOKINETICS OF FLUOROQUINOLONES

The pharmacokinetic profiles for the newer and some older fluoroquinolones arelisted in Table 2 Once a standard of care for most infections the appeal ofparenteral therapy has lost significant ground to newer equally potent and phar-macokinetically equivalent oral dosing formulations Although bacteria have be-come quite sophisticated they are still unable to discriminate between the routesby which an antibiotic is delivered to the infection site The fluoroquinoloneshave revolutionized the treatment of modern infectious diseases for a variety ofreasons none perhaps more important than their pharmacokinetic characteristicsWhether administered intravenously or orally similar pharmacodynamic rela-tionships can be achieved because of their high degree of bioavailability whichis consistent among the newer generation agents Protein binding is one character-istic that is highly variable among the quinolones The degree of protein bindingnot only influences penetration into tissues but also determines the amount ofdrug that is capable of interacting with bacteria at the site of infection [17] Theexact role of protein binding is often more controversial than not but clearlythe pharmacokinetic parameter used in pharmacodynamic analyses should reflectfree drug (active drug) rather than total drug concentrations


TA

BLE

2P

har

mac

oki

net

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les

of

Var

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uo

roq

uin

olo

nes

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amet

erN

orfl

oxa

cin

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rofl

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cin

Levo

flo

xaci

nS

par

flo

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nG

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oxa

cin

Mo

xifl

oxa

cin

Tro

vafl

oxa

cin

Gem

iflo

xaci

n

Do

se(m

g)

400

750

500

200

400

400

200

320

Bio

avai

lab

ility

()

5070

9992

9690

8880

P

rote

in-b

ou

nd

1525

3040

2055

7559

Pea

k(micro

gm

L)1

53

56

13

43

45

23

148

Free

pea

k(micro

gm

L)1

32

64

20

83

42

00

60

6A

UC

(0ndash2

4)(micro

gsdoth

mL)

136

6447

517

751

348

312

93

Free

AU

C(0

ndash24)(micro

gsdoth

mL)

116

4833

310

641

021

67

83

8T

12

33

46

209

1212

74

R

enal

elim

inat

ion

(act

ive

dru

g)40

6095

1085

206

30

Co

urt

esy

of

Ro

ber

tO

wen

sP

har

m

D


5 OUTCOME DATA

51 Animal Models and In-Vitro Models of Infection

In 1991 Leggett et al [18] published data evaluating ciprofloxacin in neutropenicmurine thigh and pulmonary infection models using strains of P aeruginosa andK pneumoniae respectively The manipulation of the dosing intervals in thesestudies minimally affected the extent of bacterial killing suggesting that theAUCMIC ratio may be most closely associated with this endpoint

Drusano et al [26] evaluated lomefloxacin in the neutropenic rat model ofPseudomonas sepsis Doses were administered in a variety of regimens to studythe effect of different pharmacodynamic indices associated with outcome ThepeakMIC ratio was significantly associated with reduced mortality comparedwith the AUCMIC ratio and the time MIC The doses used in this studyprovided peakMIC ratios of approximately 201 and 101 The reason for theclear association between the index of the peakMIC ratio and survival was statedto be the increased inoculum size used (1 109) in the study These findingsdemonstrated that with a higher burden of organisms one is more likely to en-counter resistant subpopulations and in such settings the peakMIC ratio be-comes the more appropriate pharmacodynamic index that predicts outcome

Several studies have been recently published or presented evaluating thepharmacodynamic relationships of quinolones against gram-positive pathogensparticularly S pneumoniae Onyeji et al [19] compared the efficacy of ciproflox-acin and levofloxacin in a murine model of peritoneal sepsis Clinical isolates ofS pneumoniae were used that displayed a variety of susceptibilities to penicillinand MIC values for ciprofloxacin (1 and 2 microgmL) and levofloxacin (1 and 2microgmL) were reflective of those also seen clinically Dosing regimens were variedsuch that concentrations of drug measured in the animals simulated those in hu-mans Five-day survival rates between ciprofloxacin (2ndash6) and levofloxacin(7ndash9) groups were negligible (p 005) perhaps because the 24 h AUCMICratios were not very different from those seen in humans For ciprofloxacin 24h AUCMIC ratios ranged between 175 and 35 and for levofloxacin valuesranged between 22 and 44 Neither ciprofloxacin nor levofloxacin achieved adesirable peakMIC ratio of at least 10 as expected

Studies evaluating the impact of protein binding have been conductedCraig and Andes [20] studied six fluoroquinolones in the classic nonneutropenicmurine thigh infection model in an effort to determine optimal AUCMIC ratiosin terms of survival and bactericidal activity using both free and total drug con-centrations After being infected with S pneumoniae mice were treated withciprofloxacin levofloxacin sitafloxacin moxifloxacin gemifloxacin or gati-floxacin Serum concentrations and protein binding were determined using micro-biological assay and ultrafiltration respectively The Emax model was used to cor-relate 24 h AUCMIC values using both free and total drug concentrations with


colony forming units in the thigh on day 1 and survival on day 5 Results indicatedthat the 24 h AUCMIC ratio correlated with survival on day 5 and the extentof bacterial killing on day 1 (p 0001) Additionally free drug better correlatedwith survival on day 5 than did total drug concentrations (R 2 82 and 74respectively) (see Fig 4) Survival (90 of animals) was predicted with an 24 hAUCMIC ratio of 34 4 and a similar value predicted a 25 log10 kill afterone day of treatment Again agreement existed among all quinolones tested asto the pharmacodynamic breakpoint required for endpoints of survival and theextent of bacterial killing (24 h AUCMIC somewhere between 25 and 35) andunbound drug was better correlated with these effects than total drug concentra-tions

Similarly for S pneumoniae in vitro models of infection have demon-strated that for levofloxacin and ciprofloxacin a 24 h AUCMIC ratio of approxi-mately 30 was associated with a 4 log kill whereas values less than 30 wereassociated with a significantly reduced extent of bacterial killing and in someinstances bacterial regrowth [2122] These observations are supported by datafrom nonneutropenic animal models of infection where maximal survival wasassociated with a 24 h AUCMIC ratio of 25 against the pneumococcus [23]Clinically there have been a significant number of treatment failures and superin-fections involving meningeal seeding from S pneumoniae in patients receivingciprofloxacin where the 24 h AUCMIC ratio is approximately 12 [24] Con-versely similar treatment failures or superinfections have not occurred with quin-

FIGURE 4 Correlation between survival and (a) free-drug concentration (R 2 74) and (b) total drug concentrations (R 2 82) (From Ref 20)


olones for which the 24 h AUCMIC ratios against this bacterium are greaterthan 30ndash40 [15]

52 Human Studies

Some of the first data to correlate pharmacodynamics and response in humanswere published by Peloquin et al [25] Intravenous ciprofloxacin was evaluatedin the treatment of lower respiratory tract infections in seriously ill patients hospi-talized in the intensive care unit A relationship between time of exposure andoutcome was established Interestingly against P aeruginosa a low peakMICratio (101) was observed and resulted in the development of resistance in 10of 13 pathogens Although the researchers concluded that the time duration ofexposure was the important determinant of outcome against difficult to treat andless susceptible pathogens (eg P aeruginosa) failure to obtain an optimal peakMIC ratio resulted in the emergence of resistance This is consistent with findingsfrom the animal model of infection reported by Drusano et al [26] further em-phasizing the importance of the peakMIC ratio when dealing with resistant sub-populations Eventually after the addition of 24 new patients Forrest et al [14]in 1993 published their reanalyzed data from the 1989 publication (see Fig 5)Multivariate analysis and logistic regression showed that the 24 h AUCMIC ratiopredicted outcome best rather than the time of exposure as previously reported

A relationship also exists between the 24 h AUCMIC ratio and the likli-hood of developing resistance while on therapy Thomas et al [27] describedthis relationship in 107 hospitalized patients being treated for bacterial pneumo-nia Patients were treated with a variety of dosing regimens involving a fluoro-quinolone (ciprofloxacin 200 mg q12h to 400 mg q8h) or a cephalosporin (cef-menoxime 1ndash2 g q4-6h or ceftazidime 1ndash2 g q8-12h) providing a wide rangeof exposures Gram-negative pathogens predominated as expected in nosocomialpneumonia accounting for over 90 of the organisms isolated Gram-positivepathogens such as S aureus and S pneumoniae were infrequently isolated Withthe exception of Bush group 1 β-lactamase elaborating organisms (eg Entero-bacter spp) treated with a β-lactam a pharmacodynamic breakpoint was estab-lished If a 24 h AUCMIC ratio of at least 100 was achieved the potential todevelop resistance was 10 In contrast the probability of developing a resis-tant strain during therapy was greater than 80 if the 24 h AUCMIC was 100Because the majority of pathogens encountered were gram-negative bacilli mostof which were P aeruginosa it is difficult to generalize this identified pharmaco-dynamic relationship to gram-positive pathogens More information is clearlyneeded here

Ciprofloxacin a second-generation fluoroquinolone remains the most po-tent antipseudomonal quinolone in terms of in vitro microbiological activity For


FIGURE 5 Pharmacodynamic correlates of efficacy for gram-negative patho-gens (From Ref 14)

instance ciprofloxacin is consistently 4ndash8 times as active against Pseudomonasaeruginosa in vitro as trovafloxacin and levofloxacin Along these lines ci-profloxacin is also pharmacodynamically superior to other available fluoroquino-lones against P aeruginosa in terms of an achievable 24 h AUCMIC ratio whendosed appropriately Unfortunately none of the currently available fluoroquino-lones routinely achieve a target 24 h AUCMIC ratio against P aeruginosa ofat least 125 which is one reason combination therapy is always recommendedfor the treatment of infection outside of the lower urinary tract when a quinoloneis employed

One must consider however that ciprofloxacinrsquos strength against P aeru-ginosa is counterbalanced by its poor pharmacodynamic profile against manygram-positive microorganisms For instance ciprofloxacin even when dosed at750 mg every 12 h does not reach a pharmacodynamic goal of approximately30 for S pneumoniae This poor pharmacodynamic profile is consistent withnumerous reports of failures and superinfections when ciprofloxacin has beenused in community-acquired infection due to S pneumoniae [2428ndash33]

With the advent of newer generation fluoroquinolones (eg temafloxacin)with significantly greater potency against the pneumococcus in the early 1990s


and the recent observations of increasing β-lactam resistance among pneumo-cocci it is not surprising that Daniel Musher in 1992 stated lsquolsquoThose of us whoa few years ago scoffed at the notion of using a quinolone for treatment ofpneumococcal pneumonia may be just doing that in the not-too-distant futurersquorsquo

Newer generation fluoroquinolones are filling the ever-widening niche be-ing created by pneumococci that are now demonstrating widespread resistanceto multiple drug classes including the β-lactams macrolides and azalides sulfon-amides and tetracyclines In fact with respect to the treatment of community-acquired pneumonia Marvin Turck has widely been quoted as saying lsquolsquoa fluoro-quinolone for your mother or a β-lactam for your mother-in-lawrsquorsquo Despite theirhigh degree of activity against most strains of S pneumoniae the fluoroquino-lones particularly less active agents such as ciprofloxacin ofloxacin and lev-ofloxacin are showing elevated rates (albeit small percentages) of resistance[3435] Some newer generation fluoroquinolones (eg moxifloxacin gatifloxa-cin gemifloxacin) are also affected but to a lesser degree [3436] The fluoroqui-nolones do have the potential for overuse because of their convenience and agent-specific low side effect profiles Appropriate use of these compounds and theselection of those agents with optimized pharmacodynamic profiles will mostlikely reduce the selective pressure being placed on pneumococci

Preston et al [13] evaluated the association between levofloxacinrsquos pharma-codynamic profile and clinical as well as microbiological outcomes In this studyconcentrations of levofloxacin in serum were obtained from patients being treatedfor urinary tract pulmonary and skinsoft-tissue infections after they had beengiven standard doses appropriate for the site of infection Of the initial 313 pa-tients 116 had sufficient pharmacokinetic microbiological and outcome data forevaluation Patients in whom a peakMIC ratio of 122 was achieved had a100 chance of a eradicating the infecting organism from the site of infectionConversely if the peakMIC ratio was 122 the liklihood of successful eradica-tion was 808 Although there was significant covariation between the peakMIC and 24 h AUCMIC indices on outcome predictability the peakMIC ratioreached statistical significance for all pathogens and infection sites A recent re-analysis of the infections caused by S pneumoniae however revealed that a 24h AUCMIC ratio of 30 was associated with favorable outcomes [37] Thesedata are consistent with the previous work conducted by Drusanorsquos group whichdemonstrated that when a peakMIC ratio of at least 101 could not be achievedthe index most closely associated with outcome was the 24 h AUCMIC ratio

Most recently Ambrose and Grasela [15] reported a correlation betweenthe free-drug 24 h AUCMIC ratio and microbiological eradication of S pneu-moniae in patients enrolled in phase III double-blinded randomized trials inNorth America Of the initial 778 patients enrolled in these studies involvingcommunity-acquired pneumonia and acute bacterial exacerbations of chronicbronchitis 376 patients had sufficient clinical and microbiological data for evalu-


FIGURE 6 Pharmacodynamic correlates of efficacy for Streptococcus pneu-moniae (Courtesy of Paul Ambrose)

ation Of these patients 58 were infected with S pneumoniae that was isolatedfrom either blood or sputum These data established that for gatifloxacin andlevofloxacin a 24 h AUCMIC ratio of at least 33 correlated with the eradicationof S pneumoniae in patients being treated for pneumococcal pulmonary infec-tions (Fig 6) Although not every patient with an 24 h AUCMIC ratio that didnot reach this pharmacodynamic breakpoint failed therapy these patients cer-tainly had a high probability of failure

In the clinic we are now seeing what has already been predicted pharmaco-dynamically [38] As MICs continue to increase gradually for older fluoroquino-lones the pharmacokinetics of these agents can no longer compensate for thisdecrease in activity Davidson et al [39] reported recently well documented clini-cal failures that could be traced back to the inability of a less active fluoroquino-lone to eradicate S pneumoniae When pulse field gel electropheresis and PCRwere performed on the pretherapy and post-therapy isolates it was evident thatthe organisms were in fact the same and resistance had developed during therapyPretherapy isolates in both cases were susceptible to the agent and after receivinglevofloxacin for the treatment of community-acquired pneumococcal pneumoniathe strains had acquired resistance via gyrA and parC mutations A likely expla-nation for these findings are the propensity to select for resistant strains secondaryto reduced concentrations at the end of the dosing interval There are differencesamong the newer generation fluoroquinolones in their ability to select for resistantstrains The rank order in terms of most likely to least likely to select for resis-


tance among pneumococci is as follows ciprofloxacin levofloxacin gati-floxacin moxifloxacin [40ndash43]

Finally various pharmacodynamic targets for predicting higher probabili-ties for successul outcomes in humans now exist For quinolones against gram-negative enteric bacilli and P aeruginosa a 24 h AUCMIC ratio of 125 maybe targeted for the treatment of infections by these pathogens For preventing theemergence of resistance primarily for non-Bush group 1 β-lactamase-elaboratinggram-negative bacilli a 24 h AUCMIC ratio of at least 100 has been suggestedAnd for gram-positive pathogens such as S pneumoniae an AUCMIC ratio ofat least 30 has been associated with higher probabilities of successful eradicationof these organisms More data are clearly needed to assess the effect of thesepharmacodynamic indices on newly emerging mechanisms of resistance and foranaerobic pathogens

6 PHARMACODYNAMIC ANALYSES

Once data have become available demonstrating an association between a phar-macodynamic relationship and outcome one can predict the efficacy of certaincompounds using available pharmacokinetic and microbiological data individual-ized toward specific patient and pathogen populations This can be quite useful tothe clinician in the assessment of newer agents in the formulary decision processparticularly when they are being compared to other newer agents as well as moretraditional anti-infectives The methodology of such analyses should be viewedwith a certain amount of skepticism as these analyses have now become popularamong the marketing departments of the pharmaceutical industry Moreover cli-nicians should be careful when performing these analyses to ensure that the dataare generalized to appropriate patient populations Ultimately this approachwould be most useful when performed individually at the bedside using patient-specific data in an effort to optimize treatment in real time

61 Single-Point Analyses

Because of the recent interest in pharmacodynamics and its ability to distinguishanti-infective agents it has become popular to perform so-called single pointpharmacodynamic analyses These analyses are popular because they are bothconvenient and easy to perform They rely primarily on the selection of meanpharmacokinetic values (eg 24 h AUC) collected in healthy volunteers whichcan be readily gleaned from product package inserts In addition to the meanpharmacokinetic parameters MIC90 values are obtained most often from largenational surveillance studies By integrating these data one can calculate 24 hAUCMIC ratios for various quinolonendashpathogen combinations very quickly At


best these evaluations convey what is possible rather than what is probable Atworst they are meaningless to the practicing clinician [44] Unfortunately manyhazards exist with these evaluations For instance significant variability existsin both pharmacokinetic data and MIC values local resistance trends are notrepresented when national susceptibility data are used and significant bias isoften introduced into the analysis

Considerable variability exists when fixed doses of drug are administeredto populations of individuals with varying weights heights and organ functionAdditionally healthy volunteers must be discriminated from clinically sick pa-tients Mean 24 h AUC values cannot adequately reflect the range of possibilities

Similarly the MIC90 value of a given drug versus a specific organism al-though perhaps useful for comparing susceptibility data from different locationscannot possibly be used alone to simulate the range of potential susceptibilitiesencountered clinically In fact most strains encountered would have MIC valuesfar less than the MIC90 value they are labeled with By the same token how dowe accommodate for the 10 of organisms whose MICs are in excess of theMIC90 value

Because of this rather arbitrary method for conducting a pharmacodynamicanalysis one can also foresee the introduction of bias With the availability ofa variety of surveillance studies one is left to decide which MIC value to selectA twofold variance in MIC can lead to the resulting 24 h AUCMIC ratio beingeither below or above the desired pharmacodynamic target with the agent beingdeemed either unfavorable or favorable respectively As an example for lev-ofloxacin if one selects an MIC90 of 10 or 20 microgmL keeping the mean 24 hAUC value fixed (500 mg dose administered orally 24 h AUC 475 microg sdot hmL) the conclusions are dramatically different If an MIC90 of 10 microgmL isused the resulting 24 h AUCMIC ratio is 475 whereas if 20 microgmL is usedthe 24 h AUCMIC ratio is 2375 Keeping in mind that the 24 h AUCMICratio pharmacodynamic target of 30 is desirable for S pneumoniae the seeminglyminute one-dilution difference in MIC resulted in two discordant conclusionsOne can clearly see that although this approach might seem convenient the re-sults may equate to lsquolsquogarbage in garbage outrsquorsquo

62 Monte Carlo Analysis

A departure from a past paradigm involves a novel method for conducting phar-macodynamic analyses The use of a certain methodology has received consider-able attention lately This methodology termed Monte Carlo analysis accountsfor individual variability across a wide range of input variables The results ofMonte Carlo analyses estimate what is probable rather than defining what ispossible [45ndash48] Monte Carlo simulations are sampling experiments for estimat-ing the distribution of an outcome that is dependent on multiple probabilisticinput variables For example MIC values obtained from an institution or region


FIGURE 7 Pharmacodynamics and the use of Monte Carlo methodology In-dividual AUC values obtained from patients (clinical trials) Individual MICvalues derived from local or national surveillance data

and the 24 h AUC values from patients are considered as input variables Randomvalues across input variables that conform to their probabilities are generatedand then an output is calculated (eg AUCMIC ratio) (see Fig 7) Each individ-ual output that is calculated is then plotted in a probability chart Monte Carlomethodology demonstrates the range of possible outcomes and the probabilityassociated with each

In a recent study we characterized the pharmacodynamics of levofloxacingatifloxacin and moxifloxacin using pharmacokinetic parameters and local clini-cal isolates of Streptococcus pneumoniae [47] Clinical isolates of Streptococcuspneumoniae were collected consecutively over the 1999ndash2000 respiratory infec-tion season by our laboratory (n 100) Pharmacokinetic data were obtainedfrom patients enrolled in a variety of trials for levofloxacin (172 acutely ill adultpatients with community-acquired infections enrolled in multicenter clinical trialstreated intravenously [mean AUC SD 508 358 microg(mL sdot h)]) [13]gatifloxacin (64 acutely ill adult patients with community-acquired infections en-rolled in a multicenter trial treated intravenously [mean AUC SD 41 163 microgmL sdot h)]) [46] and moxifloxacin (286 adult volunteers in phase I andII trials that received oral moxifloxacin were used [mean AUC SD 185 45 microgmL sdot h)]) [49] Twenty-four-hour AUC values were corrected for proteinbinding based on the following levofloxacin 30 protein-bound gatifloxacin20 and moxifloxacin 50 protein bound Monte Carlo simulation (1000 sub-jects 3) was used to estimate the probability of attaining a free-drug 24 h


AUCMIC ratio of at least 30 Streptococcus pneumoniae susceptibility testingresults were as follows gatifloxacin MIC5090 025025 range 0125ndash120 levo-floxacin MIC5090 07510 range 0125 to 32 moxifloxacin MIC5090 0125019 range 0023 to 60 The probabilities (mean SD) of achieving a 24 hAUCMIC ratio of at least 30 against S pneumoniae isolates were as followsgatifloxacin 988 023 moxifloxacin 979 075 and levofloxacin 811 144

Our results indicated that in our region of the country gatifloxacin andmoxifloxacin were associated with high probabilities of reaching a target 24 hAUCMIC ratio of 30 over a wide range of actual AUCs and MICs comparedwith levofloxacin The results of our regional pharmacodynamic analysis weresimilar to those previously presented using similar pharmacokinetic data and thenational SENTRY database of 1977 isolates of S pneumoniae [46] A limitationof our analysis is that the 24 h AUC values for moxifloxacin were obtained fromindividuals enrolled in phase I and II trials rather than from infected patientsHowever because moxifloxacin is considered to have a dual route of elimination(eg urinary hepatobiliary elimination) the AUC values would not be expectedto be dramatically influenced by moderate renal or hepatic impairment [49] Also24 h AUC values for gatifloxacin and levofloxacin were measured after intrave-nous administration whereas moxifloxacin AUC values were obtained after oraldosing Nevertheless because of the high degree of bioavailability of all threecompounds it is not likely that this fact would change the results of the analysissignificantly [49]

The implications of more sophisticated pharmacodynamic analyses arewide ranging but from a practical viewpoint we have been able to use these datafor formulary evaluation of new antimicrobial agents where sufficient data existto correlate findings with clinical outcome Furthermore clinicians practicing atinstitutions that perform such analyses will have an opportunity to select agentsthat demonstrate optimal pharmacodynamic profiles against specific pathogenssuch as in this case the pneumococci

7 SUMMARY

Over the last several years an amalgam of information has become available toclinicians and researchers alike illustrating the importance of pharmacodynamicsand its role in optimizing drug selection and dosing Once of putative valuerecent developments in pharmacodynamics have provided insight into far morethan the basic understanding of how antimicrobial agents actually kill bacteriamore efficiently In terms of the fluoroquinolones new pharmacodynamicbreakpoints have been discovered and validated in humans Twenty-four-hourAUCMIC ratios of 125 and around 25ndash35 have been established for many gram-


negative and gram-positive pathogens respectively More data are needed to as-sess anaerobic breakpoints in patients to clarify outcome determinants

Pharmacodynamic data have provided insight into the determination ofnew clinically meaningful breakpoints germane to patient care for other drugclasses as well One day clinicians may not ask why their patients with pulmo-nary infections caused by penicillin-resistant S pneumoniae still in fact respondto penicillin Pharmacodynamics will provide the backbone of such a monumen-tal shift in both theory and practice

REFERENCES

1 Polk R Optimal use of modern antibiotics Emerging trends Clin Infect Dis 199929264ndash274

2 Owens RC Jr Ambrose PG Clinical use of the fluoroquinolones Med Clin N Am2000841447ndash1469

3 Ambrose PG Owens RC Jr Quintiliani R Nightingale CH New generation quino-lones Conn Med 199761269ndash272

4 Owens RC Jr Ambrose PG Quintiliani R Nightingale CH Classifying quinoloneanti-infective agents by generation A pharmacodynamic approach to rational drugselection Antibio Clin 1997170ndash74

5 Ambrose PG Owens RC Jr New antibiotics in pulmonary and critical care medicineFocus on advanced generation quinolones and cephalosporins Semin Respir CritCare Med 2000 2119ndash32

6 Eagle H Fleischman R Levy M Continuous vs discontinuous therapy with penicil-lin N Engl J Med 1953238481ndash486

7 Eagle H Effect of schedule of administration on therapeutic efficacy of penicillinImportance of aggregate time penicillin remains at effectively bactericidal levelsAm J Med 19509280ndash299

8 Eagle H Fleischman R Mussleman AD Effective concentrations of penicillin invitro and in vivo for streptococci and pneumococci and Treponema J Bacteriol 195059625ndash643

9 Shah PM Junghanns W Stille W Dosis-Wirkungs-Beziehung der Bakterizidie beiE coli K pneumoniae und Staphylococcus aureus Deut Med Wochenschr 1976101325ndash328

10 Eliopoulos GM Moellering RC Jr Antibiotic synergism and antimicrobial combina-tions in clinical infections Rev Infect Dis 19824282ndash293

11 Calain P Krause K-H Vadaux P et al Early termination of a prospective random-ized trial comparing teicoplanin and flucloxacillin for treating severe staphylococcalinfections J Infect Dis 1987155187ndash191

12 Chambers HF Mills J Drake TA Sande MA Failure of a once-daily regimen ofcefonicid for treatment of endocarditis due to Staphylococcus aureus Rev Infect Dis19846(suppl 4)S870ndashS874

13 Preston SL Drusano GL Berman AL Fowler CL Chow AT Dornseif B ReichlV Natarajan J Corrado M Pharmacodynamics of levofloxacin J Am Med Assoc1998279125ndash129


14 Forrest A Nix DE Ballow CH Schentag J Pharmacodynamics of intravenous ci-profloxacin in seriously ill patients Antimicrob Agents Chemother 1993371073ndash1081

15 Ambrose PG Grasela DM Grasela TH et al Pharmacodynamics of fluoroquino-lones against Streptococcus pneumonia Analysis of phase III clinical trials In Pro-gram and Abstracts of the 40th Interscience Conference on Antimicrobial Agentsand Chemotherapy Sept 17ndash21 2000 Toronto Canada

16 Quintiliani R Owens RC Jr Grant ED Clinical role of fluoroquinolones in patientswith respiratory tract infections Infect Dis Clin Pract 19998(suppl 1)S28ndashS41

17 Craig WA Ebert SC Protein binding and its significance in antibacterial therapyInfect Dis Clin N Am 19893407ndash414

18 Leggett JE Ebert S Fantin B et al Comparative dose-effect relations several dosingintervals for β-lactam aminoglycoside and quinolone antibiotics against gram-nega-tive bacilli in murine thigh-infection and pneumonitis models Scand J Infect Dis199174179ndash184

19 Onyeji CO Bui KQ Owens RC Jr Nicolau DP Quintiliani R Nightingale CHComparative efficacies of levofloxacin and ciprofloxacin against Streptococcuspneumoniae in a mouse model of experimental septicemia Int J Antimicrob Agents199912107ndash114

20 Craig WA Andes DR Correlation of the magnitude of the AUC24MIC for 6 fluoro-quinolones against Streptococcus pneumoniae with survival and bactericidal activityin an animal model In Program and Abstracts of the 40th Interscience Conferenceon Antimicrobial Agents and Chemotherapy Sept 17ndash21 2000 Toronto Canada

21 Lacy MK Lu W Xu X Nicolau DP Quintiliani R Nightingale CH Pharmacody-namic comparison of levofloxacin ciprofloxacin and ampicillin against Streptococ-cus pneumoniae in an in vitro model of infection Antimicrob Agents Chemother199943672ndash677


23 Vesga O Craig WA Activity of levofloxacin against penicillin-resistant Streptococ-cus pneumoniae in normal and neutropenic mice In Program and Abstracts of the36th Annual Interscience Conference on Antimicrobial Agents and ChemotherapyNew Orleans LA September 1996

24 Lee BL Padula AM Kimbrough RC Infectious complications with respiratorypathogens despite ciprofloxacin therapy N Engl J Med 1991325520ndash521

25 Peloquin CA Cumbo TJ Nix DE et al Evaluation of intravenous ciprofloxacin inpatients with nosocomial lower respiratory tract infections Arch Intern Med 19891492269ndash2273

26 Drusano GL Johnson DE Rosen M et al Pharmacodynamics of a fluoroquinoloneantimicrobial agent in a neutropenic rat model of Pseudomonas sepsis AntimicrobAgents Chemother 199337483ndash490

27 Thomas JK Forrest A Bhavnani SM et al Pharmacodynamic evaluation of factorsassociated with the development of bacterial resistance in acutely ill patients duringtherapy Antimicrob Agents Chemother 199842521ndash527

28 Gordon JJ Kauffman CA Superinfections with Streptococcus pneumoniae duringtherapy with ciprofloxacin Am J Med 199089383ndash384


29 Perez-Trallero E Garcia-Arenzana JM Jimenez JA et al Therapeutic failure andselection of resistance to quinolones in a case of pneumococcal pneumonia treatedwith ciprofloxacin Eur J Clin Microbiol Infect Dis 19909905ndash906

30 Righter J Pneumococcal meningitis during intravenous ciprofloxacin therapy AmJ Med 199088548

31 Frieden TR Mangi RJ Inappropriate use of oral ciprofloxacin J Am Med Assoc19902641438ndash1440

32 Giamarrellou H Activity of quinolones against gram-positive cocci Clinical fea-tures Drugs 199549(suppl 2)58ndash66

33 Cooper B Lawlor M Pneumococcal bacteremia during ciprofloxacin therapy forpneumococcal pneumonia Am J Med 198987475

34 Chen DK McGeer A DE Azavedo JC et al Decreased susceptibility of Streptococ-cus pneumoniae to fluoroquinolones in Canada N Engl J Med 1999341233ndash239

35 Linares J De La Campa AG Palleres R Fluoroquinolone resistance in Streptococcuspneumoniae N Engl J Med 19993411546ndash1547

36 Applebaum PC Microbiological and pharmacodynamic considerations in the treat-ment of infection due to antimicrobial-resistant Streptococcus pneumoniae Clin In-fect Dis 200031(suppl 2)S29ndashS34

37 Woodnut G Pharmacodynamics to combat resistance J Antimicrob Chemother20004625ndash31

38 Fishman NO Suh B Weigel LM Lorber B Gelone S Truant AL Gootz TD Chris-tie JE Edelstein PH Three levofloxacin treatment failures of pneumococcal respira-tory tract infections In Program and Abstracts of the 39th Interscience Conferenceon Antimicrobial Agents and Chemotherapy San Francisco CA September 1999

39 Davidson RJ De Azavedo J Bast D et al Levofloxacin treatment failure of pneumo-coccal pneumonia and development of resistance during therapy In Programs andAbstracts of the 40th International Conference on Antimicrobial Agents and Chemo-therapy Toronto Canada 2000

40 Hooper DC Mechanisms of action and resistance of older and newer fluoroquino-lones Clin Infect Dis 200031(suppl 2)S24ndashS28

41 Pestova E Millichap JJ Noskin GA Peterson LR Intracellular targets of moxiflox-acin A comparison with other fluoroquinolones J Antimicrob Chemother 200045583ndash590

42 Fukuda J Hiramatsu K Primary targets of fluoroquinolones in Streptococcus pneu-moniae Antimicrob Agents Chemother 199943410ndash412

43 Zhao X Xu C Domagala J Drlica K DNA topoisomerase targets of the fluoroqui-nolones A strategy for avoiding bacterial resistance Proc Natl Acad Sci USA 19979413991ndash13996

44 Ambrose PG Quintiliani R Limitations of single point pharmacodynamic analysesPediatr Infect Dis J 200019769

45 Drusano GL Preston SL Hardalo CJ et al A model using population pharmacoki-netics of ziracin with Monte Carlo simulation for preliminary MIC breakpoint selec-tion and dose selection decision support In Program and Abstracts of the 39th An-nual Interscience Conference on Antimicrobial Agents and Chemotherapy Sept 26ndash29 1999 San Francisco CA

46 Ambrose PG Grasela DM The use of Monte Carlo simulation to examine pharma-


codynamic variance of drugs fluoroquinolone pharmacodynamics against Strepto-coccus pneumoniae Diagn Microbiol Infect Dis 2000 in press

47 Owens RC Jr Ambrose PG Piper D Thomas S Pharmacodynamic comparison ofnew fluoroquinolones against Streptococcus pneumoniae using Monte Carlo analy-sis In Program and Abstracts of the 40th Interscience Conference on AntimicrobialAgents and Chemotherapy Sept 17ndash21 2000 Toronto Canada

48 Dudley MN Ambrose PG Pharmacodynamics in the study of drug resistance andestablishing in vitro susceptibility breakpoints Ready for prime time Curr OpinionMicrobiol 20003515ndash521

49 Stass H Kubitza D Pharmacokinetics and elimination of moxifloxacin after oraland intravenous administration in man J Antimicrob Chemother 199943(suppl B)83ndash90

8

Glycopeptide Pharmacodynamics

Gigi H Ross and David H Wright

Ortho-McNeil Pharmaceutical Raritan New Jersey

John C Rotschafer and Khalid H Ibrahim

University of Minnesota Minneapolis Minnesota

1 INTRODUCTION

Pharmacodynamics represents a blending of pharmacokinetic parameters with ameasure of bacterial susceptibility the minimum inhibiting concentration (MIC)As such there is a prerequisite that the pharmacokinetic parameters of the antibi-otic be adequately defined prior to exploring the drugrsquos pharmacodynamic proper-ties This in itself has not been an easy task with a drug such as vancomycinwhich has undergone several different formulation changes to remove impuritiesand increase the drugrsquos purity

Measuring vancomycin concentrations by any method other than microbio-logical assay was not possible until the late 1970s when a radioimmunoassay wasintroduced Microbiological assays were technically challenging were accurate atbest to 10 [1] and often could not be performed if patients were receivingother antibiotics

177

178 Ross et al

Pharmacokinetically vancomycin the only commercially available gly-copeptide in the United States has been characterized using one- two- andthree-compartment models as well as noncompartmentally As a result there ismodel-dependent variability in the reporting of vancomycin pharmacokineticparameters Thus getting to a point where clinically applicable pharmacodynamicparameters could be identified and quantified has not been easy Even todaythere are extremely limited in vitro animal and human data characterizing vanco-mycinrsquos performance against only a few bacteria Clearly the characterizationand quantification of vancomycin pharmacodynamics remains a work in progressThe purpose of this review is to examine the microbiology pharmacology andpharmacokinetics of vancomycin so as to build on the data presently availablefor describing the pharmacodynamics of the drug

11 History of Vancomycin

Vancomycin was first introduced in 1956 with widespread clinical use by 1958[2] Originally the drug was isolated from the actinomycete Streptomyces orien-talis however its structure and molecular weight were not identified until 1978The compound consists of a seven-membered peptide chain and two chlorinatedβ-hydroxytyrosine moieties with a molecular weight of 1449 [2] Clinical use ofthe drug was highly prevalent in the late 1950s due to the emergence of penicil-linase-producing strains of staphylococcus but it soon lost favor with the intro-duction of methicillin Impurities in early vancomycin formulations led to anunacceptable incidence of infusion-related reactions Subsequently for 20 yearsvancomycin was used exclusively for the treatment of serious staphylococcalinfections in patients with severe penicillin allergies The current Eli Lilly formu-lation marketed in 1986 is estimated to be 93 pure factor B (vancomycin) andis the result of several production changes and improved separation techniques[2] With the enhancement in purity and the heightened frequency of methicillin-resistant staphylococci and ampicillin-resistant enterococci clinical use of vanco-mycin has significantly increased Today approximately 800000 patients receivevancomycin each year accounting for 14000 kg of drug worldwide [3]

12 Antimicrobial Spectrum

Vancomycin is primarily effective against gram-positive cocci including staphy-lococcus streptococcus and enterococcus and is considered to be bactericidal(MBCMIC 4) against most gram-positive pathogens with the exception toenterococci limited numbers of tolerant (MBCMIC 32) S pneumoniae andtolerant staphylococci The National Committee for Clinical Laboratory Stan-dards has established minimum inhibitory concentration (MIC) standards of sus-ceptibility for vancomycin against staphylococci and enterococci [4] Sensitivestrains have MICs of 4 mgL intermediate isolates have MICs of 8ndash16 mg


L and resistant strains have MICs 32 mgL Staphylococcus aureus and Staphy-lococcus epidermidis including both methicillin-susceptible and methicillin-re-sistant strains are usually sensitive with MIC90 values of 2 mgL [5] All strainsof Streptococcus are sensitive to vancomycin regardless of penicillin susceptibil-ity with MIC90 values less than 1 mgL [4] A recent report however claimsthat approximately 2 of S pneumoniae isolates have developed tolerance tovancomycin [6] Enterococcus faecalis organisms are typically susceptible tovancomycin with MIC50 1 mgL whereas Entercoccus faecium are generallynonsusceptible with MIC50 16 mgL [5] Vancomycin is also effective againstother Streptococcus spp Listeria monocytogenes Bacillus spp Corynebacteriaand anaerobes such as diphtheroids and Clostridium spp including C per-fringens and C difficile Vancomycin has no activity against gram-negative or-ganisms atypical pathogens fungi or viruses

2 PHARMACOLOGY

Vancomycin has multiple mechanisms of action preventing the synthesis andassembly of a growing bacterial cell wall altering the permeability of the bacte-rial cytoplasmic membrane and selectively inhibiting bacterial RNA synthesis[7] Vancomycin prevents polymerization of the phosphodisaccharidendashpentapep-tidendashlipid complex of the growing cell wall at the D-alanyl-D-alanine end of thepeptidoglycan precursor during the latter portion of biosynthesis [7ndash8] By tightlybinding the free carboxyl end of the cross-linking peptide vancomycin stericallyprevents binding to the enzyme peptidoglycan synthetase This activity occursat an earlier point and at a separate site from that of penicillins and cephalosporins[8] Therefore no cross resistance or competition of binding sites occurs betweenthe classes Vancomycin like β-lactams does require actively growing bacteriain order to exert its bactericidal effect However vancomycinrsquos bactericidal activ-ity is restricted to gram-positive organisms because the molecule is too large tocross the outer cell membrane of gram-negative species

Many factors appear to impede vancomycinrsquos bactericidal activity the ab-sence of environmental oxygen the size of the bacterial inoculum and the phaseof bacterial growth The antibiotic appears to kill bacteria more effectively underaerobic conditions than under anaerobic conditions [9] The fact that many gram-positive pathogens including streptococcus and staphylococcus can grow underaerobic and anaerobic conditions could prove problematic in clinical situationsVancomycin activity was reduced by 19 and 99 with increases in inoculumsize from 106 CFUmL to 107 and 108 CFUmL respectively [10ndash11] Whenvancomycin was evaluated against growing and nongrowing Staphylococcusepidermidis cells the drug was found to be effective only against actively grow-ing cultures [12] Finally activity is relatively unaffected by extremes in pH butis maximal at pH 65ndash80 [101113]

180 Ross et al

3 PHARMACOKINETICS

The pharmacokinetics of vancomycin are highly dependent upon the modelingmethod used to characterize the parameters Data can be found in the literaturethat characterize vancomycin using one- two- three-compartment and noncom-partmental pharmacokinetic models that employ different serum samplingschemes and vary in the duration of study As a result the literature varies in thereporting of vancomycin pharmacokinetic parameters

Absorption is complete only when the drug is given intravenously becauseoral absorption is poor and intramuscular administration is both erratic and pain-ful Vancomycin is readily absorbed after intraperitoneal administration also [14]

The distribution of vancomycin is a complex process and is best character-ized by using a multicompartmental approach Vancomycin has a large volumeof distribution varying from 04 to 06 Lkg in patients with normal renal functionand up to 09 Lkg in patients with end stage renal disease [131516] Distributionincludes ascitic pericardial synovial and pleural fluids as well as bone and kid-ney Penetration into bile however is generally considered poor Cerebral spinalfluid concentrations are minimal unless sufficient inflammation is present where10ndash15 of serum concentrations can be obtained [1315] Approximately 10ndash50 of vancomycin is protein-bound primarily to albumin providing a relativelyhigh free fraction of active drug [1317] Studies attempting to measure the effectof other serum proteins have reported virtually no binding to the reactive proteinα-1 glycoprotein but have noted binding to IgA [17]

Drug elimination is almost exclusively via glomerular filtration with 80ndash90 of the vancomycin dose appearing unchanged in the urine within 24 h inpatients with normal renal function [131516] The remainder of the dose is elim-inated via biliary and hepatic means Vancomycin when taken orally is excretedprimarily in the feces Vancomycin is not significantly removed by conventionalhemodialysis or peritoneal dialysis owing to its large molecular weight (2000)however high-flux dialyzers can remove vancomycin and other molecules withmolecular weights of less than 20000 [18]

The elimination of vancomycin is multicompartmental with an alpha ordistribution half-life of 06ndash3 h and a beta or elimination half-life of 4ndash8 hwith normal renal function [1516] Renal insufficiency can prolong the terminalhalf-life to as much as 7ndash12 days Due to the complexity of this biexponentialdecay attempts to utilize various modeling techniques are difficult A one-com-partment model inappropriately characterizes the distribution phase by formulat-ing a regression line that is a hybrid of the alpha and beta phases The pharmacoki-netic parameters produced are accordingly mythical values that may or may notrelate to the actual parameters The extrapolated peak concentration and the half-life can be greatly underestimated depending upon the sampling scheme usedGenerally pairing a serum concentration obtained early in the distribution phase


with a serum concentration late in the elimination phase results in the greatesterror Because one compartment modeling also underestimates the area under theserum concentrationndashtime curve this error is passed along in the calculation ofboth distribution volume and drug clearance

For a concentration-independent or time-dependent antibiotic vancomycinhas an almost ideal pharmacokinetic profile The drug has a large volume ofdistribution low serum protein binding and a long terminal half-life Addition-ally due to modest hepatic metabolism vancomycin-drug interactions are lim-ited As such vancomycin can be used effectively and conveniently to treat infec-tions in most body sites

4 GLYCOPEPTIDE RESISTANCE

Vancomycin has been in clinical use for over 40 years without the emergenceof resistance The multiple modes of action of vancomycin necessitate significantalterations in bacterial wall synthesis in order for the intrinsically susceptibleorganisms to develop resistance Thus the rarity of acquired vancomycin resis-tance led to predictions that such resistance is unlikely to occur on any significantscale [1920]

The first reports of vancomycin-resistant enterococci however began toappear in Europe in the mid-1980s [19] How the enterococci were able to de-velop resistance to vancomycin is unclear However several hypotheses havebeen elucidated ranging from the overuse of antibiotics to the incorporation ofglycopeptide antibiotics into animal feed

Enterococci are normal gut flora and the emergence of resistance has beenlinked to vancomycin overuse in the treatment of Clostridium difficile enterocoli-tis [20] Additionally the parenteral use of vancomycin has steadily increasedsince the late 1970s and may have played a role in the development of vancomy-cin-resistant enterococci (VRE) [21] The agricultural use of avoparcin a relatedglycopeptide may have been important in Europe but this drug has not beenused in the United States In any case the enterococci were the first class oforganisms to acquire vancomycin resistance and vancomycin resistance are nowproblematic in both Europe and the United States [20]

The genetic basis for glycopeptide resistance in enterococci is complexand is characterized by several different phenotypes Resistance-conferring genesencode a group of enzymes that enable the enterococci to synthesize cell wallprecursors generally ending in D-alanine-D-lactate rather than the usual D-ala-nine D-alanine vancomycin binding site [22ndash23] The affinity of vancomycin andteicoplanin for D-alanine-D-lactate is 1000-fold less than that for D-alanine-D-alanine [20]

The most frequently encountered resistance phenotype vanA consists ofhigh level vancomycin resistance (MIC 32 mgL) accompanied by high level

182 Ross et al

resistance to teicoplanin [22] The resistance found on vanA strains is vancomy-cin- andor teicoplanin-inducible The genes encoding vanA resistance are rela-tively easily transferred to other enterococcal species via conjugation [2223]Significant concern has been expressed in both the lay and professional literaturethat this plasmid mediated form of resistance could be passed on not only toother enterococci but also to gram-positive organisms such as staphylococciwhich could lead to catastrophic consequences worldwide Although this eventhas not been realized naturally the vanA plasmid has been successfully intro-duced into staphylococci in the laboratory raising concerns that given enoughtime vancomycin-resistant staphylococci will eventually become a clinical prob-lem [24]

Enterococci with vanB phenotypic resistance have variable levels of vanco-mycin resistance and are susceptible to teicoplanin The vanB phenotype is induc-ible by vancomycin but not teicoplanin and vancomycin exposure produces tei-coplanin resistance Genes that encode VanB are more commonly chromosomalbut can be transferred by conjugation [2225]

The vanC resistance phenotype consists of relatively low levels of vanco-mycin resistance (MIC 8ndash16 mgL) and is devoid of teicoplanin resistanceResistance to vanC is chromosomally produced by encoded genes found in allstrains of Enterococcus flavescens Enterococcus casseliflavus and Entercoccusgallinarum Genes encoded with vanC are not transferable [20] In 1996 Perichonet al [26] described a fourth phenotype vanD similar to vanB found in a rarestrain of Enterococcus faecium [26]

Following a steady increase of VRE prevalence in the United States overthe past 10 years almost 15 of enterococci in hospital intensive care units(participating in the National Nosocomial Infections Surveillance surveys) ex-hibit vancomycin resistance [2327] Similarly rapid increases in VRE prevalencehave also been observed outside the intensive care units in US hospitals [23]Approximately 70 of VRE found in the United States exhibit the vanA resis-tance phenotype with the remaining 25 mostly constituted by the vanB resis-tance phenotype [28]

Evidence exists for both clonal dissemination of resistant strains and rapidtransfer of vancomycin resistance genes among species of hospital enterococci[29ndash30] With the transfer of resistance genes multiple different enterococcalsubtypes carry the same vancomycin resistance genes suggesting a possiblelsquolsquoplasmid or transposon VRE epidemicrsquorsquo [20] Considerable heterogeneity in thegenetic sequence of vancomycin resistance genes found in the United States fur-ther suggest that these genes are being modified as they spread among the variousenterococcal strains [31]

The greatest threat VRE pose is the potential that they could transfer theirresistance encoding genes to other more pathogenic gram-positive bacteria Van-comycin resistance has been transferred from enterococci to streptococci listeria


and S aureus in vitro [2432] Also the recent description of a naturally occurringvancomycin-resistant strain of Streptococcus bovis harboring the vanB resistancephenotype is of significant concern [33]

Low-level vancomycin resistance was reported in clinical isolates of coag-ulase-negative staphylococci in the late 1980s and early 1990s [34ndash36] Al-though troubling these reports were not terribly feared due to the relative lack ofvirulence associated with the coagulase-negative staphylococci In vitro studieshowever demonstrated that both coagulase-negative staphylococci and Saureus isolates when exposed to increasing levels of glycopeptides demon-strated the ability to select for resistant subpopulations [3738] Given thesefindings and the spread of VRE for which excessive use of vancomycin wasidentified as an important control measure the prudent use of vancomycin wassuggested by the CDC as critical to prevent the emergence of resistance amongstaphylococci [39]

In May 1996 a methicillin-resistant Staphylococcus aureus (MRSA) clini-cal isolate that had reduced susceptibility to vancomycin (MIC 8 mgL) wasisolated from a 4 month-old boy with a sternal surgical incision site [4041] Thisisolate has been referred to as Mu50 by the investigators who isolated the organ-ism By current NCCLS standards this S aureus clinical isolate is classified ashaving intermediate resistance to vancomycin In August 1997 the first MRSAisolate intermediately susceptible to vancomycin was reported in Michigan andNew Jersey [4243] Since these reports the organism has been identified in NewYork and England The two US isolates exhibited different antimicrobial suscep-tibility patterns suggesting that these strains are developing de novo secondaryto vancomycin exposure All of these decreased susceptibility strains were iso-lated from patients who had received multiple extended courses of vancomycintherapy

The exact mechanism of resistance for these glycopeptide intermediate sus-ceptibility S aureus (GISA) strains remains largely unknown None of the GISAstrains isolated to date have carried the vanA or vanB genes as judged by PCRDNA amplification Changes in the GISA cell wall structure have been notedhowever and may be in part responsible for the decreased sensitivity to vancomy-cin This is inferred from three findings The cell wall appeared twice as thickas the wall of control strains on electron microscopy there was a three foldincrease in cell wall murein precursor production compared with vancomycin-susceptible MRSA strains and there was a threefold increase in the productionof penicillin-binding protein (PBP) 2 and PBP2prime [4041]

To date there is no evidence that vancomycin resistance genes have beennaturally transferred to the staphylococci or pneumococci however that does notpreclude this event from happening in the future If such a transfer of vancomycinresistance were to occur particularly if the S aureus strain is already methicillin-resistant the result would be an especially terrifying pathogen

184 Ross et al

5 PHARMACODYNAMICS

51 Introduction to Basic Principles

Evaluations of serum peakMIC ratios the ratio of the area under the serumconcentrationndashtime curve for 24 h to the MIC (AUCMIC24) and the length oftime for which antibiotic concentration exceeds the MIC of the infecting organ-ism (T MIC) have been employed as surrogate markers of the bactericidaleffects of antibiotics Pharmacodynamic indices for vancomycin have beenpoorly characterized and therefore most dosing strategies have been based onextrapolations from aminoglycoside studies By modifying aminoglycoside dos-ing models specific peak and trough concentrations have been proposed withthe assumption that similar clinical outcomes will be produced high peak concen-trations being essential for bacterial killing and definitive trough concentrationranges minimizing drug-related toxicity

On the basis of limited in vitro studies T MIC appears to most closelypredict efficacy of vancomycin Therefore the length of time the antibiotic con-centration exceeds the MIC of the offending organism and not the height of thepeak above the MIC as in aminoglycosides should be considered the goal ofthe dosing of vancomycin Although higher serum concentrations of vancomycinmay be helpful in driving the drug to relatively inaccessible sites of infectionsuch as endocardial vegetation or cerebrospinal fluid they are unlikely to improvethe rate of bacterial kill Attempting to push the dose of vancomycin for seriousbut relatively accessible infections will likely only expose patients to an increasedrisk of adverse reactions it is unlikely this approach will alter bacterial response

Investigations of other pharmacodynamic parameters including postantibi-otic effect (PAE) sub-MIC effect (SME) and postantibiotic sub-MIC effect (PASME) have also been undertaken to create a more informative depiction of van-comycin bactericidal activity than MICs allow alone The PAE or the continuedsuppression of microbial growth after limited antibiotic exposure of vancomycinagainst gram-positive bacteria can persist for several hours depending on theorganism and the initial antibiotic concentration [4445] This effect may inhibitregrowth when antibiotic concentrations fall below the MIC of the infecting or-ganism and may be important to consider when dosing vancomycin because ofthe extended half-life and prolonged dosing intervals The postantibiotic effectof vancomycin was evaluated against Staphylococcus epidermidis by Svenssonet al [12] The PAE was dependent upon concentration as drug concentrationincreased from 05 to 8 times the MIC of the organism the PAE increased from02 h to 19 h Another study found PAEs ranging from 06ndash20 h for S aureusto 43ndash65 h for S epidermidis [46]

Because patients receiving antibiotics will always have some amount ofdrug remaining in the body after dosing and elimination PAEs are typically stud-


ied in vitro SMEs and PA SMEs are parameters studied in vivo Generally allof these effects are longer when measured in vivo than when mesaured in vitroSMEs characterize the inhibition of bacterial regrowth following initial sub-MICconcentrations of antibiotic [46] Postantibiotic SMEs on the other hand illus-trate microbial suppression following bacterial exposure to supra-MIC concentra-tions that have declined below the MIC This phenomenon is important clinicallywhere patients given intermittent boluses will experience gradually lowered se-rum and tissue levels that will expose bacteria to both supra- and sub-MICs duringthe dosing interval [46]

52 In Vitro Studies

In vitro investigations have demonstrated that like β-lactam antibiotics vanco-mycin is a concentration-independent or time-dependent killer of gram-positiveorganisms and exhibits minimal concentration-dependent killing In vitro studieshowever can be limiting for several reasons [47]

1 One compartment models represent only concentrations that would ex-ist in the central compartment and not necessarily those that wouldexist at the site of infection

2 Typically only bacteria in log phase growth at standard inocula (105

or 106 CFUmL) are used3 The effects of the immune system or protein binding are generally not

considered

Despite the limitations in vitro studies appear to correlate well with animal andhuman studies and therefore provide useful information for optimal dosing strate-gies in clinical situations

Several investigators demonstrated the concentration-independent killingof vancomycin by exposing various bacteria to increasing amounts of the drugVancomycinrsquos killing effect against Staphylococcus aureus was investigated invitro by Flandrois et al [48] The early portion of the timendashkill curve was thefocus of the study to characterize the bactericidal activity in the initial phases ofthe dosing interval A decrease in CFU of only 1 log was obtained at the end ofthe 8 h study at concentrations of 1 25 5 and 10 times the MIC indicating aconcentration-independent slow rate of kill The killing phase occurred betweenhours 2 and 4 with the CFUmL being held constant for the remainder of thecurve Ackerman et al generated mono- and biexponential killing curves forvancomycin over a 2ndash50 microgmL concentration range to evaluate the relationshipbetween concentration and pharmacodynamic response against Staphylococcusaureus and coagulase-negative Staphylococcus species For all organisms tested

186 Ross et al

killing rates did not change with increasing concentrations of vancomycin andmaximum killing appears to be achieved once concentrations of 4ndash5 times theMIC of the pathogen are obtained

Because the pharmacokinetics of vancomycin involve at minimum biex-ponential decay further studies attempting to simulate this elimination and anyeffects on bacterial killing were investigated Utilizing an in vitro model simulat-ing mono- or biexponential decay Larrson et al [9] found no statistically signifi-cant difference in either the rate or extent of bacterial killing of Staphylococcusaureus Again varying concentrations did not induce a change in bactericidalactivity thereby demonstrating that the high drug concentrations achieved duringthe distribution phase did not enhance the bactericidal activity attained duringthe elimination phase

With the understanding that vancomycin killed staphylococci in a concen-tration-independent fashion the need to select a pharmacodynamic index thatbest predicts efficacy was warranted Duffull et al [47] used four different vanco-mycin regimens against S aureus in an in vitro dynamic model47 Three dosingschedules with different peak concentrations but the same AUC and a fourthdosing regimen with a smaller AUC were compared for efficacy The authorsfound that killing was independent of both peak concentrations and total exposureto drug (AUC) In addition maintaining a constant concentration above the MICwas equally effective even with an AUC that was half of that obtained by theother three dosing regimens This investigation thus supported T MIC as theoptimal parameter for efficacy

Greenberg and Benes [50] produced time-kill curves from experiments per-formed in a static environment with 50 bovine serum and constant antibioticconcentrations They reported a significantly increased rate and extent of killingof Staphylococcus aureus when the concentration of vancomycin increased from20 to 80 mgL even though free drug concentrations for all regimens exceededthe MIC by at least three fold This experiment is one of a few that demonstratedsignificant concentration-dependent killing with vancomycin alone with concen-trations beyond the MIC of the organism

Vancomycin in combination with other antimicrobials has also been evalu-ated Houlihan et al [51] investigated the pharmacodynamics of vancomycinalone and in combination with gentamicin at various dosing intervals againstStaphylococcus aureusndashinfected fibrin-clots in an in vitro dynamic model Van-comycin monotherapy simulations included continuous infusion 500 mg every6 h 1 g every 12 h and 2 g every 24 h all of which produced varying peaks andtroughs While all regimens produced concentrations above the MIC for 100of the dosing intervals no difference in kill was seen with higher peak concentra-tions The investigators also discovered that vancomycin killing was significantlyenhanced by the addition of gentamicin whether it was given every 12 or 24 hand in fact it killed in a concentration-dependent fashion The 2 g dosing scheme


of vancomycin significantly reduced bacterial counts to a greater extent than anyother combination regimen Whether this finding is due to augmented penetrationinto the fibrin clots in the presence of gentamicin is unknown

The vast majority of pharmacodynamic investigations with vancomycininclude the use of Staphylococcus aureus few studies involve other gram-positiveor anaerobic organisms Levett [52] demonstrated time-dependent killing of Clos-tridium difficile by vancomycin in vitro Vancomycin was sub inhibitory at con-centrations below the MIC of the organism Once concentrations at the MIC wereobtained no difference in kill was seen whether 4 mgL (at the MIC) or 1000mgL (250 MIC) was utilized Therefore as for other organisms vancomycinkills C difficile in a concentration-dependent manner until the MIC is achievedbeyond which time-dependent killing is observed

Odenholt-Tornqvist Lowdin and Cars have been the primary source ofinvestigations on the SMEs and PA SMEs of vancomycin In an initial studywith Streptococcus pyogenes and Streptococcus pneumoniae the investigatorsfound that the PA SME with concentrations as low as 03 the MIC preventedregrowth of both Steptococcus species for 24 h [53] In a recent in vitro investiga-tion of the pharmacodynamic properties of vancomycin against Staphylococcusaureus and Staphylococcus epidermidis the same authors detected no concentra-tion-dependent killing [46] Low killing rates were demonstrated by time to 3log kill (T3K) at 24 h with all strains the exception being a methicillin-sensitivestrain of Staphylococcus epidermidis (MSSE) that attained T3K at 9 h Regrowthoccurred between 12 and 24 h when drug concentration had declined to the MICPA SME SME and post-MIC effect (PME) were also evaluated in this studyLong PA-SMEs (23 to 20 h) were found with all strains while SMEs wereshorter (00ndash158 h) Both PA-SMEs and SMEs increased with increasing multi-ples of the MIC Interestingly longer PMEs lsquolsquothe difference in time for the num-bers of CFU to increase 1 logmL from the values obtained at the time when theantibiotic concentration has declined to the MIC compared with the correspond-ing time for a antibiotic-free growth controlrsquorsquo [46] were found with shorter half-lives Other investigations have suggested that the regrowth of bacteria can occurif insufficiently inhibited bacteria are allowed to synthesize new peptidoglycanto overcome the antimicrobialrsquos bactericidal effect [54] The authors assumedthat the PAE PA SME and PME would emulate the time for which the amountof peptidoglycan is kept below a critical level needed for bacterial growth [46]Subsequently the investigators postulated that longer PMEs may occur withshorter half-lives due to the fact that the MIC is obtained faster thereby notallowing adequate peptidoglycan production to initiate regrowth Converselyshorter PMEs were found with longer half-lives With a slower decline to theMIC and a longer period of time at the MIC sufficient peptidoglycan could beproduced to allow regrowth How PA-SMEs SMEs and PMEs will influencedosing schedules is unknown and further investigations are needed

188 Ross et al

53 Animal Studies

Animal studies focusing on pharmacodynamic predictors of efficacy for vanco-mycin are quite limited Peetermans et al [10] with a granulocytopenic mousethigh infection model showed concentration-dependent killing of staphylococcusfor concentrations at or below the MIC Once concentrations exceeded that valuehowever no further kill was seen with increasing doses

The activity of vancomycin was again evaluated against penicillin-resistantpneumococci using a mouse peritonitis model [55] In comparing variouspharmacokineticpharmacodynamic parameters at the ED50 values investigatorsconcluded that both T MIC and Cmax were important predictors of efficacyin their model These parameters were deemed best predictors because they variedthe least Also of significance with this study was the discovery that vancomycinactivity was not influenced by the penicillin susceptibility of the organism

Cantoni et al [56] in an attempt to compare the efficacy of amoxicillinndashclavulanic acid against methicillin-sensitive and methicillin-resistant Staphylo-coccus aureus (MSSA and MRSA respectively) versus vancomycin in a ratmodel of infection found vancomycin activity to be dependent upon strainAgainst the MSSA strain vancomycin at 30 mgkg given every 6 h was moreeffective than the same dose every 12 h Against the MRSA strain the fourtimes daily regimen only marginally improved outcome compared to the twice-daily regimen In that vancomycin concentrations were undetectable after 6 hof therapy the four times daily regimen was the only therapy that allowedconcentrations to remain above the MIC for a majority of the dosing intervalThis finding further supports the dependence of vancomycin activity upon the T MIC

54 Human Studies

In vivo serum bactericidal titers (SBTs) have been evaluated to determine antimi-crobial efficacy An SBT of 18 with vancomycin has been associated with clini-cal cure in patients with staphylococcal infections [57ndash58] This SBT was associ-ated with serum concentrations greater than 12 mgL James et al [59] conducteda prospective randomized crossover study to compare conventional dosing ofvancomycin versus continuous infusions in patients with suspected or docu-mented gram-positive infections In that the most effective concentration of van-comycin against staphylococcus is not known the investigators chose a targetconcentration of 15 microgmL via continuous infusion and peak and trough concen-trations of 25ndash35 and 5ndash10 microgmL respectively with conventional dosing of 1g every 12 h Despite variability in actual concentrations obtained continuousinfusion produced SBTs of 116 whereas conventional dosing produced troughSBTs of 18 which was not found to be statistically insignificant Concentrationsremained above the MIC throughout the entire dosing intervals for all patients


whether they received conventional dosing or continuous infusion and thereforethe authors concluded that both methods of intravenous administration demon-strated equivalent pharmacodynamic activities Although continuous infusiontherapy was more likely than conventional dosing to produce SBTs of 18 orgreater this study did not attempt to evaluate clinical efficacy associated withsuch values Therefore it is unknown whether improved patient outcome wasobtained

Klepser et al [60] in a preliminary report of a multicenter study of patientswith gram-positive infections receiving vancomycin therapy found increasedrates of bactericidal activity with vancomycin trough concentrations greater than10 mgL [60] Bacterial eradication was also correlated with trough SBTs of 18 or greater Patients that failed therapy had pathogen MICs of 1 mgL Hyattet al [61] suggest that the area under the inhibitory serum concentrationndashtimecurve (AUIC) as well as the organismrsquos MIC were associated with clinical out-come By performing a retrospective analysis of 84 patients receiving vancomy-cin therapy for gram-positive infections these authors found that therapy thatproduced AUIC 125 and pathogens with MICs 1 mgL had a higher likeli-hood of failure Therefore these two studies propose that not only T MIC butalso trough values may be important for maximum clinical efficacy

In summary vancomycin demonstrates concentration-independent killingof gram-positive bacteria and peak concentrations do not appear to correlate withrate or extent of kill Maximum killing is achieved at serum concentrations of4ndash5 times the MIC of the infecting pathogen and sustaining concentrations ator above these levels for the entire dosing interval will likely produce the bestantimicrobial effect Dosing strategies should therefore be aimed at maximizingthe time in which concentration at the site of infection remains above the MICof the pathogen Whether the most efficient killing is obtained by continuousinfusion of vancomycin or by intermittent bolus is controversial Several studiesrevealed that no difference in killing is seen between the two methods of adminis-tration [515962] however such benefits as predictable serum concentrationsand ease of administration might be advantageous [62] Conversely due to vanco-mycinrsquos long half-life and the perceived better tolerability associated with inter-mittent bolus injections continuous infusion of this drug may not be needed andis often discouraged [62]

6 CLINICAL APPLICATION

61 Clinical Uses

Vancomycin is available as vancomycin hydrochloride (Vancocin LyphocinVancoled and others) for intravenous use as powder for oral solution and ascapsules for oral use (Vancocin Pulvules) The indications for vancomycin use

190 Ross et al

are limited in relation to its strong gram-positive spectrum Although vancomycinis bactericidal against most gram-positive cocci and bacilli the intravenous prepa-ration should be reserved for serious gram-positive infections not treatable withβ-lactams or other traditional options The use of vancomycin should not precedetherapy with β-lactams for susceptible organisms Clinical outcomes in bothstaphylococci and enterococci show vancomycin inferiority as compared to naf-cillin and ampicillin regarding bactericidal rate and rapidity of blood sterility[63ndash67]

Vancomycin is the drug of choice for serious staphylococcal infections thatcannot be treated with β-lactams due to bacterial resistance [methicillin-resistantStaphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epid-ermidis (MRSE)] or to the patientrsquos inability to receive these medications [68ndash70] Staphylococcal infections include bacteremia endocarditis skin and softtissue infections pneumonia and septic arthritis Dialysis peritonitis due to staph-ylococci may also be treated with IV vancomycin Although vancomycin is indi-cated for S aureus osteomyelitis bone penetrations are extremely variable espe-cially between published studies and treatment with other options could provemore effective [71ndash75] Vancomycin is also indicated for infections due to coagu-lase-negative staphylococci including catheter-associated bacteremia prostheticvalve endocarditis vascular graft infections prosthetic joint infections centralnervous system shunt infections and other infections associated with indwellingmedical devices [68ndash70] Complete cure of most medical-device-related infec-tions usually requires the removal of the device due to the biofilm secreted bythe S epidermidis Staphylococcal treatment with vancomycin may require upto 1 week or longer for clinical response in serious infections such as MRSA[70] Courses of vancomycin that fail to cure serious staphylococcal infectionsmay require the addition of gentamicin rifampin or both [697076]

Two significant clinical issues surround the use of vancomycin for the treat-ment of staphylococcal endocarditis First controversy exists as to whether theaddition of rifampin is synergistic or antagonistic Although certain studies haveproven the combination to be more efficacious than single therapy with vancomy-cin [77ndash79] other more recent publications site the combination as antagonistic[65] Additionally clinical experience with the combination has been inconsistent[80]

The second issue that surrounds vancomycin use for staphylococcal endo-carditis is the potentially better outcome with β-lactams In addition to the invitro data that suggest that vancomycin is less rapidly bactericidal than nafcillinclinical data exist to support this conclusion [63ndash67] Although no large-scalecomparison studies exist to evaluate the efficacy of vancomycin versus β-lactamsin staphylococcal endocarditis assumptions can be formulated from publishedstudies In a study by Korzeniowski and Sande [67] the duration of bacteremiadue to S aureus endocarditis lasted a median of 34 days after treatment with


nafcillin whereas bacteremia lasted a median of 7 days for patients treated withvancomycin in a study conducted by Levine et al [65] The patients in the Levinestudy were infected with methicillin-resistant S aureus in comparison to themethicillin-sensitive organisms from the Korzeniowski study yet in general themorbidity and mortality of bacteremic infections due to MSSA and MRSA arecomparable [66] In a small study that compared vancomycin to nafcillin in Saureus endocarditis the investigators found that patients treated with nafcillinplus tobramycin had a cure rate of 94 whereas only 33 of patients treatedwith vancomycin plus tobramycin were cured [64] Worth mentioning howeveris the fact that while the nafcillin plus tobramycin group consisted of 50 patientsonly three patients received vancomycin plus tobramycin due to β-lactam allergySmall and Chambers [63] performed another study that evaluated the use of van-comycin in 13 patients with staphylococcal endocarditis five of whom failedtherapy The reason for vancomycin ineffectiveness in these cases may be theneed for prolonged high levels of a bactericidal antibiotic however with longerdurations of bacteremia and poorer clinical outcomes serious consideration needsto be given to whether vancomycin should be considered at all in patients withMSSA endocarditis who can tolerate β-lactam therapy

Streptococcal infections not treatable with β-lactams or other traditionaloptions are also proper indications for vancomycin [68ndash70] Endocarditis due toβ-lactam-resistant S viridans or S bovis is a common use of vancomycin al-though organisms with elevated MIC values may require that it be combinedwith an aminoglycoside Vancomycin is the drug of choice for pneumococcalinfections showing high-level resistance to penicillin [68ndash70] Cefotaxime orceftriaxone plus rifampin may be needed to adequately cover S pneumoniae men-ingitis due to vancomycinrsquos poor penetration in the central nervous system[81ndash82] Although penetration is enhanced while meninges are inflamed as inmeningitis and shunt infections certain cases may require intrathecal or intraven-tricular administration to obtain therapeutic levels

As for enterococcal infections vancomycin represents the treatment ofchoice for ampicillin-resistant enterococcus [68ndash70] Enterococcus endocarditisand other infections may require the addition of an aminoglycoside such as genta-micin Vancomycin is also the treatment of choice for corynebacterial infections[68ndash70]

Empirically vancomycin should be used only in limited situations Vanco-mycin can be considered for febrile neutropenic patients presenting with clinicalsigns and symptoms of gram-positive infections in areas of high MRSA preva-lence [39] Other indications for empirical use of vancomycin in neutropenicpatients with fever include the presence of severe mucositis colonization withMRSA or penicillin-resistant Streptococcus pneumoniae prophylaxis with quino-lone antibiotics or obvious catheter-related infection [83] Vancomycin shouldbe discontinued after 4ndash5 days if no infection is identified or if initial cultures

192 Ross et al

for gram-positive organisms are negative after 24ndash48 h For prophylaxis vanco-mycin may be used perioperatively with prosthesis implantation only in severelyβ-lactam allergic patients [39] Vancomycin is also used for endocarditis prophy-laxis for β-lactam allergic patients

Orally vancomycin is indicated for metronidazole-refractory antibiotic-associated colitis caused by Clostridium difficile [3968ndash70] Intravenous admin-istration of vancomycin typically does not achieve adequate levels in the colonlumen to successfully treat antibiotic-associated colitis however there are rarereports of success with this route cited in the literature84 Administration via na-sogastric tube enema ileostomy colostomy or rectal catheter may be neededif the patient presents with severe ileus Oral vancomycin has also been usedprophylactically to prevent endogenous infections in cancer and leukemia pa-tients This regimen seems to decrease the C difficile associated with the chemo-therapy [85ndash87]

62 Inappropriate Uses

Although vancomycin is an effective option for most gram-positive infectionsthe drug needs to be judiciously used to prevent the emergence and spread ofresistance Vancomycin should not be used when other drug options such as β-lactams are viable Microbial susceptibilities need to be treated to determine theappropriateness of vancomycin therapy and the antibiotic should be changed ifthe organism is susceptible to a different agent

The CDC has published guidelines for the appropriate use of vancomycin(Tables 1 and Table 2) [39] however vancomycin misuse around the nation iswidespread A retrospective study from May 1993 to April 1994 identified 61 ofvancomycin usage as inappropriate according to the CDC criteria [88] A similarevaluation published in 1997 found that only 47 of vancomycin orders pre-scribed for 7147 patients were appropriate [89] According to this study inade-

TABLE 1 Appropriate Use of Vancomycin

Treatment of serious infections due to β-lactam-resistant gram-positivepathogens

Treatment of gram-positive infections in patients with serious β-lactamallergies

Antibiotic-associated colitis failure to metronidazoleEndocarditis prophylaxis per American Heart Association

recommendationsAntibiotic prophylaxis for implantation of prosthetic devices at institutions

with a high rate of infections due to methicillin-resistant staphylococci

Source Ref 37


TABLE 2 Inappropriate Use of Vancomycin

Routine surgical prophylaxisEmpirical treatment for febrile neutropenic patients without strong

evidence of gram-positive infection and high prevalence of β-lactamresistant organisms in the institution

Treatment in response to a single positive blood culture for coagulase-negative staphylococci when other blood cultures taken appropriately inthe same time frame are negative

Continued empirical use without positive culture for β-lactam-resistantgram-positive pathogen

Systemic or local prophylaxis for central or peripheral catheterSelective gut decontaminationEradication of methicillin-resistant Staphylococcus aureus colonizationPrimary treatment of antibiotic-associated colitisRouting prophylaxis for patients on chronic ambulatory peritoneal dialysisRoutine prophylaxis for very low birthweight infantsTopical application or irrigation

Source Ref 37

quate use and inappropriate control patterns were similar whether large teachingcenters or small rural hospitals were evaluated As such alternative methods ofvancomycin control need to be implemented to ensure adequate use and limitresistance

63 Toxicity and Adverse Drug Reactions

A variety of adverse reactions have been associated with vancomycin includingfever rash phlebitis neutropenia nephrotoxicity auditory toxicity interstitialnephritis and infusion-related reactions Many of the infusion-related reactionswere likely due to impurities in the initial formulations and have been signifi-cantly reduced with the newer formulations The red man or red neck syndromeis an anaphylactoid reaction related to rapid infusion of large doses typically12 mg(kg sdot h) [1369ndash70] The reaction begins 10 min after infusion and gener-ally resolves within 15ndash20 min after stopping the dose Patients may experiencetachycardia chest pain dyspnea urticaria and swelling of the face lips andeyelids Additionally patients may experience a hypotensive episode with a 25ndash50 reduction in systolic blood pressure Interestingly volunteers receiving van-comycin infusions have a higher propensity toward the reaction than patients [62]The reason is unknown Symptoms of red nan syndrome appear to be histamine-mediated however investigations are inconclusive Extending the administrationof vancomycin to 1 h or a maximum of 15 mgmin should prevent most infusion-related reactions

194 Ross et al

Vancomycin toxicity was retrospectively studied by Farber and Moellering[90] in 98 patients They noted a 13 incidence of phlebitis a 3 incidence offever and rash and a 2 incidence of neutropenia However this report mayoverestimate true adverse reactions because of the inclusion of many potentiallyhigh-risk patients Interestingly whereas other studies have shown that concomi-tant aminoglycosides are not a risk factor for nephrotoxicity [91] patients receiv-ing both vancomycin and an aminoglycoside experienced a 35 incidence ofreversible nephrotoxicity which is more than expected from either antibioticalone Only 5 of patients receiving vancomycin alone experienced nephrotoxic-ity The authors also found that patients with nephrotoxicity had trough concen-trations of 20ndash30 mgL

Vancomycin ototoxicity has been reported with peak serum concentrationsof 80ndash100 mgL [92] Geraci [92] identified two patients with vancomycin-induced ototoxicity one of whom had a history of renal disease an elevatedblood urea nitrogen on admission and a recorded diastolic blood pressure ofzero Serum concentrations determined 3ndash6 h after the dose was administeredranged from 80 to 95 mgL Due to the biexponential nature of the vancomycinserum concentrationndashtime curve the true vancomycin peak was likely near 200ndash300 mgL Farber and Moellering [90] also reported the occurrence of ototoxicityin a patient who at 1 h postinfusion had serum concentrations of 50 mgLhowever the true peak was likely in the toxic range as defined by Geraci [92]

In summary the incidence of adverse reactions associated with vancomycinare relatively infrequent Only approximately 40 cases of oto- and nephrotoxicitywere reported in the medical literature in the years 1956ndash1984 despite incessantuse Most of these cases were complicated by concomitant aminoglycoside ther-apy and pre-existing renal problems as well as investigator discrepancies in inter-preting serum levels

64 Dosing and Therapeutic Monitoring

Medical literature abounds that questions the need to therapeutically monitor van-comycin concentrations Cantu et al [93] suggest that monitoring vancomycinconcentrations is unnecessary in that no correlation has been demonstrated be-tween drug levels toxicity and clinical response Opponents propose that vanco-mycin can be dosed using published nomograms based on the the patientrsquos ageweight and estimated creatinine clearance Conversely Moellering et al [94]argue that therapeutic vancomycin monitoring would in fact be prudent for opti-mal clinical response and restriction of toxicity in such situations as patients onhemodialysis patients with rapidly changing renal function and patients receiv-ing high dose vancomycin or concomitant aminoglycoside therapy

Numerous strategies do exist for empirically dosing vancomycin Adminis-tering 500 mg every 6 h 1 g every 12 h or 20ndash40 mgkg body weightday are


commonly employed In addition nomograms exist such as those established byMatzke et al [95] Moellering et al [94] Lake and Peterson [96] and Nielsenet al [97] Serious faults lie in the dependence of these nomograms on efficacioususe of vancomycin however because the authors assume rather than prove thattheir method of pharmacokinetically modeling the data was appropriate Mostempirical regimens were designed to provide peak concentrations of 20ndash40 mgL and trough concentrations of 5ndash10 mgL (or approximately 5 times the MICof the infecting pathogen) however such practices place only 3ndash23 of patientsin this therapeutic range according to one published study [98] Unfortunatelyalthough such goals in serum levels are set no solid data are available to supportthis therapeutic range and accordingly serum peak and trough concentrationshave been selected somewhat arbitrarily based on speculations from retrospec-tive studies case reports and personal opinions Peak concentrations appear toplay little to no role in the efficacy of the drug and appear to have limited involve-ment in toxicity unless exceedingly large peak values are obtained On the otherhand trough concentrations may be useful monitoring parameters Because van-comycin is a concentration-independent killer the goal of therapy should be tomaintain the unbound concentration above the microbial MIC for a significantportion of the dosing interval because regrowth of most organisms will beginshortly after drug concentrations fall below the MIC A depiction of predictedvancomycin pharmacodynamic indices obtained from a typical intravenous doseusing various pathogen MICs is presented in Table 3

The role of vancomycin degradation products also needs to be consideredwhen interpreting levels in patients with renal failure where half-lives are signifi-cantly extended [99ndash100] In vitro and in vivo vancomycin breaks down overtime to form crystalline degradation products Antibodies in commercial assayssuch as TDx fluorescence polarization immunoassay cross react with major and

TABLE 3 Estimated Vancomycin PharmacodynamicRatios for Various MIC Valuesa

MIC (mgL) CpmaxMIC T MIC (h) AUC24MIC

025 140 12 78405 70 12 39210 35 12 19620 175 12 9840 875 12 4980 438 11 245

a Calculations based on a 1 g dose given every 12 h to a 70 kgpatient with normal renal function

196 Ross et al

minor degradation products thereby overstating factor B (active drug) content inthe level This can result in an overstated vancomycin concentration of 20ndash50

In summary trough concentrations of 5ndash10 mgL appear to be reasonablegoals for vancomycin therapy in that MICs of most gram-positive pathogens are1 mgL Such concentrations would allow the unbound concentrations to re-main above the MIC of the organism for the entire dosing interval Administering10ndash15 mgkg per dose and adjusting the dosing interval per renal function basedupon numerous published nomograms is not likely to produce lsquolsquotoxicrsquorsquo peak con-centrations and should allow lsquolsquotherapeuticrsquorsquo concentrations throughout the dosinginterval in the majority of patients with normal renal function Loading dosesare not typically needed because transiently high distribution phase concentra-tions are unlikely to enhance bacterial killing However loading doses may bereasonable in patients in whom the site of infection is distal to the central compart-ment or poorly accessible Until a relationship among clinical efficacy toxicityand vancomycin concentration is established vancomycin therapy will inevitablycontinue to be monitored in an attempt to improve patient outcome Whethertherapeutic monitoring of vancomycin should be a standard of practice or is nec-essary only in patients receiving high dose therapy patients on concomitantaminoglycoside therapy or patients with renal insufficiency or failure on dialysisis likely to remain a personal preference until further studies establish guidelinesHowever if the CDC guidelines for appropriate vancomycin usage were strin-gently followed at least half of vancomycin use could be eliminated leaving theremaining patients to be monitored

7 OTHER GLYCOPEPTIDES

71 Teicoplanin

Teicoplanin like vancomycin binds to the terminal D-alanyl-D-alanine portionof the peptidoglycan cell wall of actively growing gram-positive bacteria to exertits bactericidal activity [101] Currently available only in Europe teicoplanin canbe used to treat infections caused by both methicillin-sensitive and -resistantstrains of Staphylococcus aureus S epidermidis streptococci and enterococciClinical trials have demonstrated teicoplanin to be a safe well tolerated agentwith reports of side effects occurring in 6ndash13 of recipients [101] The mostprevalent adverse reactions reported are pain at the injection site and skin rashNephro- and ototoxicity are uncommon even when the drug is used concomitantlywith other nephro- and ototoxic drugs Pharmacokinetically teicoplanin differsfrom vancomycin The half-life is considerably longer (47 h) and the percentprotein-bound nears 90 [101] Also teicoplanin can be administered by eitherthe intravenous or intramuscular route as opposed to vancomycin which is lim-ited parenterally to the intravenous route Pharmacodynamic evaluations virtually


duplicate those of vancomycin once the heightened protein binding of teicoplaninand subsequent lower active free concentrations are accounted for [102] Furtherreviews of teicoplanin can be found elsewhere [101103]

72 LY333328

LY333328 (Eli Lilly and Company) is a synthetic glycopeptide that is currentlybeing developed to treat gram-positive bacterial infections including those resis-tant to vancomycin Because it is still in the early stages of development littleis known about the antibiotic The drug acts on the same molecular target asvancomycin and other glycopeptide antibiotics [104] however LY333328 ap-pears to display concentration-dependent bactericidal activity against gram-positive pathogens [102ndash106] The half-life is long approaching 105 dayswhich may allow for infrequent dosing [107] Pharmacodynamic investigationsand clinical efficacy trials are needed prior to drug approval and utilization

8 CONCLUSION

With years of clinical experience vancomycin has proven to be a safe and effica-cious agent against gram-positive pathogens including many multidrug-resistantstrains Despite this history to date the therapeutic range has not been rigorouslydefined however going beyond the currently suggested therapeutic range is notlikely to improve antibiotic performance The accumulation of in vitro and invivo studies suggests that vancomycin is a concentration-independent killer ofgram-positive organisms with maximum killing occurring at serum concentra-tions of 4ndash5 times the MIC of the infecting organism High peak concentrationsare not associated with an improved rate or extent of kill and therefore therapyshould be targeted toward sustaining serum concentrations above the MIC for alarge portion of the dosing interval With the high level of vancomycin use thedevelopment and spread of vancomycin-resistant organisms is a formidable andpredictable occurrence At a time when we are attempting to be more prudentand judicious in the use of vancomycin we also find ourselves more dependenton the drug Unfortunately this combination of factors may drive bacterial resis-tance and ultimately nullify a drug that has been a gold standard product for ahalf a century

REFERENCES

1 KB Crossley JC Rotschafer MM Chern KE Mead DE Zaske Comparison of aradioimmunoassay and a microbiological assay for measurement of serum vanco-mycin concentrations Antimicrob Agents Chemother 198017654ndash657

2 GL Cooper DB Given The development of vancomycin In GL Cooper and DB

198 Ross et al

Given eds Vancomycin A Comprehensive Review of 30 Years of Clinical Re-search Park Row Publ 19861ndash6

3 HA Kirst DG Thompson TI Nicas Historical yearly usage of vancomycin Antimi-crob Agents Chemother 1998421303ndash1304

4 National Committee for Clinical Laboratory Standards Performance standards forantimicrobial susceptibility testing 9th Informational Supplement M100-S9 1999Vol 19(1) National Committee for Clinical Laboratory Standards Wayne PA

5 RN Jones CH Ballow DJ Biedenbach JA Deinhart JJ Schentag Antimicrobialactivity of quinupristindalfopristin (RP 59500 Synercid) tested against over28000 recent clinical isolates from 200 medical centers in the United States andCanada Diagn Microbiol Infect Dis 199831437ndash451

6 Nature 1999399524ndash526590ndash5937 PE Reynolds Structure biochemistry and mechanism of action of glycopeptide

antibiotics Eur J Clin Microbiol Infect Dis 19898943ndash9508 PE Reynolds EA Somner Comparison of the target sites and mechanisms of glyco-

peptide and lipoglycodepsipeptide antibiotics Drugs Under Exp Clin Res 199016385ndash389

9 AJ Larsson KJ Walker JK Raddatz JC Rotschafer The concentration-independenteffect of monoexponential and biexponential decay in vancomycin concentrationon the killing of Staphylococcus aureus under aerobic and anaerobic conditionsJ Antimicrob Chemother 199638589ndash597

10 WE Peetermans JJ Hoogeterp AM Hazekamp-VanDokkum P Van Den BroekH Mattie Antistaphylococcal activities of teicoplanin and vancomycin in vitro andin an experimental infection Antimicrob Agents Chemother 1990341869ndash1874

11 KC Lamp MJ Rybak EM Bailey GW Kaatz In vitro pharmacodynamic effectsof concentration pH and growth phase on serum bactericidal activities of dapto-mycin and vancomycin Antimicrob Agents Chemother 1992362709ndash2714

12 E Svensson H Hanberger LE Nilsson Pharmacodynamic effects of antibiotics andantibiotic combinations on growing and nongrowing Staphylococcus epidermidiscells Antimicrob Agents Chemother 199741107ndash111

13 TS Lundstrom JD Sobel Vancomycin trimethoprim-sulfamethoxazole and rifam-pin Infect Dis Clin N Am 19959747ndash767

14 GD Morse MA Apicella JJ Walshe Absorption of intraperitoneal antibiotics DrugIntell Clin Pharm 19982258ndash61

15 RC Moellering Pharmacokinetics of vancomycin J Antimicrob Chemother 198414(suppl D)43ndash52

16 JC Rotschafer K Crossley DE Zaske K Mead RJ Sawchuk LD Solem Pharmaco-kinetics of vancomycin Observation in 28 patients and dosage recommendationsAntimicrob Agents Chemother 198222391ndash394

17 H Sun EG Maderazo AR Krusell Serum protein-binding characteristics of vanco-mycin Antimicrob Agents Chemother 1993371132ndash1136

18 GR Matzke RF Frye Drug therapy individualization for patients with renal insuf-ficiency In JT DiPiro RL Talbert GC Yee GR Matzke BG Wells LM Poseyeds Pharmacotherapy A Physiologic Approach 3rd ed Stamford CT Appleton ampLange 19971083ndash1103


19 N Woodford AP Johnson D Morrison DCE Speller Current perspectives on gly-copeptide resistance Clin Microbiol Rev 19958585ndash615

20 RC Moellering Vancomycin-resistant enterococci Clin Infect Dis 1998261196ndash1199

21 J Ena RW Dick R Jones RP Wenzel The epidemiology of intravenous vancomy-cin usage in a university hospital J Am Med Assoc 1993269598ndash602

22 M Arthur PE Reynolds F Depardieu et al Mechanisms of glycopeptide resistancein enterococci J Infect Dis 19963211ndash16

23 HS Gold RC Moellering Jr Drug therapy Antimicrobial-drug resistance N EnglJ Med 19963351445ndash1454

24 WC Noble Z Virani RGA Cree Co-transfer of vancomycin and other resistancegenes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus FEMSMicrobiol Lett 199293195ndash198

25 R Quintiliani S Evers P Courvalin The vanB gene confers various levels of self-transferable resistance to vancomycin in enterococci J Infect Dis 1993161220ndash1223

26 B Perichon PE Reynolds P Courvalin VanD-type glycopeptide-resistant Entero-coccus faecium (abst LB12) In Program and Abstracts of the 36th InterscienceConference on Antimicrobial Agents and Chemotherapy (New Orleans) Washing-ton DC Am Soc Microbiol 19965

27 Centers for Disease Control and Prevention Nosocomial enterococci resistant tovancomycinmdashUnited States 1989ndash1993 MMWR Morb Mortal Wkly Rep 199342597ndash599

28 NC Clark RC Cooksey BC Hill JM Swenson FC Tenover Characterization ofglycopeptide-resistant enterococci from US hospitals Antimicrob Agents Chemo-ther 199342597ndash599

29 LG Rubin V Tucci E Cercenado GM Eliopoulos HD Isenberg Vancomycin resis-tant Enterococcus faecium in hospitalized children Infect Control Hosp Epidemiol199213700ndash705

30 JF Boyle SA Saumakis A Rendo et al Epidemiologic analysis and genotypiccharacteristic of a nosocomial outbreak of vancomycin-resistant enterococci J ClinMicrobiol 1993311280ndash1285

31 S Handwerger J Skoble LF Discotto MJ Pucci Heterogeneity of the vanA genecluster in clinical isolates of enterococci from the northeastern United States Anti-microb Agents Chemother 199539362ndash368

32 F Biavasco E Giovanetti A Miele C Vignaroli B Facinelli PE Varalso In vitroconjugative transfer of vanA vancomycin resistance between enterococci and lister-iae of different species Eur J Clin Microbiol Infect Dis 19961550ndash59

33 C Poyart C Pierre G Quesne et al Emergence of vancomycin resistance in thegenus Streptococcus Characterization of a vanB transferable determinant in Strep-tococcus bovis Antimicrob Agents Chemother 19974124ndash29

34 RS Schwalbe JT Stapleton PH Gilligan Emergence of vancomycin resistance incoagulase-negative staphylococci N Engl J Med 1987316927ndash931

35 LA Veach MA Pfaller M Barrett FP Koontz RP Wenzel Vancomycin resistancein Staphylococcus haemolyticus causing colonization and bloodstream infectionJ Clin Microbiol 1990282064ndash2068

200 Ross et al

36 D Sanyal AP Johnson RC George BD Cookson AJ Williams Peritonitis dueto vancomycin-resistant Staphylococcus epidermidis Lancet 199133754

37 RS Schwalbe WJ Ritz PR Verma EA Barranco PH Gilligan Selection for van-comcyin resistance in clinical isolates of Staphylococcus haemolyticus J Infect Dis199016145ndash51

38 RS Daum S Gupta R Sabbagh WM Milewski Characterization of Staphylococcusaureus isolates with decreases in susceptibility to vancomycin and teicoplanin Iso-lation and purification of a constitutively produced protein associated with de-creased susceptibility J Infect Dis 19921661066ndash1072

39 Hospital Infection Control Practices Advisory Committee Recommendations forpreventing the spread of vancomycin resistance Recommendations of the HospitalInfection Control Practices Advisory Committee (HICPAC) MMWR Morb MortalWkly Rep 199544(12)

40 K Hiramatsu H Hanaki T Ino K Yabuta T Oguri FC Tenover Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin suscepti-bility J Antimicrob Chemother 199740135ndash136

41 Centers for Disease Control and Prevention Reduced susceptibility of Staphylococ-cus aureus to vancomycinmdashJapan 1996 MMWR Morb Mortal Wkly Rep 199746(27)624ndash628

42 Centers for Disease Control and Prevention Staphylococcus aureus with reducedsusceptibility to vancomycinmdashUnited States 1997 MMWR Morb Mortal WklyRep 199746(33)756ndash766

43 Centers for Disease Control and Prevention Update Staphylococcus aureus withreduced susceptibility to vancomycinmdashUnited States 1997 MMWR Morb MortalWkly Rep 199346(35)813ndash815

44 WA Craig B Vogelman The post-antibiotic effect Ann Intern Med 1987106900ndash902

45 MA Cooper YF Jin JP Ashby JM Andrews R Wise In vitro comparison of thepostantibiotic effect of vancomycin and teicoplanin J Antimicrob Chemother 199026203ndash207

46 E Lowdin I Odenholt O Cars In vitro studies of pharmacodynamic properties ofvancomycin against Staphylococcus aureus and Staphylococcus epidermidis Anti-microb Agents Chemother 1998422739ndash2744

47 SB Duffull EJ Begg ST Chambers ML Barclay Efficacies of different vancomy-cin dosing regimens against Staphylococcus aureus determined with a dynamic invitro model Antimicrob Agents Chemother 1994382480ndash2482

48 JP Flandrois G Fardel G Carret Early stages of in vitro killing curve of LY146032and vancomycin for Staphylococcus aureus Antimicrob Agents Chemother 198832454ndash457

49 Ackerman AM Vannier E Eudy Analysis of vancomycin time-kill studies withStaphylococcus species by using a curve stripping program to describe the relation-ship between concentration and pharmacodynamic response Antimicrob AgentsChemother 1992361766ndash1769

50 RN Greenberg CA Benes Time-kill studies with oxacillin vancomycin and tei-coplanin versus Staphylococcus aureus J Infect Dis 1611036ndash1037 1990

51 HH Houlihan RC Mercier MJ Rybak Pharmacodynamics of vancomycin alone


and in combination with gentamicin at various dosing intervals against methicillin-resistant Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infectionmodel Antimicrob Agents Chemother 1997412497ndash2501

52 PN Levett Time-dependent killing of Clostridium difficile by metronidazole andvancomycin J Antimicrob Chemother 19912755ndash62

53 I Odenholt-Tornqvist E Lowdin O Cars Postantibiotic sub-MIC effects of vanco-mycin roxithromycin sparfloxacin and amikacin Antimicrob Agents Chemother1992361852ndash1858

54 D Greenwood K Bidgood M Turner A comparison of the responses of staphylo-cocci to teicoplanin and vancomycin J Antmicrob Chemother 198720155ndash164

55 JD Knudsen K Fuursted F Espersen N Frimodt-Moller Activities of vancomycinand teicoplanin against penicillin-resistant pneumococci in vitro and in vivo andcorrelation to pharmacokinetic parameters in the mouse peritonitis model Antimi-crob Agents Chemother 1997411910ndash1915

56 L Cantoni A Wenger MP Glauser J Bille Comparative efficacy of amoxicillin-clavulanate cloxacillin and vancomycin against methicillin-sensitive and methicil-lin-resistant Staphylococcus aureus endocarditis in rats J Infect Dis 1989159989ndash993

57 UB Schadd GH McCracken JD Nelson Clinical pharmacology and efficacy ofvancomycin in pediatric patients J Pediatr 198096119ndash126

58 DB Louria T Kaminski J Buchman Vancomycin in severe staphylococcal infec-tions Arch Intern Med 1961107225ndash240

59 JK James SM Palmer DP Levine MJ Rybak Comparison of conventional dosingversus continuous-infusion vancomycin therapy for patients with suspected or doc-umented gram-positive infections Antimicrob Agents Chemother 199640696ndash700

60 ME Klepser SL Kang BJ McGrath et al Influence of vancomycin serum concen-tration on the outcome of gram-positive infections Presented at The American Col-lege of Clinical Pharmacy Annual Winter Meeting Feb 6ndash9 1994 San Diego

61 JM Hyatt PS McKinnon GS Zimmer JJ Schentag The importance ofpharmacokineticpharmacodynamic surrogate markers to outcome Clin PharmConcepts 199528143ndash160

62 ME Klepser KB Patel DP Nicolau R Quintiliani CH Nightingale Comparison ofbactericidal activities of intermittent and continuous infusion dosing of vancomycinagainst methicillin-resistant Staphylococcus aureus and Enterococcus faecalisPharmacotherapy 1998181069ndash1074

63 PM Small HF Chambers Vancomycin for Staphylococcus aureus endocarditis inintravenous drug users Antimicrob Agents Chemother 1990341227ndash1231

64 HF Chambers RT Miller MD Newman Right sided Staphylococcus aureus endo-carditis in intravenous drug abusers Two-week combination therapy Ann InternMed 1998109619ndash624

65 DP Levine BS Fromm BR Reddy Slow response to vancomycin or vancomycinplus rifampin in methicillin-resistant Staphylococcus aureus endocarditis Ann In-tern Med 1991115674ndash680

66 AW Karchmer Staphylococcus aureus and vancomycin The sequel Ann InternMed 1991115739ndash741

202 Ross et al

67 O Korzeniowski MA Sande National Collaborative Endocarditis Study GroupCombination antimicrobial therapy for Staphylococcus aureus endocarditis in pa-tients addicted to parenteral drugs and in nonaddicts Ann Intern Med 198297496ndash503

68 Anonymous The choice of antibacterial drugs Med Lett Drugs Ther 19984033ndash42

69 RH Glew MA Keroack Vancomycin and teicoplanin In SL Grobach JG BartlettNR Blacklow eds Infectious Diseases WB Saunders Philadelphia 1998260ndash269

70 R Fekety Vancomycin and teicoplanin In GL Mandell JE Bennett R Dolin Prin-ciples and Practice of Infectious Diseases 4th ed Churchill Livingstone NewYork 1995346ndash353

71 CW Norden K Niederreiter EM Shinners Treatment of experimental chronic os-teomyelitis due to staphylococcus aureus with vancomycin and rifampin J InfectDis 1983147352ndash357

72 C Martin M Alaya MN Mallet X Viviand K Ennabli R Said PD Micco Penetra-tion of vancomycin into mediastinal and cardiac tissues in humans AntimicrobAgents Chemother 199438396ndash399

73 L Massias C Dubois P de Lentdecker O Brodaty M Fischler R Farinotti Penetra-tion of vancomycin in uninfected sternal bone Antimicrob Agents Chemother1993362539ndash2541

74 AL Graziani LA Lawson GA Gibson MA Steinberg RR MacGregor Vancomy-cin concentrations in infected and noninfected human bone Antimicrob AgentsChemother 1998321320ndash1322

75 JR Torres CV Sanders AC Lewis Vancomycin concentrations in human tissuePreliminary report J Antimicrob Chemother 19795475

76 V Gopal AL Bisno FJ Silverblatt Failure of vancomycin treatment in Staphylococ-cus aureus endocarditis In vivo and in vitro observations J Am Med Assoc 19762361604ndash1606

77 AS Bayer K Lam Efficacy of vancomycin plus rifampin in experimental aortic-valve endocarditis due to methicillin-resistant Staphylococcus aureus In vitro-invivo correlations J Infect Dis 1985151157ndash165

78 RM Massanari ST Donta The efficacy of rifampin as adjunctive therapy in selectedcases of staphylococcal endocarditis Chest 197873371ndash375

79 RJ Faville DE Zaske EL Kaplan K Crossley LD Sabath PG Quie Staphylococ-cus aureus endocarditis Combined therapy with vancomycin and rifampin J AmMed Assoc 19782401963ndash1965

80 DP Levine RD Cushing J Ji WJ Brown Community-acquired methicillin-resistant Staphylococcus aureus endocarditis in the Detroit Medical Center AnnIntern Med 198297330

81 PF Viladrich F Gudiol J Linares R Pallares I Sabate G Rufi J Ariza Evaluationof vancomycin for therapy of adult pneumoccocal meningitis Antimicrob AgentsChemother 1991352467ndash2472

82 JS Bradley WM Scheld The challenge of penicillin-resistant Streptococcus pneu-moniae meningitis current antibiotic therapy in the 1990s Clin Infect Dis 199724(suppl 2)S213ndashS221


83 WT Hughes D Armstrong GP Bodey AE Brown JE Edwards R Feld P PizzoKVI Rolston JL Shenep LS Young 1997 guidelines for the use of antimicrobialagents in neutropenic patients with unexplained fever Clin Infect Dis 199725551ndash573

84 ST Donta GM Lamps RW Summers TD Wilkins Cephalosporin-associated coli-tis and Clostridium difficile Arch Intern Med 1980140574ndash576

85 JG Bartlett Antibiotic-associated colitis Disease-A-Month 1984301ndash5486 SD Miller HJ Koornhof Clostridium difficile colitis associated with the use of

antineoplastic agents Eur J Clin Microbiol 1984310ndash1387 MA Cudamore J Silva R Fekety MK Liepman KH Kim Clostridium difficile

colitis associated with cancer chemotherapy Arch Intern Med 1982142333ndash33588 SV Johnson LL Hoey K Vance-Bryan Inappropriate vancomycin prescribing

based on criteria from the Centers for Disease Control and Prevention Pharmaco-therapy 199515579ndash585

89 C Gentry Wide overuse of antibiotic cited in study Wall St J 19974B-190 BF Farber RC Moellering Retrospective study of the toxicity of preparations of

vancomycin from 1974 to 1981 Antimicrob Agents Chemother 19832313891 K Vance-Bryan JC Rotschafer SS Gilliland KA Rodvold CM Fitzgerald DR

Guay A comparative assessment of vancomycin-associated nephrotoxicity in theyoung versus the elderly hospitalized patient J Antimicrob Chemother 199433811ndash821

92 JE Geraci Vancomycin Mayo Clin Proc 19775263193 TG Cantu NA Yamanaka-Yuen PS Lietman Serum vancomycin concentrations

Reappraisal of their clinical value Clin Infect Dis 199418533ndash54394 RC Moellering DJ Krogstad DJ Greenblatt Vancomycin therapy in patients with

impaired renal function A nomogram for dosage Ann Intern Med 198194343ndash346

95 GR Matzke JM Kovarik MJ Rybak SC Boike Evaluation of the vancomycin-clearance Creatinine-clearance relationship for predicting vancomycin dosageClin Pharm 19854311ndash315

96 KD Lake CD Peterson A simplified dosing method for initiating vancomycin ther-apy Pharmacotherapy 19855340ndash344

97 HE Nielsen HE Hansen B Korsager PE Skov Renal excretion of vancomycin inkidney disease Acta Med Scand 1975197261ndash264

98 HZ Zokufa HA Rodvold RA Blum LJ Riff JH Fischer KB Crossley JCRotschafer Simulation of vancomycin peak and trough concentrations using fivedosing methods in 37 patients Pharmacotherapy 1989910ndash16

99 NJ Saunders SV Want DJ Adams Vancomycin monitoring in renal failure Varia-tion between assays In Program and Abstracts of the 34th Interscience Conferenceof Antimicrobial Agents and Chemotherapy 1994 Orlando FL Am Soc Microbi-ology Washington DC A-31

100 AL Somerville DH Wright JC Rotschafer Implications of vancomycin degrada-tion products on therapeutic drug monitoring in patients with end-stage renal dis-ease Pharmacotherapy 199919702ndash707

101 KW Shea BA Cunha Teicoplanin Med Clin N Am 199579833ndash 844102 H Lagast P Dodion J Klastersky Comparison of pharmacokinetics and bacteri-

204 Ross et al

cidal activity of teicoplanin and vancomycin J Antimicrob Chemother 198618513ndash520

103 AP MacGowan Pharmacodynamics pharmacokinetics and therapeutic drug moni-toring of glycopeptides Therap Drug Monit 199820473ndash477

104 NE Allen DL LeTourneau JN Hobbs Molecular interactions of a semisyntheticglycopeptide antibiotic with D-alanyl-D-alanine and D-alanyl-D-lactate residuesAntimicrob Agents Chemother 19974166ndash71

105 TI Nicas JE Flokowitsch DA Preston DL Mullen J Grissom-Arnold NJ Snyderet al Semisynthetic glycopeptides active against vancomycin-resistant enterococciActivity against staphylococci and streptococci in vitro and in vivo In Abstractsof the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy1995F-248

106 M Robbins D Felmingham Cidal activity of LY 333328 a new glycopeptideagainst Enterococcus spp In Program and Abstracts of the 20th International Con-ference of Chemotherapy Syndey Australia 19974292

107 J Chien S Allerheiligen D Phillips B Cerimele HR Thomasson Safety and phar-macokinetics of single intravenous doses of LY333328 diphosphate (glycopeptide)in healthy men In Programs and Abstracts of the 38th Interscience Conferenceon Antimicrobial Agents and Chemotherapy San Diego CA 1999A-55

9

Macrolide Azalide and KetolidePharmacodynamics



Holly M Mattoes


1 INTRODUCTION

The macrolides and azalides have activity against gram-positive bacteria and arerelatively weakly active against many gram-negative bacteria These agents alsopenetrate well into mammalian tissue and achieve high concentrations in mamma-lian cells and are therefore very useful in the treatment of infections caused byintracellular pathogens Their spectrum of activity makes them a good choicefor the treatment of community acquired respiratory tract infections because theorganisms associated with such diseases usually involve Streptococcus pneumon-iae Haemophilus influenzae and Moraxella cattarhalis and frequently involveintracellular organisms (Table 1) [1ndash3] The macrolides and azalides (either asthe parent compound or in combination with a microbiologically active metabo-lite) have adequate activity against these pathogens and have emerged as usefuland popular agents for the treatment of milder forms of these diseases

205


TABLE 1 MIC90 Values of Organisms Commonly Associatedwith Community Acquired Respiratory Tract Infections

Organism Erythromycin Clarithromycin Azithromycin

S pneumoniae 003 0015 012S pyogenes 003 0015 012H influenzae 4 1(w14-OH) 05M catarrhalis 025 025 006M pneumoniae 001 003 0001C pneumoniae 006 0015 05Legionella sp 1 0125 0125

The macrolides and azalides bind to the 50S ribosome in the bacterial celland therefore interfere with the production of bacterial protein [45] The proteinis essential for the bacterial life cycle and because of the presence of the antibi-otic the bacteria cannot make the needed protein and eventually die or stop repro-ducing Although it is essential for bacterial life apparently existing stores ofthe protein must be depleted before effects of the antibiotic are noticeable As aresult many studies that use concentrations in the therapeutic range classify thesecompounds as bacteriostatic and pharmacodynamic studies have shown them tobe concentration independent (time-dependent) bacterial killers [6]

The first commercially available antibacterial agent in popular worldwideuse was erythromycin It is used as a single agent for the treatment of a varietyof community associated respiratory tract infections and as a second agent (dueto its effectiveness against intracellular pathogens) as part of a combination regi-men for hospitalized patients Although still popular as a less costly macrolideoption it does cause gastrointestinal side effects that many patients cannot toler-ate for a complete course of therapy This has implications for the selection ofmacrolide resistant organisms which is the topic of another chapter in this bookAdditionally its half-life is shorter than those of the newer agents requiring dailyfrequent multi-dosing The drug is also owing to its metabolic pathway of elimi-nation involved in a number of drug interactions [78] The popularity of erythro-mycin is decreasing and it is being replaced in clinical use with the newer macro-lide and azalide drugs (clarithromycin roxithromycin and azithromycin) As aresult less attention will be devoted to erythromycin in this chapter and the phar-macodynamics of the macrolidesazalides will be discussed with a focus on thenewer drugs

Information on the new ketolide class of antibiotics semisynthetic macro-lide characterized by the replacement of the clandinose moiety with a ketonegroup will also be discussed [9] The ketolides are effective in treating grampositive pathogens especially those exhibiting resistance to macrolides by utiliz-


ing the same mechanism of action as the macrolide and azalide compounds [10]Due to the high achievable drug concentrations in white blood cells and broncho-pulmonary tissues the ketolides are especially effective against common respira-tory pathogens and intracellular organisms [11]

Because the purpose of this chapter is to discuss the pharmacodynamicsof these agents issues such as utility in clinical use side effect profiles and druginteractions will not be discussed The reader is referred to standard textbooksand primary literature references on these topics Additionally no attempt willbe made to provide every detail on pharmacodynamic properties of each drugRather there are certain principles that are important for this class of agents froma pharmacodynamic perspective and these principles will be discussed and illus-trated using some (but not all) of the newer agents

2 OVERVIEW OF MACROLIDE AND AZALIDE

PHARMACODYNAMICS

As discussed in an earlier chapter antibiotics can be classified as either bacterio-static or bactericidal drugs and further subcategorized as concentration dependentor time dependent bacterial killers In reality every antibiotic has all of theseproperties however the property that emerges and upon which the classificationis dependent is a function of the concentrations achieved after clinical dosingrelative to its activity against the pathogen For the macrolides the currentlyavailable data indicate that when the serum plasma concentrations are highenough the pharmacodynamic parameter that correlates best with bacterial eradi-cation is the time for which the organism is exposed to adequate concentrationsof these agents (clarithromycin roxithromycin) [12] Obviously if the concentra-tion of the drug is not high enough one cannot make the simplifying assumptionthat the concentration part of the AUC (Cp time) contributes little to the bacte-rial eradication process and can be ignored In such a case bacterial eradicationis related not just to the time of exposure of the bacteria to the drug (T MIC)but also to the concentration of drug to which the bacteria are exposed (AUC)Bacteria that have relatively high MICs may correlate clinical cure rates betterto the pharmacodynamic parameter AUCMIC This may become an issue as theMICs of pathogens increase owing to the emergence of resistant strains At thepresent time however most bacteria worldwide for which the macrolides havebeen useful agents still have MICs low enough such that the pharmacodynamicparameter of T MIC is appropriate This will probably change as resistanceincreases and may already have changed in some countries Although the exactduration of exposure that is adequate to cure or manage an infection in the clinicalsetting is not known animal experiments and in vitro data [12] suggest that agood working number is a time above the MIC of approximately 50 of thedosing interval for patients with a functioning host defense system [12ndash15] Ani-


mal studies in which neutropenia was induced indicate that superior animal sur-vival was associated with drug concentrations of 4ndash5 times the MIC [1213]This suggests that more appropriate pharmacodynamic criteria for neutropenicpatients might be a T MIC of 100 of the dosing interval

It has been reported that azithromycin has a prolonged post antibiotic effect(PAE) [1213] It is controversial whether this PAE is sufficiently long to signifi-cantly affect clinical outcome The usual regulatory agency approved doses ofdrugs are based on clinical studies that are derived after treatment with a particu-lar dose and dosing regimen This incorporates the total properties of the drugsand is then correlated to clinical outcome To state this in another way goodclinical outcomes are due to the dose and dosing regimen of the drug used Thistakes into account concentration issues and drug exposure issues which includeissues of protein binding and PAE It has been stated however that because ofthe long PAE of azithromycin the proper pharmacodynamic parameter wouldnot be the T MIC but rather the AUCMIC ratio Unfortunately there areinsufficient data to substantiate this assumption in humans Another assumptionalso unsubstantiated is that azithromycin like the macrolides eradicates bacteriaby having an adequate exposure time (T MIC) For the macrolides we sug-gested a T MIC of approximately 50 of the dosing interval Because of thePAE of azithromycin an appropriate number might be somewhat less than 50ie 35 or 40 This hypothesis is also possible however further investigationis needed to clarify this issue

3 TISSUE DISTRIBUTION

The macrolides and azalides share the common property of extensive distributioninto mammalian tissues Other drug classes such as the quinolones also have thisproperty but not to the extent exhibited by the macrolides and azalides Oneconsequence of this as noted above is that the latter particularly useful in thetreatment of infections caused by intracellular pathogens Another consequenceof this property is that it is responsible for the drugsrsquo longer serum half-life com-pared to erythromycin (Table 2) [1617] Examination of Table 2 reveals that allof the newer agents have a longer half-life than erythromycin The azithromycinhalf-life is particularly long (actually this is a working half-life the true half lifeis even longer than shown in this table) [41618ndash20] The reason for the longazithromycin half-life is not the fact that the normal excretory routes of the bodyhave difficulty in eliminating this drug Rather the body can only eliminate whatis available to it The rate of appearance to excretory organs is a function of itsrelease from tissue storage sites Kinetically we can calculate only the sloweststep in the elimination process which in the case of azithromycin is release fromtissue The long half-life of azithromycin therefore is really an estimate of thehalf-life of tissue elimination


TABLE 2 Important Parameters of the Pharmacokinetics of Macrolides

Erythromycin Clarithromycin Azithromycin RoxithromycinParameter (250 mg) (500 mg) (250 mg) (150 mg)

Half-life 2ndash4 3ndash4 68 8ndash15Cpmax

(microgmL) 025ndash05 2 024 66ndash79

Absorption 35ndash40 50ndash55 37 mdashAUC (microgmL) mdash 1214 21 726ndash81

Distribution into tissue sites has ramifications related to the clinical use ofthese agents This can be illustrated by referring to two studies of a similar nature[1213] In these studies human volunteers were dosed to steady state with clar-ithromycin and azithromycin Plasma samples were obtained and bronchial la-vage was performed allowing the calculation of epithelial lining fluid (ELF) drugconcentrations and determination of alveolar macrophage (AM) concentrationsof both drugs The results of both experiments demonstrate the same relationshipsbetween drug concentrations in these sites as they relate to the microbiologicalactivity of target pathogens

It is interesting to examine three figures that emanate from these studiesFigure 1 shows the plasma concentrations of both agents at steady state and in

FIGURE 1 Plasma concentrations of clarithromycin and azithromycin with Spneumoniae MIC90


this figure the typical MIC for S pneumoniae is superimposed upon the kineticcurves One can see that for clarithromycin concentrations in plasma at steadystate yield a T MIC of 100 of the dosing interval The azithromycin curveindicates that this drug stays above the MIC of this pathogen (when a 250 mgonce per day dose is used) for somewhat less than 50 of its dosing intervalOf course if all the pathogens are eradicated or if the remaining pathogens canbe eliminated by the patientrsquos host defense mechanisms then this drug would beexpected to be clinically useful against this organism However if any bacteriasurvive such a dose and dosing regimen they are exposed to subtherapeutic con-centrations of active azithromycin for long periods of time Theoretically this isa condition that can select for resistant organisms and exists because of the exten-sive tissue distribution properties of azithromycin [12] Resistance issues are dis-cussed more completely in another chapter of this book

As described in an earlier chapter the drug and bacteria need to be in thesame place at the same time in order for the antibiotic to function by interferingwith the bacterial life cycle The question of where the bacteria reside in relation-ship to the drug is an important and yet unresolved issue This affects the applica-tion of pharmacodynamics to any agent that has extensive tissue distribution andis a topic of discussion in the next section Assuming however that in a lunginfection the extracellular bacteria reside in some interstitial fluid such as theELF it is the concentration in the ELF that might be of clinical importanceFigure 2 shows the calculated ELF concentrations for each agent and again theMIC for S pneumoniae is superimposed on the kinetic curves In this case bothdrugs clearly show that the T MIC is 100 of the dosing interval This analysis

FIGURE 2 Epithelial lining fluid concentrations of clarithromycin and azithro-mycin with S pneumoniae MIC90


needs to be determined with each target pathogen to predict clinical differencesin the treatment of respiratory infections between these drugs

Finally as mentioned previously the macrolides and azalides have utilityin the treatment of respiratory tract infections caused by intercellular organismsExamination of Fig 3 allows one to understand the relationship between penetra-tion into mammalian tissue and the MIC of these target organisms Figure 3shows the penetration of these drugs into the alveolar macrophage which is usedas a marker for mammalian tissue uptake It can be seen that the AM concentra-tions for both drugs are well above the MIC for some typical intracellular patho-gens (T MIC for 100 of the dosing interval) One would predict that the twoagents would be equally effective in eradicating intracellular bacteria in a clinicalsetting

Pharmacodynamically the ketolide telithromycin demonstrates a concen-tration dependent kill rather than a time dependent kill In vitro studies also havesupported the theory that telithromycin exhibits primarily concentration depen-dent and inoculum dependent bacteriostatic activity [21] Additionally animalstudies have determined the AUCMIC ratio to be the best predictor of in vivoefficacy for telithromycin and therefore once daily dosing is an appropriate dos-ing regimen for this ketolide [22] A study evaluating telithromycinrsquos pharmaco-kinetics after an 800 mg oral dose demonstrated a Cmax of 190 mgl AUC of896 mghl and Tmax of 10 h [23] As a result of this favorable kinetic profileeffective AUCMIC ratios are observed for respiratory pathogens A summaryof MICs of telithromycin for these organisms is listed in Table 3 H influenzae

FIGURE 3 Alveolar macrophage concentrations of clarithromycin and azithro-mycin with S pneumoniae MIC90


TABLE 3 Ketolide Pharmaacodynamics

Telithromycin ABT773

MIC90 MIC90

Streptococcus pneumoniae 006 mgL 003mgLHaemophilus influenzae 2 mgL 4 mgLMoraxella cattarhalis 006 mgL 012 mgLMycoplasma pneumoniae 0004 mgL NRChlamydia pneumoniae 025 mgL NRLegionella spp 003 mgL 1 mgL

NR Not reported at this time

has an increased MIC for telithromycin but still demonstrates similar in vitroactivity to azithromycin and clarithromycin against this organism Treatmentsuccess relies heavily on factors other than the interaction of the antibiotic withthe bacteria These were previously described and include the patientrsquos immuno-competency ABT 773 demonstrates time dependent kill but unfortunately thereare few pharmacokinetic data published and therefore the TMIC cannot beaccurately determined at the present time However it is expected to demonstratean adequate pharmacodynamic profile against its target organisms ie thosecausing community-acquired respiratory infections

For good clinical cure of respiratory intracellular organisms the alveolarmacrophage drug concentration is an important consideration because this iswhere the organism is believed to reside A study evaluating steady state pharma-codynamics of the ketolide telithromycin in healthy volunteers demonstratedgood penetration into the bronchopulmonary tissues surpassing the plasma con-centration in these individuals The mean concentrations of this ketolide in thealveolar macrophages epithelial lining fluid and bronchial mucosa exceeds theMIC for most common respiratory pathogens [24] High intracellular concentra-tions of this drug were also observed in the white blood cells of healthy volun-teers again exceeding the MIC for the typical intracellular organisms for 100of the dosing interval [25]

It is unfortunate that one is exposed to the pseudo pharmacodynamic termpenetration ratio We call the term pseudo pharmacodynamic because the wayit is used implies that it has pharmacodynamic meaning but usually insufficientinformation is given to understand its meaning To illustrate this point we referyou to Table 4 which came from the same datasets Examination of this tablereveals that the clarithromycin AMplasma ratio goes from approximately 340to 1300 whereas that of azithromycin goes from approximately 1300 to 15000Also note that these data were obtained after the last dose of the drug was givenGenerally the ratio increases as a function of time after the last dose This is


TABLE 4 ClarithromycinAzithromycin AlveolarMacrophagePlasma Ratio

AM cell ratioTime afterdosing (h) Clarithromycin Azithromycin

4 543 12928 465 15237

12 1041 1280724 1265 14386

intuitively problematic because one expects to see concentrations decrease afterdosing Upon reflection one must realize that the ratio has a numerator and de-nominator To make the ratio larger one can increase the numerator or decreasethe denominator or both Usually insufficient data are presented to determinewhich is occurring and the meaning of the ratio is therefore unknown It is indeedunfortunate that when one sees a high ratio one assumes that the numerator islarge Examination of this table and the plasma and AM concentration data (Figs1 and 3) reveals that the ratio is increasing because the denominator (plasmaconcentrations) is decreasing much faster than the numerator (AM concentra-tions) after dosing is stopped The high ratios in this case are not a function ofsuperior tissue concentration (Fig 3) but of differences between the drugsrsquo AMand plasma concentrations (Fig 3) ie the lower the plasma concentration thehigher the ratio As a result one must evaluate lsquolsquopenetrationrsquorsquo data carefully tofully understand that importance of delivering drug to the body site where thebacteria may reside

4 APPROPRIATE PHARMACODYNAMIC MODEL

To understand the practical applicability of the current pharmacodynamic modelto drugs such as the macrolides and azalides we must remember two importantcriteria First the pharmacodynamic models index microbiological activity toserum or plasma concentrations The question is whether this is appropriate fora drug class that has a large degree of tissue distribution The second factor iswhere the pathogen reside in the body Figure 4 helps clarify this latter issue[1617] Bacteria that are not in intimate contact with mammalian cells eg ina mucus layer of the respiratory tract are not pathogens They may be detectedafter laboratory tests but they are colonizers For bacteria to be pathogens theyusually need to attach to a mammalian cell membrane (extracellular organisms)or penetrate into the mammalian cell (intracellular organisms) Let us considerextracellular organisms that act as pathogens and examine the existing pharmaco-dynamic model to see how it applies to this class of agents


FIGURE 4 Proposed stages of infection of the bronchial mucosa IgA immu-noglobulin A

Figure 5 schematically shows the typical time dependent pharmacodynamicmodel as it applies to β-lactam antibiotics These drugs do not penetrate intomammalian cells (the square boxes) The drug concentration shown by the Arsquosin the schematic represent the average interstitial fluid concentration which weassume is represented by the average serum or plasma concentration (which iswhat we usually measure) If this average concentration is high enough (2ndash4

FIGURE 5 Time-dependent pharmacodynamic model


FIGURE 6 Macrolide pharmacodynamic model in mammalian cells

times the MIC) and remains above the MIC for a long enough period of time (T MIC for 50 of the dosing interval) then the drug is expected to be clinicallyeffective in eradicating the pathogens A similar model is schematically shownin Fig 6 In this figure the macrolides and azalides penetrate into the mammaliancells If the average drug concentration is high enough (2ndash4 times the MIC) andis exposed to the bacteria for a long enough period of time (T MIC for 50of the dosing interval) then the bacteria will be exposed to lethal concentrationsof drug This occurs when the MIC of the bacteria is very low ie the bacteriais sensitive to the drug In essence the model collapses to the β-lactam modelIf the MIC of the bacteria were high relative to the average serum concentrationsthen this model would predict that the bacterial would not be eradicated This isillustrated in Table 5 which shows the pharmacodynamic prediction when the

TABLE 5 Macrolide PharmacodynamicAnalysismdashH influenzae

Macrolide T MIC CpkMIC AUCMIC

Erythromycin 250 mg 0 013 mdashClarithromycin 500 mg 3 025 12Azithromycin 250 mg 0 048 5


macrolides or azalides are targeted against an organism with a higher MIC suchas H influenzae Whether one prefers the pharmacodynamic parameter of AUCMIC T MIC or CpMIC the result is the same The pharmacodynamic parame-ters are too low to predict clinical success of these drugs against this pathogenClinical studies indicate that macrolidesazalides are effective in respiratory tractinfections when H influenzae is involved Obviously there is a mismatch betweenwhat nature is telling us and what the existing model is predicting It is our beliefthat this model does not fully apply when the pathogen has a relatively high MICSimply put if the model indexes microbiological data to average serum or plasmaconcentrations and the organism resides elsewhere (attached to the outer mem-brane of the bacteria) indexing microbiological activity to serum concentrationmay not be correct if the MIC is high To explain this further one must recognizethat although these drugs tend to easily move into tissues they do not remainthere forever They slowly (azithromycin) or not so slowly (clarithromycin) comeout of these tissue sites A concentration gradient probably exists with the highestconcentrations just outside the mammalian cell membrane the place where patho-gens are believed to reside These pathogens could be exposed to high concentra-tions of active antibiotic and be killed as a result This could explain the apparentparadox between clinical results and pharmacodynamic predictions for bacteriawith higher MICs As time goes by and resistance continues to develop the MICswill continue to increase At some point the concentrations will be too high to

FIGURE 7 Macrolide pharmacodynamic model in the presence of white bloodcells


even evoke this expanded model and we will clinically observe patient failuresupon therapy Of course the entire situation is further complicated by the hostrsquosimmune system (Fig 7) White blood cells are attracted to the site of infectionbecause the bacteria secrete chemotactic factors They are also capable of en-gulfing bacteria and killing them and it is believed that they actually bring activedrug to the lsquolsquobattlefieldrsquorsquo None of this has been adequately studied and representshypothesis to some extent It is necessary however to evoke an explanationlike the one presented above to explain the discrepancy between the existingpharmacodynamic models and human clinical empirical data The point is thatthe macrolide and azalide pharmacodynamic situation is rather complex and asimple pharmacodynamic model that indexes microbiological activity to serumconcentrations cannot fully explain such a complex situation

5 FUTURE CONSIDERATIONS

The macrolides azalides and ketolides are a useful class of drugs for the treat-ment of respiratory tract infections For these agents however owing to theirability to easily penetrate into mammalian tissue serum concentrations appearto be predictive of clinical outcome for only very sensitive organisms For organ-isms with higher MICs the drug concentrations at the site in the body where thebacteria reside may be of more clinical relevance however these data are difficultor even impossible to obtain in humans It is indeed unfortunate that the reportingof resistant organisms is not strongly base on the pharmacodynamic characteris-tics of these drugs They are determined by the setting of so-called breakpointswhich are an interpretation of mostly in vitro and some clinical and pharmacody-namic data These breakpoints can be somewhat arbitrarily set in spite of thegood intentions and hard work of the officials setting them It is possible as inthe case of macrolides to report many isolates as resistant but the reportingsystem does not provide any information about the magnitude of the resistanceKetolides which in a sense are macrolides with activity against macrolide resis-tant agents are an exception to this phenomenon at the present time Whetherthe drug (macrolide or azalide) can eradicate the organism depends on the serumand tissue concentrations achieved in relationship to the MIC If drug concentra-tions are high enough for a long enough time after the usual clinical doses then theorganism will be adversely affected (die) regardless of whether one classifies it asresistant or sensitive Clarithromycin has been developed in an extended releaseform in an attempt to increase drug concentrations over a longer period of timeThese tablets are taken once daily and have a steady state AUC equivalent to con-ventional immediate release clarithromycin which is taken twice daily [26]

Unfortunately the reporting of in vitro resistance data has a strong impacton the drug selection process because for the most part macrolide and azalidetherapy is empirical This issue is not easy to address because the existing phar-macodynamic data use serum or plasma as their index to microbiological efficacy


which may not be appropriate for the macrolides when they are targeted againstorganisms with higher MICs that reside in bodily fluids whose drug concentrationprofiles are not the same as that of serum As time passes and more organismsbecome resistant and therefore have higher MICs this situation will become moreconfused and one can predict that the macrolide class of antibiotics will probablybe replaced by the lsquolsquorespiratoryrsquorsquo quinolones for the empirical therapy of respira-tory tract infections This may be an unfortunate occurrence that may relegatethe macrolides to the role of adjunctive agent in the treatment or prophylaxisof infections believed to be caused by intracellular pathogens This would beunfortunate if it is based upon erroneous resistance data and as a result deprivespatients of an acceptable and somewhat unique (due to its high tissue penetrationand immunological properties) class of agents

REFERENCES

1 LA Mandell Community-acquired pneumonia Etiology epidemiology and treat-ment Chest 1995108(2)35Sndash41S

2 D Lieberman F Schlaeffer I Boldur D Lieberman S Horowitz MG Friedman MLeiononen O Horovitz E Manor A Porath Multiple pathogens in adult patientsadmitted with community-acquired pneumonia A one year prospective study of 346consecutive patients Thorax 199651179ndash184

3 HA Cassiere MS Niederman Community-acquired pneumonia Disease of theMonth 1998(Nov)622ndash675

4 SC Piscitelli LH Danziger KA Roldvold Clarithromycin and azithromycin Newmacrolide antibiotics Clin Pharm 199211137ndash152

5 MS Whitman AR Tunket Azithromycin and clarithromycin overview and compari-osn with erythromycin Infection Control Hosp Epidemiol 199213357ndash368

6 A Bauernfeind R Jungwirth E Eberlein Comparative pharmacodynamics of clar-ithromycin and azithromycin against respiratory pathogens Infection 199523(5)316ndash321

7 TM Ludden Pharmacokinetic interactions of the macrolide antibiotics Clin Pharma-cokinet 19851063ndash79

8 P Periti T Mazzei E Mini A Novelli Pharmacokinetic drug interactions of macro-lides Clin Pharmacokinet 199223106ndash131

9 C Agouridas A Bonnefoy JF Chanton In vitro antibacterial activity of HMR 3647a novel ketolide highly active against respiratory pathogens [abstract F-112] InProgram and Abstracts of the 37th Interscience Conference on AntimicrobialAgents and Chemotherapy (Toronto Ontario) Washington DC Am Soc Microbiol1997

10 C Marie-Bigdot D Decre C Muller E Salgado E Bergogne-Berezin In vitro activ-ity of a novel ketolide antimicrobial agent HMR 3647 compared to erythromycinroxithromycin azithromycin and clarithromycin [abstract F-117] In Program andAbstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemo-therapy (Toronto Ontario) Washington DC Am Soc Microbiol 1997


11 RP Smith AL Baltch W Ritz M Franke P Michelson Antibacterial effect ofketolides HMR 3004 and HMR 3647 against intracellular Legionella pneumophilia[abstract F-249] In Program and Abstracts of the 37th Interscience Conference onAntimicrobial Agents and Chemotherapy (Toronto Ontario) Washington DC AmSoc Microbiol 1997

12 RC Owens DP Nicolau R Quintiliani CH Nightingale Bactericidal activity of clar-ithromycin azithromycinand cefuroxime against penicillin-susceptible -intermedi-ate and -resistant pneumococci 20th Int Congress of Chemotherapy (Sydney Aus-tralia) June 29ndashJuly 3 1997

13 JG Den Hollander JD Knudsen JW Mouton F Fuursted N Frimodt-Moller HAVerbrugh F Espersen Comparison of pharmacodynamics of azithromycin anderythromycin in vitro and in vivo Antimicrob Agents Chemother 199842377ndash382

14 WA Craig Interrelationship between pharmacokinetics and pharmacodynamics indetermining dosage regimens for broad-spectrum cephalosporins Diagn Microb In-fect Dis 19952289ndash96

15 B Vogelman S Gudmundson J Leggett J Turnidge S Ebert WA Craig Correlationof antimicrobial pharmacokinetic parameters with therapeutic efficacy in animalmodel J Infect Dis 1998158831ndash847

16 F Kees M Wallenhofer H Grobecker Serum and cellular pharmacokinetics of clar-ithromycin 500mg qd and 250mg bid in volunteers Infection 199523168ndash172

17 G Foulds RM Shepard RB Johnson The pharmacokinetics of azithromycin in hu-man serum and tissues J Antimicrob Chemother 199025(suppl A)73ndash82

18 A Wildfeuer H Laufen T Zimmermann Uptake of azithromycin by various cellsand its intracellular activity under in vivo conditions Antimicrob Agents Chemother19964075ndash79

19 GW Amsden AN Nafziger G Foulds Pharmacokinetics in serum and leukocyteexposure of oral azithromycin 1500 milligrams given over a 3- or 5-day periodin healthy subjects Antimicrob Agents Chemother 199943163ndash165

20 G Panteix B Guillaumond R Harf A Desbos V Sapin M Leclercq M Perrin-Fayolle In-vitro concentration of azithromycin in human phagocytic cells J Antimi-crob Chemother 199331(suppl E)1ndash4

21 FJ Boswell JM Andrews R Wise Pharmacodynamic properties of HMR 3647 anovel ketolide demonstrated by studies of time-kill kinetics and post antibiotic effect[abstract F-254] In Program and Abstracts of the 37th Interscience Conference onAntimicrobial Agents and Chemotherapy (Toronto Ontario) Washington DC AmSoc Microbiol 1997

22 O Vesga C Bonnat WA Craig In vivo pharmacodynamic activity of HMR 3647a new ketolide [abstract F-255] In Program and Abstracts of the 37th InterscienceConference on Antimicrobial Agents and Chemotherapy (Toronto Ontario) Wash-ington DC Am Soc Microbiol 1997

23 F Namour DH Wessels MH Pascual D Reynolds E Sultan Dose proportionalityof the pharmacokinetics of HMR 3647 a new ketolide antimicrobial in healthy adultmales [abstract 79] In Program and Abstracts of the 37th Annual Meeting of theInfectious Diseases Society of America (Philadelphia PA) Alexandria VA InfectDis Soc America 1999


24 CM Serieys C Cantalloube P Soler HP Gia F Brunner HMR 3647 achieves highand sustained concentrations in broncho-pulmonary tissues [abstract P78] In Ab-stracts of the 21st International Congress of Chemotherapy (Birmingham UK) Ox-ford UK Br Soc Antimicrob Chemother 1999

25 HP Gia V Roeder F Namour E Sultan B Lenfant HMR 3647 achieves high andsustained concentrations in white blood cells in man [abstract P79] In Abstractsof the 21st International Congress of Chemotherapy (Birmingham UK) OxfordUK Br Soc Antimicrob Chemother 1999

26 LE Gustavson GX Cao SJ Semla RN Palmer Pharmacokinetics of a new extended-release clarithromycin tablet at doses of 500 and 1000 mg daily [abstract A-1191]In Program and Abstracts of the 39th Interscience Conference on AntimicrobialAgents and Chemotherapy (San Francisco CA) Washington DC Am Soc Micro-biol 1999

10

Metronidazole Clindamycin andStreptogramin Pharmacodynamics

Kenneth Lamp

Bristol-Myers SquibbPlainsboro New Jersey

Melinda K Lacy

University of Kansas Medical CenterKansas City Kansas

Collin Freeman

Bayer Corporation West Haven Connecticut

1 METRONIDAZOLE

Metronidazole (Flagyl) is a nitroimidazole antibiotic that has been available foruse in clinical practice for over 25 years Its original indication was for treatmentof infections caused by Trichomonas vaginalis but over the years it has beendiscovered to be useful in treating a variety of infections caused by various organ-isms Although the nitroimidazole class of antibiotics includes tinidazole secni-dazole and ornidazole the wealth of information about the pharmacokineticsand pharmacodynamics of these agents comes from published data about metroni-dazole However even with the vast amount of information about metronidazolethere is very little information regarding its pharmacodynamic properties com-

221

222 Lamp et al

pared to other antimicrobial agents such as the β-lactams aminoglycosides andfluoroquinolones The only distinguishing features of tinidazole ornidazole orsecnidazole are prolonged T12 values compared to metronidazole

11 Chemistry and Mechanism of Action

Metronidazole is a nitroimidazole (chemical name 1-[β-hydroxyethyl]-2-methyl-5-nitromidazole) that belongs to the same chemical class as tinidazole and ornida-zole (structure shown in Fig 1) The compound was discovered in the late 1950swhen researchers at RhonendashPoulenc Research Laboratories in France were tryingto create a synthetic product from a Streptomyces sppndashderived compound calledazomycin that would have appreciable activity against Trichomonas vaginalis[1]

From the research of different investigators metronidazolersquos mechanismof action is believed to involve four phases (1) entry into the bacterium cell (2)reduction of the nitro group cytotoxic effect of the reduced product and (4)liberation of end products that are inactive [2] It is the redox intermediate intra-cellular metabolites that are believed to be the key components of microorganismkilling for metronidazole The intracellular targets for these intermediates couldbe organismsrsquo RNA DNA or cellular proteins


Following their work in 1959 Cosar and Julou and other researchers over thenext several years found a variety of potential human uses for metronidazole thatwent beyond treatment for T vaginalis infection The following is a summaryof metronidazolersquos antimicrobial activity

FIGURE 1 Metronidazole molecule


121 Protozoa

In addition to T vaginalis metronidazole has exhibited activity against otherprotozoan organisms including Giardia lamblia and Entamoeba histolytica [3ndash15]

122 Bacteria

Gram-Negative Bacteria Shinn [8] and later Tally et al [9] discoveredthe potential of using metronidazole for anaerobic infections Metronidazole hassince been used and studied extensively for treatment of various anaerobic infec-tions [10ndash12] Even today metronidazole is considered the gold standard bywhich other antimicrobials with perceived antianaerobic activity are comparedThis is due primarily to metronidazolersquos rapid killing of Bacteroides species andthe very low rate of resistance acquired by these bacteria [13ndash15] Other Bacteroi-des species and Fusobacterium spp have been reported as being at least as sus-ceptible to metronidazole as Bacteroides as well as Helicobacter (formerlyCampylobacter) pylori Actinobacillus and Prevotella although resistance tometronidazole may be common in various regions of the world [9131416ndash20]Minimum bactericidal concentrations (MBCs) of metronidazole when reportedin these studies are the same or within one dilution of the reported MICs

Gram-Positive Bacteria As for gram-positive anaerobes the peptostrep-tococci have usually been reported to exhibit MICs in the range of 025ndash40mgL [132122]

Susceptibility of Clostridium perfringens to metronidazole is also in theMIC range of 10ndash40 mgL although not all clostridia are so susceptible[13212421] Metronidazole is also considered to be one of the few drugs withactivity against Clostridium difficile [212526]

123 Other Organisms

Metronidazole has also been reported to have in vitro activity against Campylo-bacter fetus Gardnerella vaginalis Leptotrichia buccalis and Treponema pal-lidum to varying degrees [27ndash32]

13 Pharmacokinetic and Pharmacodynamic Properties

The following is a brief summary of the many trials that have studied metronida-zolersquos kinetics

131 Absorption

The absorption of metronidazole has been studied for oral tablets vaginal andrectal suppositories and topical gel

224 Lamp et al

Oral Absorption The oral absorption of metronidazole is excellent withbioavailability often reported as 90 [37ndash39] The oral Cmax with a 500 mgdose is approximately 80ndash130 mgL with a corresponding Tmax of 025ndash40 h[33ndash36]

Rectal Absorption Rectal administration of metronidazole 500 mg hasproduced Cmax values of approximately 40ndash55 mgL at Tmaxrsquos of 05ndash10 fora retention enema to 40ndash12 h for suppositories [11333738] Metronidazoleabsorption from the rectum has been reported as being approximately 67ndash82based upon calculations with equivalent intravenous (IV) doses used as references[38]

Intravaginal Absorption Intravaginal absorption of metronidazole hasvaried considerably depending on the vehicle used The 075 metronidazoleintravaginal gel at a dose of 50 g has produced Cmax values of 02ndash03 mgLwith Tmax values of 83ndash85 h [39] For vaginal suppositories and inserts the 500mg dose Cmax values have been approximately 19 mgL with corresponding Tmax

values of 77ndash20 h [3640] The bioavailability of vaginal suppositories is 25of an oral 500 mg dose but the vaginal gelrsquos bioavailability was 56 that of a500 mg IV dose [3639]

Topical Absorption Systemic absorption of 10 g of 075 metronidazolegel is reportedly quite low After 10 g of the gel was applied to the face of adultswith rosacea the resulting serum concentrations ranged from undetectable to 66ngmL in the following 24 h [4142]

132 Distribution

Protein binding of metronidazole is 20 [3543] Metronidazole crosses cellmembranes well in general and distributes well into a variety of tissues and fluidsThe reported volumes of distribution (Vd) in studies for various age groups haveranged from 051 to 11 Lkg [4344]

Single-dose studies using metronidazole 500 mg IV or oral doses havedetermined AUCs to be approximately 100ndash159 mg(L sdot h) [36] Oral or IV dosesof 10 g have displayed AUCs of 214ndash257 mg(L sdot h) [3345] The penetrationof metronidazole into polymorphonuclear leukocytes has been described asequivalent to the concentration found in the extracellular fluid [46]

133 Metabolism

Metronidazole undergoes hepatic metabolism forming five metabolites The twomajor metabolites are an acid metabolite and a hydroxy metabolite The acidmetabolitersquos activity is considered to be clinically negligible having only 5 ofthe activity of metronidazole [47]


Hydroxymetronidazole The hydroxy metabolite of metronidazole is themost clinically important because it also has antimicrobial activity but to a lesserextent than the parent compound Hydroxymetronidazole has been reported ashaving 30ndash65 of the activity of the parent compound with respect to similarisolates [192447]

134 Excretion

Metronidazole is primarily excreted in the bile as the parent drug and in the urineas its various metabolites Only about 6ndash18 of metronidazole is excreted in theurine unchanged [35] Hydroxymetronidazole is entirely excreted in the urinebeing approximately 25 of the original dose [48]

The elimination half-life (T12) has been reported to range from 6 to 10 h formetronidazole in volunteers and patients with normal hepatic and renal functions[3744] The hydroxy metabolitersquos T12 reportedly ranges from 8 to 12 h [36]Patients may have altered clearance values such as T12 values that are 10 h[49] Metronidazole appears to exhibit dose-dependent clearance in doses rangingfrom 250 to 2000 mg [4449]

14 Pharmacodynamics


Metronidazole appears to have an extremely rapid rate of killing against suscepti-ble anaerobes [1550]

Outcomes associated with the use of metronidazole in mixed aerobicndashan-aerobic infections such as intra-abdominal infections have yielded excellent re-sults in terms of bacterial eradication when an antianaerobic agent such as metro-nidazole was included as part of the therapeutic regimen [5152]

142 Concentration-Dependent Killing

Nix et al [53] discovered that metronidazole had a concentration-dependent kill-ing effect against T vaginalis under anaerobic conditions at concentrations thatranged from 01 to 80 mgL [53] Time-kill kinetic studies of ciprofloxacinalone (concentration 05 mgL MIC 025 mgL) compared to a combination ofciprofloxacin with metronidazole (100 and 400 mgL) displayed more rapidkilling with the combined antibiotics against C perfringens If this concentration-dependent effect truly exists for metronidazole it would suggest that the generaloptimal dosing strategy for metronidazole against organisms like T vaginalis andB fragilis would be to give higher doses less frequently rather then smaller dosesmore frequently similar to the use of aminoglycosides and fluoroquinolonesagainst susceptible gram-negative aerobic bacteria

226 Lamp et al


Like aminoglycosides and fluoroquinolones metronidazole appears to exhibit apostantibiotic effect (PAE) extending over 3 h [54]

144 Serum Bactericidal Titers

Serum bactericidal titers (SBTs) have been used in studies as an ex vivo methodof assessing metronidazolersquos effect on anaerobes primarily B fragalis Resultsfrom an SBT study reported a median metronidazole SBT of 12 against variousBacteroides species at 05 10 and 60 h after administration of a single doseof metronidazole 500 mg IV [55] Results with metronidazole against two strainsof B fragilis at the 6 h time point were 12 but titers related to FusobacteriumPeptostreptococcus and Eubacterium lentum were all 18 at all time points

Fluoroquinolones have often been studied for use in combination with met-ronidazole for antibacterial activity against gram-negative aerobes and anaerobesBoeckh et al [56] studied the effect of fluoroquinolones (ofloxacin ciprofloxacinenoxacin fleroxacin) in combination with antianaerobic antibiotics includingmetronidazole via SBTs SBTs were performed from serum samples obtainedat 1 2 6 and 8 h depending upon the antibiotic combination against variousgram-positive and gram-negative aerobic pathogens as well as strains of B frag-ilis and B thetaiotaomicron None of the combinations appeared to interferewith the individual antibioticsrsquo antibacterial activities and mean SBTs for theciprofloxacin-metronidazole ofloxacin-metronidazole enoxacin-metronidazoleand fleroxacin-metronidazole combinations were all in the 12ndash14 dilutionrange for the various strains and species of Bacteroides Similarly Pefanis etal [57] established greater effectiveness at eradicating mixed aerobicndashanaerobicgram-negative pathogens from experimentally induced abscesses in a rat modelbased upon reduced CFU counts

Serum bactericidal titer studies involving the combination of metronidazole10 g with extended spectrum cephalosporins have also yielded results in the14ndash18 range for B fragilis at the end of 12 or 24 h for ceftizoxime or ceftriax-one respectively [58ndash60]

15 SummarymdashMetronidazole

Although data are scarce metronidazolersquos concentration-dependent bactericidalactivity prolonged T12 and sustained bactericidal activity in plasma support theclinical evaluation of higher doses and longer dosing intervals In vitro and exvivo human studies have corroborated metronidazolersquos effectiveness as an anti-protozoal and antianaerobic agent

The rapid concentration-dependent killing of anaerobes by metronidazoleits active metabolite which helps to extend its effective duration of action thepenetrability of metronidazole into various tissues the near lack of resistance of


anaerobes like B fragilis to metronidazole and its low cost make metronidazolethe preferred agent for antianaerobic therapy in many cases

The other nitroimidazoles doubtless have identical pharmacodynamic prop-erties although their respective pharmacokinetics may be somewhat differentfrom those of metronidazole As newer nitroimidazoles are brought to market andare used with greater frequency metronidazolersquos use may continue to graduallydecline However the value of metronidazole is still readily apparent and it stillhas an important role in todayrsquos antimicrobial armamentarium

2 CLINDAMYCIN

Clindamycin (Cleocin Pharmacia amp Upjohn Co) a lincosamide antibiotic hasbeen used in clinical practice for over 30 years Compared to its parent compoundlincomycin clindamycin has improved oral absorption and a wider antibacterialspectrum of activity making it the most popular agent in this classification ofantibiotics It is commercially available as an intravenous formulation oral cap-sules and suspension as well as in several topical preparations (solution gellotion individual pledgets and vaginal cream) Clinically clindamycin is usedin the treatment of gram-positive and anaerobic bacterial infections of the abdo-men bone lung skin and soft tissues and pelvis and is often an alternative agentin those who are allergic to β-lactam antibiotics Additionally it is used in thetreatment of opportunistic protozoal infections of the lung and central nervoussystem in patients infected with the human immunodeficiency virus

Usual oral doses in adults range from 150 to 450 mg every 6 h whereasparenteral doses are generally 600ndash900 mg every 8 h [61]

21 Chemistry and Mechanism of Action of Clindamycin

Clindamycin (7-chloro-7-deoxylincomycin) is a semisynthetic derivative of lin-comycin and is therefore classified as a lincosamide antibiotic Similar to macro-lides streptogramins and chloramphenicol clindamycin exerts its activityagainst susceptible pathogens by binding to the 50S ribosomal subunit whichinhibits microbial protein synthesis through its effects on peptide chain initiation


Clindamycin is active against a variety of gram-positive and anaerobic bacterialpathogens [62] Most streptococci including Streptococcus pneumoniae andStreptococcus pyogenes are susceptible as are most methicillin-susceptiblestrains of Staphylococcus aureus However it is not active against enterococcior aerobic gram-negative organisms Clindamycin is active against a broad rangeof anaerobic gram-negative bacilli including Bacteroides species Fusobacteriumspecies anaerobic gram-positive bacilli such as Propionibacterium Eubacte-

228 Lamp et al

rium and Actinomyces and anaerobic gram-positive cocci such as PeptococcusPeptostreptococcus microaerophilic streptococci and most strains of Clostrid-ium perfringens It is also active against pathogens that cause bacterial vaginosiswhich include Bacteroides Gardnerella vaginalis Mobiluncus and Mycoplasmahominus

Additionally many protozoa including Toxoplasma gondii Plasmodiumfalciparum Babesia and Pneumocystis carinii are susceptible to clindamycin[6263]

Two recent surveillance studies in the United States demonstrated thatgreater than 95 of all pneumococcal strains were susceptible to clindamycinwith reported MIC90 values of 012 mgL [6465] In one of the studies 76 of969 S aureus isolates were susceptible with reported MIC50 and MIC90 valuesof 025 and 8 mgL [65] The susceptibility breakpoint is 05 mgL for Staphy-lococcus spp and 025 mgL for Streptococcus spp and S pneumoniae [66]

23 Pharmacokinetic Properties of Clindamycin

Clindamycin oral preparations are rapidly absorbed and reported oral bioavail-ability is around 90 Other notable pharmacokinetic properties include 90protein binding wide distribution in body fluids and tissues except for cerebralspinal fluid extensive hepatic metabolism to active metabolites and a half-lifeof around 20ndash24 h [616267ndash69] For severe liver dysfunction clindamycindoses should be reduced

For 600 mg intravenous doses peak serum concentrations are around 109gmL while trough concentrations are 20 and 11 mgL at 6 and 8 h respectively[61] Peak concentrations after intramuscular doses of 600 mg are 90 mgLwhich is slightly lower than those reported after intravenous dosing For 900 mgintravenous doses reported peak and trough concentrations at 8 h are 141 and17 mgmL respectively [61]

24 Pharmacodynamics of Clindamycin

Until recently little was known about the pharmacodynamic characteristics ofclindamycin Recent studies have demonstrated that clindamycin displays time-dependent pharmacodynamics against S pneumoniae methicillin-susceptible Saureus and B fragilis [6870] Furthermore clindamycin has an extended post-antibiotic effect against Staphylococcus [5471]

241 In Vitro Studies

Two time-kill studies have evaluated clindamycinrsquos activity against B fragilis[7172] Clindamycin was shown to display concentration-independent activityin a study conducted by Klepser et al [70] In this study clindamycin concentra-tions were evaluated over a range of 1ndash64 times the MIC Five strains of B


fragilis with clindamycin MICs of 003ndash80 mgL were studied Bactericidal ac-tivity (3 log decrease in the starting inoculum) was independent of clindamycinconcentration for three of the five strains A bacteristatic effect was noted for theisolate with an MIC of 80 mgL for all tested concentrations Against anotherstrain with an MIC of 10 mgL regrowth was observed at the MIC concentrationand bactericidal activity was observed for all other concentrations

Aldridge and Stratton [72] described concentration-dependent activity forclindamycin ceftizoxime and cefotetan against B fragilis However these inves-tigators studied only antibiotic concentrations over a range of 05ndash4 times theMIC In this study metronidazole chloramphenicol and cefoxitin were also eval-uated and reported clindamycin MIC values against the study isolates were 05and 10 mgL As described in the above study clindamycin clearly displaysconcentration-independent pharmacodynamic activity at concentrations that ex-ceed 4 times the MIC [70] Furthermore several in vitro and in vivo studiesdemonstrate that cephalosporins have time-dependent and not concentration-dependent pharmacodynamics which differs from the results reported by Al-dridge [54]

242 Serum Inhibitory and Bactericidal Titers

Klepser et al [68] evaluated the duration of serum bactericidal activity for 300mg oral and intravenous doses of clindamycin dosed every 8 h and every 12 hagainst two isolates each of S aureus S pneumoniae and B fragilis at steadystate in healthy volunteers Median MICs for the study isolates were 0125 and025 mgL for S aureus 0125 mgL for both S pneumoniae isolates and 05mgL for the B fragilis isolates For the every 8 h regimens measurable activitywas observed for 875ndash100 of the dosing interval against all study isolates Forthe every 12 h regimen bactericidal activity was observed for 50ndash77 of thedosing interval against S aureus 100 for S pneumoniae and 80ndash88 for Bfragilis These investigators concluded that dosing intervals of every 8 or every12 h for 300 mg oral or intravenous doses of clindamycin provided adequatecoverage against S aureus S pneumoniae and B fragilis except that an every8 h interval may be necessary for treatment of S aureus infections

Serum inhibitory titers were measured by Flaherty et al [67] in six volun-teers participating in a multiple dose intravenous clindamycin study against areference strain of B fragilis with an MIC of 025ndash05 mgL Clindamycin regi-mens assessed in this study were as follows 600 mg every 6 h 900 mg every8 h and 1200 mg every 12 h When considering the doses used in this study itis not surprising that activity was observed throughout the entire dosing intervalfor all regimens and that reciprocal inhibitory titers at the trough ranged from 5to 12

The synergistic effects of clindamycin with fluoroquinolones have alsobeen studied [7374] Increased serum bactericidal activity was observed when

230 Lamp et al

clindamycin was combined with ciprofloxacin ofloxacin and fleroxacin againstS aureus and S pneumoniae [73] This single-dose study evaluated 600 mg intra-venous and 300 mg oral doses of clindamycin given in combination with thequinolone test agents against both gram-positive organisms and anaerobes En-hanced activity was observed when clindamycin (600 mg IV) was given in combi-nation with ciprofloxacin (200 mg IV) and ofloxacin (200 mg IV) for S aureusand S pneumoniae Against S aureus reported reciprocal titers at 6 h were 54for ciprofloxacin-clindamycin compared to 23 for ciprofloxacin alone Similarlyofloxacin-clindamycin reciprocal titers of 41 were observed compared to 2 forofloxacin alone at 6 h Against S pneumoniae reciprocal titers at 6 h were 2and 136 for ciprofloxacin alone and ciprofloxacin-clindamycin respectively Forofloxacin and ofloxacin-clindamycin reciprocal titers of 2 and 135 were notedat 6 h Less activity was observed when oral clindamycin (300 mg) was givenin combination with oral enoxacin (400 mg) and fleroxacin (400 mg) than eitherquinolone alone at 8 h against S aureus

The serum bactericidal activity of an oral combination of ciprofloxacin (750mg every 12 h 3 doses) and clindamycin (300 mg every 6 h 5 doses) wasstudied by Weinstein et al [74] in healthy elderly volunteers Unlike the resultsreported by Boeckh et al [73] the bactericidal activity of ciprofloxacin againstS aureus was antagonized by clindamycin when clindamycin-susceptible strainswere tested However the reported MBCs for clindamycin against the three testS aureus strains were either 40 or 80 mgL This antagonistic effect was notobserved for the combination against S pyogenes and S pneumoniae in thisstudy


Several investigators have studied the postantibiotic effect (PAE) for clindamycinagainst S aureus [7576] Xue et al [76] reported that the duration of the PAEfor clindamycin is dependent on both concentration and duration of exposure toclindamycin with longer PAEs observed at higher concentrations In this studywhich used 21 clinical osteomyelitis isolates of S aureus observed PAEs rangedfrom 04 to 39 h Reported PAE values were not influenced by multiple dosingWhen considering clinically achievable concentrations and estimated PAE valuesof 24 h these investigators concluded that oral clindamycin could be adminis-tered at 300 mg every 8 h for the treatment of S aureus whereas recommendedintravenous dosing is 300 mg every 6 h

25 SummarymdashClindamycin

Similar to that observed for β-lactams macrolides vancomycin and others onlyrecently have the time-dependent pharmacodynamics of clindamycin been dis-covered Studies demonstrating serum bactericidal activity against susceptible


bacteria for either the majority or the entire dosing interval has been demonstratedwith lower doses than are currently recommended The application of the pharma-codynamics of clindamycin needs to be more fully evaluated in actual clinicalsituations

3 STREPTOGRAMIN

The streptogramin class of antibiotics are new introductions into the UnitedStates however they have been available in Europe for many years for mild tomoderate staphylococcal infections Recent modifications to the structure allowfor formulation of water-soluble forms of the compounds and subsequent usein intravenous solutions [77] These efforts have led to the combination drugquinupristin-dalfopristin (RP 59500 Synercid) and its evaluation for treatmentof severe antibiotic-resistant infections

31 Chemistry and Mechanism of Action of Streptogramin

Streptomyces pristinaespiralis produces two clinically unrelated antibiotics(streptogramin A and B) which in combination exert their antibacterial actionThe streptogramin A antibiotics are polyunsaturated cyclic macrolactones andinclude pristinamycin IIA virginiamycin M and dalfopristin Streptogramin Bantibiotics are cyclic depsipeptides and include pristinamycin IA quinupristinand virginiamycin S [78] Quinupristin was produced by modifying pristinamycinIA to quinuclindinylthiomethyl pristinamycin IA Dalfopristin is diethylamino-ethyl-sulfonyl-pristinamycin IIA [79] A mixture of quinupristin a streptograminB and dalfopristin a streptogramin A in a 3070 ratio is capable of synergisti-cally inhibiting protein synthesis Streptogramin A binds to the 50S and 70Ssubunits of the ribosome when they are not actively synthesizing protein It hasbeen suggested that they block the elongation phase of bacterial protein synthesisConversely streptogramin B can bind to active ribosomes at the 50S subunitThe synergistic activity of the streptogramins is achieved through a conforma-tional change in the 50S subunit after binding a type A streptogramin whichleads to a strengthening of the binding of the type B streptogramin Ultimatelythe streptogramin combination inhibits early- and late-stage protein synthesis[80]

32 Antimicrobial Activity of Streptogramin


The quinupristin-dalfopristin combination inhibits growth of most gram-positivebacteria It has useful activity against Staphylococcus aureus Staphylococcusepidermidis Streptococcus pneumoniae Streptococcus agalactiae Streptococcus

232 Lamp et al

pyogenes viridans streptococci and Enterococcus faecium Enterococcus fae-calis is resistant to the streptogramins [81]


Quinupristin-dalfopristin has acceptable activity for many respiratory pathogensMost Moraxella spp Legionella spp Mycoplasma spp and Neisseria spp havean MIC90 of 40 mgL Against Haemophilus influenzae it has less activitywith MIC90 values of 40ndash80 mgL [7981] Quinupristin-dalfopristin has noactivity against Enterobacteriaceae or Pseudomonas aeruginosa [81]

322 Other Bacteria

Quinupristin-dalfopristin maintains useful activity against both Bacteroides frag-ilis (MIC90 40 mgL) and other Bacteroides species (MIC90 4ndash8 mgL) [8283]

33 Pharmacokinetic Properties of Streptogramin

The pharmacokinetics of quinupristin-dalfopristin are reported in a very smallnumber of studies Further data is needed for complete dosing recommendationsespecially in patients

331 Absorption

Quinupristin-dalfopristin is available only as an injectable product although oralderivatives are being developed

Etienne et al [84] studied the pharmacokinetics in doses ranging from 14to 294 mgkg administered as a 1 h intravenous infusion Maximum concentra-tions at the end of the infusion rose linearly from 095 to 242 mgL for thedoses studied As would be expected the area under the timendashconcentration curve(AUC) also increased linearly from 159 to 377 mgsdothL for the 126 and 294mgkg doses Antibacterial activity was present for up to 6 h after the dosesmicrogsdothml or mgsdothL is the most frequently used units for AUC despite plasmaconcentrations below the typical MIC for staphylococci and streptococci prob-ably indicating the importance of active metabolites [84]

A more extensive study by Bergeron and Montay [85] used single dosesof 5 10 and 15 mgkg given intravenously over 1 h to 18 healthy volunteersAt the 3 doses quinupristin Cmax increased linearly (from 12 to 23 to 36 mgL) and dalfopristin Cmax values were 46 64 and 85 mgL The dalfopristinmetabolite RP 12536 appeared rapidly and increased disproportionally from 09to 25 to 38 mgL However a combined AUC0ndashinfin for both dalfopristin and itsmetabolite did increase linearly Quinupristin AUC values increased from 14 to30 to 47 mgsdothL [85]

332 Distribution

In the Etienne et al study the mean T12 for quinupristindalfopristin ranged from127 to 153 h The RP 12536 metabolite of dalfopristin had a T12 of 075ndash084 h


[84] Bergeron and Montay [85] reported that following biphasic plasma elimina-tion the elimination T12 values for quinupristin and dalfopristin were 093ndash096h and 039ndash091 h respectively Variability was higher for dalfopristin andT12 increased with increasing doses

Quinupristin and dalfopristin are highly protein bound in animal infectionmodels Quinupristin in plasma was bound approximately 80 in rats and 90in monkeys Dalfopristin binding appears to increase over time from 45 to90 or greater in rats and monkeys [85] Protein binding ranges from 23 to32 for quinupristin and from 50 to 56 for dalfopristin in humans [86]Volume of distribution (Vd) and Vd at steady state (Vdss) were 137ndash144 Lkgand 079ndash083 Llg respectively for quinupristin and were unaffected by doseDalfopristin Vd and Vdss rose with each dose For the 5 10 and 15 mgkg dosesVd was 054 088 and 18 Lkg while Vdss was 033 043 and 070 Lkg respec-tively [85]

Radiolabeled quinupristin and dalfopristin were injected into rats and mon-keys and radioactivity was assessed for distribution Either drug was rapidly andextensively distributed to tissues in both species High concentrations were seenin the gall bladder and in bile indicating biliary excretion Both componentsreached high concentrations in gastrointestinal tissues kidneys and liver Thebrain and spinal cord had very low concentrations of quinupristin or dalfopristin[85]

Bernard measured blister fluid penetration after a single 12 mgkg IV dosein six healthy male volunteers Although a microbiological assay method wasemployed that is incapable of differentiating between the two components ormetabolites the mean percent tissue penetration was 824 indicating good tis-sue penetration Six hours after the end of the infusion the combination wasdetectable at a concentration of 092 mgL which should be sufficient for bacte-rial activity [87]

333 Metabolism

Bergeron and Montay [85] reported that clearance rates were high and approxi-mated hepatic blood flow 11 L(hsdotkg) and 10 to 12 L(hsdotkg) for quinupristinanddalfopristin respectively Bernard et al [87] found similar values after a sin-gle 12 mgkg IV infusion reported as 74 Lh for quinupristin-dalfopristin

A single intravenous infusion of 430 mg for over 1 h was administered tosix healthy male volunteers The radioactivity of quinupristin and dalfopristinwas measured to determine their metabolic fate The two drugs are both metabo-lized to active metabolites in humans Quinupristinrsquos cysteine derivative (RP100391) accounts for 38 and 75 of total radioactivity in urine and fecesrespectively No unchanged dalfopristin was recovered in either urine or fecesIn urine dalfopristin was recovered as pristinamycin IIA (RP 12536) 70 andcysteine derivatives 30 [8588]

234 Lamp et al

334 Excretion

After IV administration both quinupristin and dalfopristin are excreted primarilyin the feces Fecal excretion as measured by radioactivity of quinupristin anddalfopristin accounted for 747 and 775 respectively The remainder of quin-upristin and dalfopristin was recovered in urine 15 and 187 respectivelyThirty-five percent of quinupristin in urine was recovered unchanged with a cor-responding value of 15 in feces [88]

Renal Dysfunction The pharmacokinetics of quinupristin and dalfopristinare not altered by the presence of continuous ambulatory peritoneal dialysis Theconcentrations of quinupristin and dalfopristin in dialysis effluent were very lowand below the MIC of most pathogens [86] Patients with severe renal failuremay have impaired clearance of quinupristin metabolites and Cmax and AUCvalues for dalfopristin were elevated 30 over those in healthy volunteers [86]

34 Pharmacodynamic Properties of Streptogramin


The MBC for streptogramin can be up to 5 dilutions higher than the MIC for Efaecium and 3ndash6 dilutions higher against S aureus Against streptococci theMIC and MBC were within one dilution for each other [89]

Quinupristin-dalfopristin has been evaluated in several studies using time-kill studies It exerts a time-dependent bacteriostatic and what is sometimesreferred to as slow bactericidal activity against E faecium and S aureus [8990]However in several studies quinupristin-dalfopristin has a good bactericidal ef-fect on certain strains of both organisms [9192] Further studies with manystrains of E faecium have shown that quinupristin-dalfopristin at a concentrationof 60 mgL can have a concentration-dependent bactericidal effect for isolateswith MBCs of 40 mgL however most strains appear to possess higher MBCs[91] The quinupristin-dalfopristin concentrationMBC ratio and quinupristinMICs were significantly correlated with the bactericidal rate [91] Other investi-gators have found that bactericidal activity is more likely to be demonstrated forenterococci in the log growth phase and those with at least intermediate suscepti-bility to erythromycin [93] Against S aureus quinupristin-dalfopristin also dem-onstrates a variable bactericidal rate presumably as a result of differences inMBCs Against methicillin-susceptible S aureus (MSSA) and methicillin-resis-tant S aureus (MRSA) quionupristin-dalfopristin decreased the inoculum 1ndash2log10 over 24 h compared to 3ndash4 log10 for oxacillin vancomycin or gentamicin[89] In other experiments quinupristin-dalfopristin has shown the ability to re-duce the inoculum by 2ndash3 log10 in only 6ndash12 h [92] The presence of a constitu-tively erythromycin-resistant phenotype is predictive of bacteriostatic activity[94]


Quinupristin-dalfopristin in early studies showed a consistent rapid bacteri-cidal activity against penicillin-susceptible S pneumoniae The inoculum wasdecreased by 2ndash3 log10 in only 10 min This bactericidal rate was faster than thatof all comparators including penicillin G erythromycin ciprofloxacin sparflox-acin and vancomycin However three out of ten strains only a bacteriostaticeffect was seen [95] Later studies with larger numbers of S pneumoniae isolatesdemonstrated its rapid bactericidal effect against all S pneumoniae regardless ofpenicillin or erythromycin susceptibility at 2 times the MIC [9697] Againstviridans streptococci quinupristin-dalfopristin exerts a bactericidal effect over12ndash24 h The bactericidal rate against viridans streptococci is less when the iso-lates are erythromycin-resistant through loss of quinupristin activity [98]


The influence of postantibiotic effect (PAE) on antibiotic dosing is controversialWith quinupristindalfopristin possessing short half-lives long PAEs may influ-ence the dosing schedule

The PAE against E faecium in in vitro experiments has produced variableresults Against E faecium the PAE after a 1 h exposure to 025 times the MICwas 85 and 26 h for vancomycin-susceptible and -resistant strains respectively[99] However others have found the PAE 4 times the MIC to be as short as02ndash30 h for vancomycin-resistant strains and inversely related to the MBC andquinupristin MIC [91] The presence of multiple and unmeasured antibiotic resis-tance may affect the results of PAE determinations for enterococci

Staphylococci are also reported to possess a long concentration-dependentPAE after exposure to quinupristindalfopristin The in vitro PAE at 10 timesthe MIC was 46ndash70 h for MSSA and MRSA either erythromycin-susceptibleor inducibly resistant For MRSA containing the constitutively erythromycin-resistant phenotype the PAE was reduced to 24 h [29] Other investigators havealso found a longer PAE in MSSA compared to MRSA at 4 times the MIC 7h versus 5 h respectively [100]

Pneumococci show concentration-dependent PAEs to quinupristin-dalfo-pristin The rapid bactericidal rate complicates the determination of PAE how-ever at 05 times the MIC the PAE ranges from 17 to 41 h [99]

In the mouse thigh infection model the PAEs against S aureus and Spneumoniae were 10 and 91 h respectively [101]

343 Experimental Infection Models

Few studies have been published with quinupristin-dalfopristin and limited infor-mation is available from in vitro or in vivo models These models are also limitedthrough the use of only a few bacterial strains in primarily endocarditis and maynot be completely predictive of results in humans for other types of infections

236 Lamp et al

In Vitro Models Quinupristindalfopristin was evaluated in an in vitroinfection model of simulated endocardial vegetations The drug was dosed tosimulate a human dose of 75 mgkg every 8 h for 96 h low-dose continuousinfusion (Css 15 mgL) and high-dose continuous infusion (Css 92 mgL)Two vancomycin-resistant and constitutively erythromycin-resistant E faeciumstrains were used and one of the isolates possessed an elevated MBC Againstthe isolate with a low MBC all regimens were bacteriostatic with the exceptionof the high-dose continuous infusion regimen which achieved bactericidal activ-ity after 90 h The addition of doxycycline to the every 8 h regimen resulted inincreased killing although it did not reach a value indicating synergy The isolatewith an elevated MBC was unaffected by quinupristindalfopristin as an every8 h regimen or low-dose continuous infusion Doxycycline possessed the greatestactivity alone and no increased killing was observed when it was administeredwith quinupristin-dalfopristin The use of a high-dose continuous infusion regi-men or the addition of doxycycline reduced or eliminated the appearance of resis-tant isolates during the model [102]

Quinupristin-dalfopristin activity against S aureus in a fibrin clot modelwas compared to vancomycin over 72 h A 75 mgkg every 8 h dose was simu-lated Quinupristin-dalfopristin was more active than vancomycin however nei-ther reached bactericidal activity This was despite both the MSSA and MRSAisolate possessing MBCs close to the MIC The combination of quinupristin-dalfopristin and vancomycin led to greater decreases in log10 counts (not syner-gistic) and prevented the emergence of quinupristin-dalfopristin resistance [103]

This same model further evaluated quinupristin-dalfopristin in simulateddoses of 75 mgkg given every 6 8 and 12 h and by continuous infusion (60mgL) against S aureus One strain was an MRSA with constitutive erythromy-cin resistance and the other was an MSSA and erythromycin-susceptible Allregimens were bactericidal against the MSSA strain but no regimens reachedthis level of activity against the MRSA isolate The AUC0-24 was significantlycorrelated with activity against the MRSA strain [104]

In Vivo Models Two inducibly erythromycin-resistant and one sensitiveE faecium strain were studied in a rabbit endocarditis model for 4 days Quinu-pristin-dalfopristin was given in a dose of 30 mgkg IM evert 8 h and compared tothrice daily regimens of amoxicillin or gentamicin or combinations Quinupristin-dalfopristin when given alone had equivalent activity to amoxicillin against theerythromycin-susceptible strain For the erythromycin-resistant strains quinu-pristin-dalfopristin was not effective Additional experiments against one of theerythromycin-resistant strains showed that combinations of quinupristin-dalfo-pristin with gentamicin or amoxicillin were more active than either of the drugsalone No information was presented on pharmacodynamic parameters [105]

Quinupristin-dalfopristin has been studied in a rabbit infected fibrin clotmodel against two strains each of S aureus and S epidermidis A rapid bacteri-


cidal rate was observed after the single dose of 50 mgkg for S aureus howeveronly bacteriostatic activity was seen against S epidermidis The elevated MBCsfor S epidermidis at the inoculum tested may explain the disparate results Alsothe Cmax obtained with this dose is considerably higher than that observed inhumans and may not be predictive of human experience [106]

An early rabbit endocarditis model administered quinupristin-dalfopristinin a dose of 20 mgkg intramuscularly (IM) every 6 h versus vancomycin 25 mgkg IV every 12 h for 4 days Three susceptible S aureus strains with increasingquinupristin-dalfopristin MICs (05ndash20 mgL) and MBCs (40ndash80 mgL) wereused The peak serum concentration of quinupristin-dalfopristin measured by bio-assay was 19 mgL with a T12 of 13 h Vancomycin was effective against allstrains whereas quinupristin-dalfopristin was effective against the strains withMICs of 05 and 10 mgL although less effective at sterilizing vegetations at anMIC of 10 mgL It failed against the strain with an MIC of 20 mgL Higherdoses of 40 mgkg in this model resulted in animal toxicity and death [107]Given the pharmacokinetic data these regimens would be expected to producea time above the MIC during the dosing interval (T MIC) of approximately57 36 and 0 for S aureus with MICs of 05 10 and 20 mgL respec-tively Therefore it appeared that quinupristin-dalfopristin required a T MICof approximately 50 for equivalent bactericidal activity

Quinupristin-dalfopristin was compared to vancomycin in another Saureus rabbit endocarditis model The isolate had an MIC and MBC of 05 mgL to quinupristin-dalfopristin Vancomycin and quinupristin-dalfopristin weredosed at 30 mgkg IM every 12 h for 4 days Quinupristin-dalfopristin was aseffective as vancomycin and no resistant isolates were recovered from vegeta-tions The T MIC for quinupristin-dalfopristin was 33 Concentrations werealso measured in the vegetations and at 1 h after a single injection the ratio ofvegetation to serum concentration was 41 These data along with a prolongedPAE may explain the efficacy of quinupristin-dalfopristin [108] However thisS aureus strain may not be representative of most clinical isolates due to itsequivalent MIC and MBC

A S aureus rat endocarditis model demonstrated that quinupristin-dalfo-pristin in a regimen simulating a human dose of 7 mgkg every 12 h was aseffective as vancomycin only against S aureus strains that were erythromycin-susceptible For constitutively erythromycin-resistant strains that had quinupristinMICs of 64 mgL the shorter dalfopristin T12 in rats did not allow for the T MIC (20) to be sufficient for efficacy A regimen that prolonged the dalfo-pristin concentration did allow the regimen to be effective for these macrolide-resistant strains The authors recommended that efficacy would be improvedthrough use of an every 8 h regimen or continuous infusion [24] Similar findingswere found in a rabbit endocarditis model evaluating the efficacy of quinupristin-dalfopristin against S aureus with variable erythromycin susceptibilities Quinu-pristin-dalfopristin doses were 30 mgkg IM every 8 or 12 h and 10 mgkg IV

238 Lamp et al

every 12 h These regimens were as effective as vancomycin against erythromy-cin-susceptible or inducibly resistant strains regardless of oxacillin susceptibilityQuinupristin-dalfopristin was less active in all doses against the two strains withconstitutively erythromycin-resistant phenotypes The AUC for quinupristin-dal-fopristin in plasma divided by the quinupristin MIC was most predictive of activ-ity The determination of quinupristin MICs may be necessary prior to beginningtherapy with quinupristin-dalfopristin for endocarditis [77]

A mouse S aureus septicemia model compared quinupristin-dalfopristinto vancomycin Only one S aureus strain was used and no MBC or other drugresistant data were reported Quinupristin-dalfopristin regimens of a single doseof 120 mgkg 60 mgkg every 12 h 40 mgkg every 8 h and 30 mgkg every6 h were administered for 24 h Quinupristin-dalfopristin in all doses demon-strated a faster bactericidal rate than vancomycin with some dose-dependent bac-tericidal activity being noted No data were presented on which parameter wasmore closely associated with activity because all AUCs were similar [109]

In a mouse thigh infection model the influence of dosing interval on theactivity of quinupristin-dalfopristin was evaluated against S aureus and S pneu-moniae The dose ranged from 125 to 800 mgkg and administered in one twoor four doses over 24 h A sigmoidal dosendashresponse model indicated that theAUC was best correlated with efficacy [101]

344 Synergy

The importance of synergistic combinations is well documented for certain en-terococcal and staphylococcal infections Given quinupristin-dalfopristinrsquos slowbactericidal activity against many of these strains synergy testing has been evalu-ated

As already mentioned quinupristin and dalfopristin are synergistic whenused in combination The IV product contains a quinupristin-dalfopristin ratioof 3070 The more variable T12 of dalfopristin and information on decreasedpenetration of dalfopristin into cardiac vegetations underlies the importance ofinvestigating the effect of different ratios on activity [104] Studies of in vitroand in vivo activity show that the combination remains synergistic and effectivein a range from 1684 to 8416 which should accommodate most clinical situa-tions [110]

Quinupristin-dalfopristin has no effect on the MIC of ciprofloxacin genta-micin or cefotaxime against E coli K pneumoniae C freundii E cloacae Smarcescens or P aeruginosa Quinupristin-dalfopristin increased the MIC forciprofloxacin against B fragilis in nine out of 20 strains but had no effect onother Bacteroides species or S aureus [82] The quinupristin-dalfopristin MICfor vancomycin-resistant E faecium was decreased when the combination com-bined with ciprofloxacin teicoplanin and tetracycline [100]

Using time-kill methods quinupristin-dalfopristin was not found to have


synergy against E faecium when combined with clinafloxacin LY333328 oreperozolid These isolates had quinupristin-dalfopristin MBCs of 80 mgL[111] No synergy was seen defined by an FIC index of 05 when quinupristin-dalfopristin was combined with vancomycin however time-kill studies indicatedsynergy against S aureus at higher inocula (5 107 CFUmL) [103] An induci-bly erythromycin-reistant gentamicin-resistant amoxicillin-sensitive E faeciumstrain was exposed to quinupristin-dalfopristin with and without gentamicin oramoxicillin but the combinations did not demonstrate faster killing rates [105]

35 SummarymdashStreptogramins

Quinupristin-dalfopristin has been shown effective against E faecium S aureusand S pneumoniae The pharmacodynamic parameter most associated with effi-cacy in animal models is the AUC The presence of erythromycin resistance hasa dramatic effect on the activity of the combination drug quinupristin-dalfopristinThis phenotype will lead to elevated quinupristin MICs and may need to be deter-mined prior to beginning therapy Further data are needed from patients to deter-mine the most appropriate dosing requirements that maximize its bactericidaleffect

REFERENCES

1 C Cosar L Joulou Activite de lrsquo(hydroxy-2-ethyl)-1-methyl-2-nitro-5-imidazole(8823 RP) vis-a-vis des infections experimentales a trichomonas vaginalis AnnInst Pasteur 195996238ndash241

2 M Muller Mode of action of metronidazole on anaerobic bacteria and protozoaSurgery 198393165ndash171

3 B Korner HK Jensen Sensitivity of Trichomonas vaginalis to metronidazole tini-dazole and nifuratel in vitro Br J Vener Dis 197652404ndash408

4 SD Sears J OrsquoHare In vitro susceptibility of Trochomonas vaginalis to 50 antimi-crobial agents Antimocrob Agents Chemother 198832144ndash146

5 L Jokipii AMM Jokipii In vitro susceptibility of Giardia lamblia trophozoites tometronidazole and tinidazole J Infect Dis 1980141317ndash325

6 VK Vinayak RC Mahajan NL Chitkara In-vitro activity of metronidazole on thecysts of Entamoeba histolytica Ind J Pathol Bacteriol 19751861ndash64

7 RC Mahajan NL Chitkara VK Vinayak DV Dutta In vitro comparative evaluationof tinidazole and metronidazole on strains of Entamoeba histolytica Indian J PatholBacteriol 197417226ndash228

8 DLS Shinn Metronidazole in acute ulcerative gingivitis Lancet 1962111919 FP Tally VL Sutter SM Finegold Metronidazole versus anaerobes In vitro data

and initial clinical observations Calif Med 197211722ndash2610 JP Rissing WL Moore Jr C Newman JK Crockett TB Buxton HT Edmondson

Treatment of anaerobic infections with metronidazole Curr Ther Res 198027651ndash663

240 Lamp et al

11 Study Group An evaluation of metronidazole in the prophylaxis and treatment ofanaerobic infections in surgical patients J Antimicrob Chemother 19751393ndash401

12 SJ Eykyn I Phillips Metronidazole and anaerobic sepsis Br Med J 197621418ndash1421

13 B Olsson-Liljequist CE Nord In vitro susceptibility of anaerobic bacteria to nitro-imidazoles Scand J Infect Dis 198126(suppl)42ndash45

14 KE Aldridge M Gelfand LB Reller LW Ayers CL Pierson F Schoenknect FTilton J Wilkins A Henderberg DD Schiro M Johnson A Janney CV SandersA five-year multicenter study of the susceptibility of the Bacteroides fragilis groupisolates to cephalosporins cephamycins penicillins clindamycin and metronida-zole in the United States Diagn Microbiol Infect Dis 199418235ndash241

15 JB Selkon The need for and choice of chemotherapy for anaerobic infectionsScand J Infect Dis 198126(suppl)19ndash23

16 GA Pankuch MR Jacobs PC Appelbaum Susceptibilities of 428 gram-positiveand -negative anaerobic bacteria to Bay y3118 compared with their susceptibilitiesto ciprofloxacin clindamycin metronidazole piperacillin piperacillin-tazobactamand cefoxitin Antimicrob Agents Chemother 1993371649ndash1654

17 CAM McNulty J Dent R Wise Susceptibility of clinical isolates of Campylobacterpyloridis to 11 antimicrobial agents Antimicrob Agents Chemother 198528837ndash838

18 M Lopez-Brea E Martin C Lopez-Lavid JC Sanz Susceptibility of Helicobacterpylori to metronidazole Eur J Clin Microbiol Infect Dis 1991101082ndash1083

19 MJAMP Pavicic AJ van Winkelhoff J de Graaff Synergistic effects betweenamoxicillin metronidazole and the hydroxymetabolite of metronidazole againstActinobacillus actinomycetemcomitans Antimicrob Agents Chemother 199135961ndash966

20 MJAMP Pavicic AJ van Winkelhoff J de Graaff In vitro susceptibilities of Actino-bacillus actinomycetemcomitans to a number of antimicrobial combinations Anti-microb Agents Chemother 1992362634ndash2638

21 AW Chow V Patten LB Guze Susceptibility of anaerobic bacteria to metronida-zole Relative resistance of non-spore-forming gram-positive bacilli J Infect Dis1975131182ndash185

22 AMM Jokipii L Jokipii Comparative activity of metronidazole and tinidazoleagainst Clostridium difficle and Peptostreptococcus anaerobius Antimicrob AgentsChemother 198731183ndash186

23 K Dornbusch CE Nord B Olsson-Liljeqvist Antibiotic susceptibility of anaerobicbacteria with special reference to Bacteroides fragilis Scand J Infect Dis 197919(suppl 19)17ndash25

24 JP OrsquoKeefe KA Troc KA Thompson Activity of metronidazole and its hydroxyand acid metabolites against clinical isolates of anaerobic bacteria AntimicrobAgents Chemother 198222426ndash430

25 JL Whiting N Cheng AW Chow Interactions of ciprofloxacin with clindamycinmetronidazole cefoxitin cefotaxime and mezlocillin against gram-positive andgram-negative anaerobic bacteria Antimicrob Agents Chemother 1987311379ndash1382


26 HM Wexler SM Finegold In vitro activity of cefotetan compared with that ofother antimicrobial agents against anaerobic bacteria Antimicrob Agents Chemo-ther 198832601ndash604

27 H Hof V Sticht-Groh Antibacterial effects of niridazole Its effect on microaero-philic campylobacter Infection 19841236ndash39

28 AM Freydiere Y Gille S Tigaud P Vincent In vitro susceptibilities of 40 Campy-lobacter fetus subsp jejuni strains to niridazole and metronidazole AntimicrobAgents Chemother 198425145ndash146

29 BM Jones I Geary AB Alawattegama GR Kinghorn BI Duerden In-vitro andin-vivo activity of metronidazole against Gardnerella vaginalis Bacteroides sppand Mobiluncus spp in bacterial vaginosis J Antimicrob Chemother 198516189ndash197

30 ABM Kharsany AA Hoosen J van den Ende Antimicrobial susceptibilities ofGardnerella vaginalis Antimicrob Agents Chemother 1993372733ndash2735

31 M Weinberger T Wu M Rubin VJ Gill PA Pizzo Leptotrichia buccalis bacter-emia in patients with cancer Report of four cases and review Rev Infect Dis 199113201ndash206

32 AH Davies Metronidazole in human infections with syphilis Br J Vener Dis 196743197ndash200

33 T Bergan E Arnold Pharmacokinetics of metronidazole in healthy volunteers aftertablets and suppositories Chemotherapy 198026231ndash241

34 GW Houghton J Smith PS Thorne R Templeton The pharmacokinetics of oraland intravenous metronidazole in man J Antimicrob Chemother 19795621ndash623

35 ED Ralph JT Clarke RD Libke RP Luthy WMM Kirby Pharmacokinetics ofmetronidazole as determined by bioassay Antimicrob Agents Chemother 19746691ndash696

36 J Mattila PT Mannisto R Mantyla S Nykanen U Lamminsivu Comparative phar-macokinetics of metronidazole and tinidazole as influenced by administration routeAntimicrob Agents Chemother 198323721ndash725

37 T Bergan O Leineboslash T Blom-Hagen B Salvesen Pharmacokinetics and bioavail-ability of metronidazole after tablets suppositories and intravenous administrationScand J Gastroenterol 198419(suppl 91)45ndash60

38 GW Houghton PS Thorne J Smith R Templeton Plasma metronidazole concen-trations after suppository administration In Phillips and Collier eds Metronida-zole Proceedings of the Second International Symposium on Anaerobic InfectionsGeneva April 197941ndash44

39 FE Cunningham DM Kraus L Brubaker JH Fischer Pharmacokinetics of intra-vaginal metronidazole gel J Clin Pharmacol 1994341060ndash1065

40 MM Alper N Barwin WM McLean IJ McGilveray S Sved Systemic absorptionof metronidazole by the vaginal route Obstet Gynecol 198565781ndash784

41 IK Aronson JA Rumsfield DP West J Alexander JH Fischer FP Paloucek Evalu-ation of topical metronidazole gel in acne rosacea Drug Intell Clin Pharm 198721346ndash351

42 LK Schmadel GK McEvoy Topical metronidazole A new therapy for rosaceaClin Pharm 1990994ndash101

242 Lamp et al

43 DE Schwartz F Jeunet Comparative pharmacokinetic studies of ornidazole andmetronidazole in man Chemotherapy 19762219ndash29

44 AH Lau K Emmons R Seligsohn Pharmacokinetics of intravenous metronidazoleat different dosages in healthy subjects Int J Clin Pharmacol Ther Toxicol 199129386ndash390

45 I Amon K Amon H Huller Pharmacokinetics and therapeutic efficacy of metroni-dazole at different dosages Int J Clin Pharmacol Ther Toxicol 197816384ndash386

46 WL Hand N King-Thompson JW Holman Entry of roxithromycin (RU 965) imi-penem cefotaxime trimethoprim and metronidazole into human polymorphonu-clear leukocytes Antimicrob Agents Chemother 1987311553ndash1557

47 KE Andersson Pharmacokinetics of nitroimidazoles Spectrum of adverse reac-tions Scand J Infect Dis 198126(suppl 26)60ndash67

48 I Nilsson-Ehle B Ursing P Nilsson-Ehle Liquid chromatographic assay for metro-nidazole and tinidazole Pharmacokinetic and metabolic studies in human subjectsAntimicrob Agents Chemother 198119754ndash760

49 TY Ti HS Lee YM Khoo Disposition of intravenous metronidazole in Asian sur-gical patients Antimicrob Agents Chemother 1996402248ndash2251

50 SK Spangler MR Jacobs PC Appelbaum Time-kill study of the activity of tro-vafloxacin compared with ciprofloxacin sparfloxacin metronidazole cefoxitinpiperacillin and piperacillintazobactam against six anaerobes J Antimicrob Che-mother 199739(suppl B)23ndash27

51 H Mattie AC Dijkmans C van Gulpen The pharmacokinetics of metronidazoleand tinidazole in patients with mixed aerobic-anaerobic infections J AntimicrobChemother 198210(suppl A)59ndash64

52 JG Bartlett TJ Louie SL Gorbach AB Onderonk Therapeutic efficacy of 29 anti-microbial regimens in experimental intraabdominal sepsis Rev Infect Dis 19813535ndash542

53 DE Nix R Tyrrell M Muller Pharmacodynamics of metronidazole determined bya time-kill assay for Trichomonas vaginalis Antimicrob Agents Chemother 1995391848ndash1852

54 WA Craig SC Ebert Killing and regrowth of bacteria in vitro A review ScandJ Infect Dis 199174(suppl 74)63ndash70

55 P Van der Auwera Y Van Laethem N Defresne M Husson J Klastersky Compar-ative serum bactericidal activity against test anaerobes in volunteers receiving imi-penem clindamycin latamoxef and metronidazole J Antimicrob Chemother 198719205ndash210

56 M Boeckh H Lode KM Deppermann S Grineisen F Shokry R Held K WernickeP Koeppe J Wagner C Krasemann K Borner Pharmacokinetics and serum bacteri-cidal activities of quinolones in combination with clindamycin metronidazole andornidazole Antimicrob Agents Chemother 1990342407ndash2414

57 A Pefanis C Thauvin-Elipoulos J Holden GM Eliopoulos MJ Ferraro RC Moell-ering Jr Activity of fleroxacin alone and in combination with clindamycin or metro-nidazole in experimental intra-abdominal abscesses Antimicrob Agents Chemother199438252ndash255

58 SF Kowalsky RM Echols EM McCormick Comparative serum bactericidal activ-


ity of ceftizoximemetronidazole ceftizoxime clindamycin and imipenem againstobligate anaerobic bacteria J Antimicrob Chemother 199025767ndash775

59 CD Freeman CH Nightingale DP Nicolau PP Belliveau PR Tessier Q Fu DXuan R Quintiliani Bactericidal activity of low-dose ceftizoxime plus metronida-zole compared with cefoxitin and ampicillin-sulbactam Pharmacotherapy 199414185ndash190

60 CD Freeman CH Nightingale DP Nicolau PP Belliveau R Quintiliani Serumbactericidal activity of ceftriaxone plus metronidazole against common intra-ab-dominal pathogens Am J Hosp Pharm 1994511782ndash1787

61 Pharmacia amp Upjohn Cleocin Phosphate manufacturerrsquos package insert Phar-macia amp Upjohn 1998

62 ME Falagas SL Gorbach Clindamycin and metronidazole Med Clin N Am 199579845ndash867

63 PG Kremsner Clindamycin in malaria treatment J Antimicrob Chemother 1990259ndash14

64 GV Doern MA Pfaller K Kugler J Freeman RN Jones Prevalence of antimicro-bial resistance among respiratory tract isolates of Streptococcus pneumoniae inNorth America 1997 results from the SENTRY antimicrobial surveillance pro-gram Clin Infect Dis 199827764ndash770

65 MA Pfaller RN Jones GV Doern K Kugler and the SENTRY Participants GroupBacterial pathogens isolated from patients with bloodstream infection Frequenciesof occurrence and antimicrobial susceptibility patterns from the SENTRY antimi-crobial surveillance program (United States and Canada 1997) Antimicrob AgentsChemother 1998421762ndash1770

66 National Committee for Clinical and Laboratory Standards Performance standardsfor antimicrobial susceptibility testing Eighth informational supplement NCCLSdocument M100-S8 Wayne PA 1998

67 JF Flaherty LC Rodondi BJ Guglielmo JC Fleishaker RJ Townsend JG Gamber-toglio Comparative pharmacokinetics and serum inhibitory activity of clindamycindifferent dosing regimens Antimicrob Agents Chemother 1988321825ndash1829

68 ME Klepser DP Nicolau R Quintiliani CH Nightingale Bactericidal activity oflow-dose clindamycin administered at 8- and 12-hour intervals against Staphylo-coccus aureus Streptococcus pneumoniae and Bacteroides fragilis AntimicrobAgents Chemother 199741630ndash635

69 JD Smilack WR Wilson FR Cockerill III Tetracyclines chloramphenicol erythro-mycin clindamycin and metronidazole Mayo Clin Proc 1991661270ndash1280

70 ME Klepser MA Banevicius R Quintiliani CH Nightingale Characterization ofbactericidal activity of clindamycin against Bacteroides fragilis via kill curve meth-ods Antimicrob Agents Chemother 1996401941ndash1944

71 WA Craig S Gudmundsson Postantibiotic effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams and Wilkins Baltimore 1996296ndash329

72 KE Aldridge CW Stratton Bactericidal activity of ceftizoxime cefotetan and clin-damycin against cefoxitan-resistant strains of the Bacteroides fragilis group J Anti-microb Chemother 199128701ndash705

73 M Boeckh H Lode KM Deppermann S Grineisen F Shokry R Held K WernickeP Koeppe J Wagner C Krasemann et al Pharmacokinetics and serum bactericidal

244 Lamp et al

activities of quinolones in combination with clindamycin metronidazole and orni-dazole Antimicrob Agents Chemother 1990342407ndash2414

74 MP Weinstein RG Deeter KA Swanson JS Gross Crossover assessment of serumbactericidal activity and pharmacokinetics of ciprofloxacin alone and in combina-tion in healthy elderly volunteers Antimicrob Agents Chemother 1991352352ndash2358


76 IB Xue PG Davey G Phillips Variations in postantibiotic effect of clindamycinagainst clinical isolates of Staphylococcus aureus and implications for dosing ofpatients with osteomyelitis Antimicrob Agents Chemother 1996401403ndash1407

77 B Fantin R Lequercq Y Merle L Saint-Julien C Veyrat J Duval C CarbonCritical influence of resistance to streptogramin B-type antibiotics on activity ofRP 59500 (quinupristin-dalfopristin) in experimental endocarditis due to Staphylo-coccus aureus Antimicrob Agents Chemother 1995 39400ndash405

78 R Rende-Fournier R Leclercq M Galimand J Duval P Courvalin Identificationof the satA gene encoding a streptogramin A acetyltransferase in Enterococcusfaecium BM4145 Antimicrob Agents Chemother 1993372119ndash2125

79 JM Andrews R Wise The in-vitro activity of a new semi-synthetic streptogramincompound RP 59500 against staphylococci and respiratory patohgens J Antimi-crob Chemother 199433849ndash853

80 C Cocito M Di Giambattista E Nyssen P Vannuffel Inhibition of protein synthesisby streptogramins and related antibiotics J Antimicrob Chemother 199739(supplA)7ndash13

81 DH Bouanchaud In-vitro and in-vivo antibacterial activity of quinupristindalfo-pristin J Antimicrob Chemother 199739(suppl A)15ndash21

82 HC Neu NX Chin JW Gu The in-vitro activity of new streptogramins RP 59500RP 57669 and RP 54476 alone and in combination J Antimicrob Chemother 199230(suppl A)83ndash94

83 PC Appelbaum SK Spangler MR Jacobs Susceptibility of 539 gram-positive andgram-negative anaerobes to new agents including RP59500 biapenem trospecto-mycin and piperacillintazobactam J Antimicrob Chemother 199332223ndash231

84 SD Etienne G Montay A Le Liboux A Frydman JJ Garaud A phase I double-blind placebo-controlled study of the tolerance and pharmacokinetic behaviour ofRP 59500 J Antimicrob Chemother 199230(suppl A)123ndash131

85 M Bergeron G Montay The pharmacokinetics of quinupristindalfopristin in labo-ratory animals and in humans J Antimicrob Chemother 199739(suppl A)129ndash138

86 CA Johnson CA Taulor SW Zimmerman WE Bridson P Chevlaier O PasquierRI Baybutt Pharmacokinetics of quinupristin-dalfopristin in continuous ambula-tory peritoneal dialysis patients Antimicrob Agents Chemother 1999 43152ndash156

87 E Bernard M Bensoussan F Bensoussan S Etienne I Cazenave E Carsenti-EtesseY Le Roux G Montay P Dellamonica Pharmacokinetics and suction blister fluidpenetration of a semisynthetic injectable streptogramin RP 59500 (RP 57669RP54476) Eur J Clin Microbiol Infect Dis 199413768ndash771


88 C Gaillard J Van Cantfort G Montay D Piffard A Le Liboux S Etienne AScheen A Frydman Disposition of the radiolabelled streptogramin RP 59500 inhealthy male volunteers In Programs and Abstracts of the 32nd Interscience Con-ference on Antimicrobial Agents and Chemotherapy Anaheim 1992 Abstract1317 Am Soc Microbiol Washington DC

89 RJ Fass In vitro activity of RP 59500 a semisynthetic injectable pristinamycinagainst staphylococci streptococci and enterococci Antimicrob Agents Chemo-ther 199135553ndash559

90 RL Hill CT Smith M Seyed-Akhavani MW Casewell Bactericidal and inhibitoryactivity of quinupristindalfopristin against vancomycin- and gentamicin-resistantEnterococcus faecium J Antimicrob Chemother 199739(suppl A)23ndash28

91 JR Aeschlimann MJ Rybak Pharmacodynamic analysis of the activity of quinu-pristin-dalfopristin against voncomycin-resistant Enterococcus faecium with dif-fering MBCs via time-kill-curve and postantibiotic effect method AntimicrobAgents Chemother 1998422188ndash2192

92 DE Low HL Nadler A review of in-vitro antibacterial activity of quinupristindalfopristin against methicillin-susceptible and -resistant Staphylococcus aureus JAntimicrob Chemother 199739(suppl A)53ndash58

93 F Caron HS Gold CB Wennersten MG Farris RC Moellering Jr GM EliopoulosInfluence of erythromycin resistance inoculum growth phase and incubation timeon assessment of the bactericidal activity of RP 59500 (quinupristin-dalfopristin)against vancomycin-resistant Enterococcus faecium Antimicrob Agents Chemo-ther 1997412749ndash2753

94 JM Entenza H Drugeon MP Glauser P Moreillon Treatment of experimental en-docarditis due to erythromycin-susceptible or -resistant methicillin-resistant Staph-ylococcus aureus with RP 59500 Antimicrob Agents Chemother 1995391419ndash1424

95 GA Pankuch MR Jacobs PC Applebaum Study of comparative antipneumococcalactivities of penicillin G RP 59500 erythromycin sparfloxacin ciprofloxacin andvancomycin by using time-kill methodology Antimicrob Agents Chemother 1994382065ndash2072

96 GA Pankuch MR Jacobs PC Applebaum MIC and time-kill study of antipneumo-coccal activities of RPR 106972 (a new oral streptogramin) RP 59500 (quinupris-tin-dalfopristin) pyostacine (RP 7293) penicillin G cefotaxime erythromycin andclarithromycin against 10 penicillin-susceptible and -resistant pneumococci Anti-microb Agents Chemother 1996402071ndash2074

97 GA Pankuch C Lichtenberger MR Jacobs PC Appelbaum Antipneumococcal ac-tivities of RP 59500 (quinupristin-dalfopristin) penicillin G erythromycin andsparfloxacin determined by MIC and rapid time-kill methodologies AntimicrobAgents Chemother 1996401653ndash1656

98 F LrsquoHeriteau JM Entenza F Lacassin C Leport MP Glauser P Moreillon RP59500 prophylaxis of experimental endocarditis due to erythromycin-susceptibleand -resistant isogenic pairs of viridans group streptococci Antimicrob AgentsChemother 1995391425ndash1429

99 GA Pankuch MR Jacobs PC Appelbaum Postantibiotic effect and postantibioticsub-MIC effect of quinupristin-dalfopristin against gram-positive and -negative or-ganisms Antimicrob Agents Chemother 1998423028ndash3031

246 Lamp et al

100 A Nougayrede N Berthaud DH Bouanchaud Post-antibiotic effects of RP 59500with Staphylococcus aureus J Antimicrob Chemother 199230(suppl A)101ndash106

101 W Craig S Ebert Pharmacodynamic activities of RP 50500 in an animal infectionmodel In Programs and Abstracts of the 33rd Interscience Conference on Antimi-crobial Agents and Chemotherapy New Orleans 1993 Abstract 470 Am Soc Mi-crobiol Washington DC

102 JR Aeschlimann MJ Zervos MJ Rybak Treatment of vancomycin-resistant En-terococcus faecium with RP 59500 (quinupristin-dalfopristin) administered by in-termittent or continuous infusion alone or in combination with doxycycline in anin vitro pharmacodynamic infection model with simulated endocardial vegetationsAntimicrob Agents Chemother 1998422710ndash2717

103 SL Kang MJ Rybak Pharmacodynamics of RP 59500 alone and in combinationwith vancomycin against Staphylococcus aureus in an in vitro-infected fibrin clotmodel Antimicrob Agents Chemother 1995391505ndash1511

104 MJ Rybak Houlihan RC Mercier GW Kaatz Pharmacodynamics of RP 59500(quinupristin-dalfopristin) administered by intermittent versus continuous infusionagainst Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infectionmodel Antimicrob Agents Chemother 1997411359ndash1363

105 B Fantin R Leclercq L Garry C Carbon Influence of inducible cross-resistanceto macrolides lincosamides and streptogramin B-type antibiotics in Enterococcusfaecium on activity of quinupristin-dalfopristin in vitro and in rabbits with experi-mental endocarditis Antimicrob Agents Chemother 199741931- 935

106 A Turcotte MG Bergeron Pharmacodynamic interaction between RP 59500 andgram-positive bacteria infecting fibrin clots Antimicrob Agents Chemother 1992362211ndash2215

107 HF Chambers Studies of RP 59500 in vitro and in a rabbit model of aortic valveendocarditis caused by methicillin-resistant Staphylococcus aureus J AntimicrobChemother 199230(supp A)117ndash122

108 B Fantin R Leclercq M Ottaviani JM Vallois B Maziere J Duval JJ PocidaloC Carbon In vivo activities and penetration of the two components of the strepto-gramin RP 59500 in cardiac vegetations of experimental endocarditis AntimicrobAgents Chemother 199438432ndash437

109 N Berthaud G Montay BJ Conard JF Desnottes Bactericidal activity and kineticsof RP 59500 in a mouse model of Staphylococcus aureus septicaemia J AntimicrobChemother 199536365ndash373

110 DH Bouanchaud In-vitro and in-vivo synergic activity and fractional inhibitoryconcentration (FIC) of the components of a semisynthetic streptogramin RP 59500J Antimicrob Chemother 199230(suppl A)95ndash99

111 RC Mercier SR Penzak MJ Rybak In vitro activities of an investigational quino-lone glycylcycline glycopeptide streptogramin and oxazolidinone tested aloneand in combinations against vancomycin-resistant Enterococcus faecium Antimi-crob Agents Chemother 1997412573ndash2575

11

Tetracycline Pharmacodynamics

Burke A Cunha

Winthrop-University Hospital Mineola and theState University of New York School of Medicine Stony Brook New York

Holly M Mattoes


1 INTRODUCTION

The first tetracycline to be discovered chlortetracycline was isolated fromStreptomyces aureufaciens in 1944 Since 1944 several tetracycline analogs havebeen developed including oxytetracycline which was introduced in 1950 tetracy-cline hydrochloride (1953) and demethylchlortetracycline (demeclocycline) Inthe late 1950s it was discovered that the 6-hydroxyl group could be removedfrom the basic tetracycline group which resulted in 6-deoxytetracyclines withsignificantly different microbiological and pharmacokinetic properties In the1960s the long-acting tetracyclines were introduced Doxycycline was isolatedin 1962 and minocycline was introduced in 1967 Although all tetracyclinesinhibit bacterial protein synthesis there are significant differences in inherentantibacterial activity between short-acting tetracyclines eg tetracycline andlong-acting second generation tetracyclines eg doxycycline and minocycline[1ndash3] The lsquolsquosecond generationrsquorsquo tetracyclines have better activity excellent phar-

247


macokinetics better intestinal absorption better tissue penetration and decreasedtoxicity [4]

Recently the glycylcyclines minocycline derivatives have demonstratedpotent activity against a wide range of pathogens including vancomycin-resistantenterococcus (VRE) [5] The glycylcyclines have enhanced activity against aero-bic and anaerobic bacteria that are typically resistant to tetracycline antimicrobi-als [6] This enhanced activity is due to the agentsrsquo ability to overcome both ofthe major resistant mechanisms ribosomal protection and active efflux whichresult in tetracycline resistance As a result the glycylcycline spectrum of activityincludes penicillin-resistant Streptococcus pneumoniae VRE and anaerobes likedoxycycline [1ndash35]

2 PHARMACOKINETICS OF TETRACYCLINES

21 Tetracycline

The tetracycline class of antibiotics are variably absorbed and their pharmacoki-netic properties are often due to the differing relative lipid solubilities and protein-binding capacities Tetracycline has a shorter serum half-life (6 h) than that ofthe second-generation antimicrobials doxycycline (22 h) and minocycline (11ndash33 h) As a result the short half-life of tetracycline means it needs to be given asfour divided doses daily In patients with renal failure the half-life of tetracyclinedramatically increases and in patients with severe renal failure the half-life canbe prolonged to 57ndash120 h Doxycycline does not accumulate in renal failure andthe dose of doxycycline remains unchanged even in dialysis patients with renalfailure Among the class of tetracyclines protein binding is the lowest with tetra-cycline demonstrated to be 20ndash65 depending on the method of analysis [3]Unlike doxcycline the absorption of tetracycline is significantly affected by foodand milk decreasing by up to 50 Additionally tetracycline is chelated by ca-tions and therefore should not be used concurrently with antacids

Pharmacokinetic studies of tetracycline found that serum concentrationsafter a single 500 mg oral dose peaked at 3ndash43 microgmL after 2ndash4 h whereas inpatients with normal renal function at steady state the same dose achieved a peakconcentration of 2ndash5 microgmL A 250 mg oral dose had an average peak serumconcentration of 24 microg at 3 h and this dropped to 1 microgmL at 12 h An in vitromodel determined that at therapeutic doses the tissue distribution in the lungachieved concentrations of 02ndash2 microgmL Tissue penetration into the lungs ap-pears to be the same whether the lungs are healthy or diseased [7] Tetracyclinehas the ability to penetrate into reticuloendothelial cells which allows for its useagainst infections by intracellular pathogens such as Rickettsia Chlamydia andLegionella [8] Gram-positive resistance to tetracycline has dramatically limited


its use to treat common infections however doxycycline and minocycline haveno resistance after decades of extensive use [210]

22 Minocycline

Minocycline and doxycycline are considered second generation tetracyclinesowing to their pharmacokinetic advantages over tetracycline which result in en-hanced tissue and fluid penetration and more convenient once or twice daily dos-ing regimens Fewer data have been published regarding the pharmacokineticsof minocycline than that of doxycycline Minocycline may be given PO or IVand serum concentrations following a typical dosing regimen of an initial 200mg dose followed by 100 mg every 12 h results in steady state of 23ndash35 microgmL The half-life is approximately 17 h after a single dose and 21 h after multipledosing and unlike tetracycline the half-life is not prolonged or substantiallyincreased in renal failure [8] The absorption of minocycline is decreased byapproximately 20 following ingestion of food andor milk but is not clinicallysignificant Chelation occurs with minocycline therefore concomittant antaciduse should be avoided Similar to tetracycline doxycycline minocycline demon-strate high concentrations intracellularly which is useful in treating Chlamydiaand Legionella infections

23 Doxycycline

Doxycycline is highly protein-bound (82) and has the advantage of being 5times as lipid-soluble as tetracycline It is rapidly and almost completely (93)absorbed from the upper portion of the gastrointestinal tract following oral admin-istration tetracycline is less efficiently (25ndash80) absorbed Food does not havean important effect on the absorption of orally administered doxycycline andserum levels are not decreased in the presence of antacids gluten or metalliccations The absorption half-life of doxycycline is approximately 50 min anddetectable serum levels are achieved within 30 min following oral administrationPeak levels occur 2 h after dosing and the serum t12 is 22 h Oral antacidshowever have been found to increase the total clearance of IV doxycycline re-sulting in a decreased half-life [28]

Peak serum levels of doxycycline following a single 200 mg oral dose are27 microgmL (range 21ndash44 microgmL) A single 200 mg dose of doxycycline re-sults in serum levels of 16 microgmL (range of 15ndash17 microgmL) After a 400 mgoral dose doxycycline serum concentrations are approximately 40 microgmL Se-rum levels of doxycycline are the same with either oral or intravenous administra-tion after a steady state is achieved Intravenous administration of doxycyclineresults in higher initial serum concentrations 100 mg (IV) q12h 4 microgmL100 mg (IV) q8h 7 microgmL 200 mg (IV) q12h 15 microgmL Doxycycline is


widely distributed throughout body tissues Lung levels after 200 mg of doxycy-cline (IV) achieve concentrations of 68 microgg Average levels of doxycycline are59 microgg Doxycycline achieves therapeutic levels in the gallbladder bile ductwall and bile and readily penetrates respiratory secretions In patients with acutemaxillary sinusitis doxycycline reaches concentrations of 21 microgmL in sinussecretion Mean prostatic concentration of doxycycline is 163 microgg and doxy-cycline accumulates in prostatic tissue In patients given 200 mg doxycyclineorally followed by 100 mgday average ovarian levels ranged from 304 to 324microgg Levels of doxycycline in fallopian tubes were 321 microgg Mean doxycyclinelevels in breast milk at 24 h after drug administration were about 40 of serumlevels Moderate doxycycline concentrations are also found in umbilical bloodand amniotic fluid [1ndash3910]

Based on doxycyclines pharmacokinetics therapy should be initiated witha 72 h loading regimen because of its high lipid solubility for serious infectionseg Legionnairersquos disease A dose of 200 mg IV q12h provides rapid tissuesaturation of doxycycline and is effective in achieving maximum serum concen-trations rapidly because 5 serum half-lives are required before steady-state ki-netics are achieved Doxycycline 100 mg (IV) q12h basis requires 4ndash5 days oftherapy before a therapeutic effect can be achieved Since the serum t12 of doxy-cycline is 22 h an initial 72 h loading regimen is required if a rapid therapeuticeffect is desired [927] Doxycycline does not significantly accumulate in patientswith renal insufficiency Blood levels of doxycycline in patients with severelyimpaired renal function are the same as in patients with normal kidneys Thusdoxycycline should be administered in the usual dosage to patients with renalimpairment without risk of accumulation Hemodialysis has a negligible effecton the serum half-life of doxycycline and a dosage adjustment is not neededwhen doxycycline is administered in patients undergoing hemodialysis [1ndash3910]

24 Glycylcyclines

The glycylcyclines are a derivative of minocycline and have good activity againstgram-positivegram-negative pathogens GAR-936 a member of the glycylcy-cline class has good penetration into tissues with a high tissueplasma ratio anda large volume of distribution 101 Lkg in rats [11] A study evaluating healthysubjects found that the maximal serum concentration (Cmax) and area under theserum concentration versus time curve (AUC) values of this antimicrobial agentwas of proportional following a 1 h IV infusion of 125 mg resulting in a Cmax

of 011 microgmL and an AUC of 09 microgsdothmL Following a 300 mg dose theCmax and AUC increased to 28 and 179 microgsdothmL respectively When GAR-936 was dosed at 50 mgkg subcutaneously in a murine model of Pseudomonas


aeruginosa AUC values reached 633 microgsdothmL achieving better concentra-tions than a similarly dosed gentamicin which demonstrated an AUC of 511microgsdothmL [12] Additionally the half-life of GAR-936 was found to be long at36 h [13] Similarly to tetracycline and minocycline GAR-936 chelates cal-cium [11]

3 PHARMACODYNAMICS OF TETRACYCLINES

The tetracyclines are believed to be bacteriostatic when used in vitro howeverin high concentrations are bacteriocidal [3] Tetracyclines to have time-dependentkill meaning that the parameter of the time above the minimum inhibitory con-centration of the pathogen (T MIC) best describes the killing by these drugsFor time-dependent killing the killing is dependent on the length of time thebacteria are exposed to the antibiotic As the area under the concentration versustime curve for plasma (AUC) approaches 2ndash4 times the MIC value of the organ-ism the effect is maximized Here the rate of killing plateau and additional serumdrug concentration have a negligible effect and it is the time above the MIC isthe important parameter related to bacterial kill and antibacterial activity Gener-ally for these agents the T MIC should be at least 50 of the dosing intervalfor immunocompetent patients whereas immunocompromised patients may re-quire the T MIC 100 of the dosing interval

Tetracycline was once useful for a variety of gram-positive and gram-negative pathogens However as a result of increased resistance it is now typi-cally reserved for uncommon infections Additionally plasma-mediated resis-tance is occurring as a result of overuse in animal feeds containing tetracyclineand it appears that most acquired resistance of gram-positive and -negativepathogens is a result of this plasma-mediated problem [3] This makes it diffi-cult to treat infections by common gram-positive organisms which currentlydemonstrate a high level of resistance to tetracycline Tetracycline resistance isusually a result of active drug efflux due to an exogenous metalndashtetracyclineH antiporter Similarly doxycycline and minocycline do not share this resistanceproblem these second generation tetracyclines have excellent antimicrobial activ-ity [14]

Doxycycline is useful against a broad range of pathogens However it wasintroduced prior to the appreciation of current pharmacodynamic concepts andas a result it is only recently that the optimal dosing regimen for doxycyclinewas determined [15ndash20] Time-kill kinetic studies demonstrated that at low con-centrations ie at 2ndash4 times the MIC 100 mg (IVPO) q12h of doxycyclinekills the organisms tested in a time-dependent manner At higher serum concen-trations 200 mg (IVPO) q12h or 400 mg (IVPO) q24h ie 8ndash16 times the MIC


of the organisms doxycycline exhibits concentration-dependent killling [17ndash20](Fig 1) Additionally the postantibiotic effect (PAE) of doxycycline influencesthe pharmacodynamics of this agent PAE T C where T is the time requiredfor the counts of CFUmL in the test culture to increase by 1 log10 above thecount observed immediately after antibiotic removal and c is the time requiredfor the count of CFUmL in an untreated control culture to increase 1 log10 abovethe count observed immediately after completion of the same procedure used onthe test culture for antibiotic removal Doxycycline has a PAE for gram-positiveand gram-negative aerobic organisms that was found to be concentration-depen-dent Doxycycline has a PAE of 21ndash42 h with gram-positive and gram-negativeorganisms [20] (Fig 2) This is similar to the PAE of tetracycline which variesbetween 2 and 3 h

Glycylcyclines demonstrate time-dependent (concentration-independent)kinetics This was demonstrated in a thigh infection model which found thatT MIC was the best correlated pharmacodynamic model [21] The studycompared two glycylcyclines GAR 936 and WAY 152288 against a variety ofcommon gram-positive and gram-negative pathogens Whereas the glycylcy-cline GAR 936 only required the concentration of free drug to remain above theMIC for 50 of the dosing interval for effective eradication of Streptococcuspneumoniae Escherichia coli and Klebsiella pneumoniae similar killing withWAY 152288 required the MIC to be exceeded for at least 75 of the dosinginterval using the same isolates Because of the long half-lives and PAEs of theseagents the AUC also may play a predictive role in the killing of organisms[21]

It has been determined that for GAR 936 a single dose of 300 mg IV wouldprovide a concentration of 2 microgmL for 75 of the time [22] The glycylcyclineswould also have excellent activity against Staphylococcus aureus (MSSA) whichfor GAR 936 demonstrates MICs ranging from 006 to 1 microgmL and the MIC90

values for MSSA is 05 microgmL [23] An endocarditis model [24] found that whenserum levels fell below the MIC before 50 of the dosing interval GAR 936was as effective in eradicating VanA-resistant Enterococcus faecium as whenserum levels were constantly above the MIC throughout the dosing interval Theauthors concluded that this result may be due to the low clearance of the agentGAR 936 from the endocarditis vegetations as well as the long PAE againstenterococci [24]

Glycylcyclines has good activity against anaerobic bacteria covering mostcommon anerobic pathogens Unlike doxycycline and minocycline glycylcyineshave litle activity against Bacteroides fragilis and possibly Clostridium per-fringens [25] For Mycoplasma pneumoniae the susceptibility of GAR 936 mea-sured by the MIC50 was 02 microgmL twice that of doxycyclineminocycline andtwofold less than that of tetracycline [26]


FIGURE 1 Doxycycline kill curves for (a) Staphylococcus aureus (b) Strepto-coccus pneumoniae (c) Pasteurella multocida (d) Escherichia coli (With per-mission from Ref 20)


FIGURE 2 Doxycycline postantibiotic effects (PAEs) for (a) Staphylococcusaureus (b) Streptococcus pneumoniae (c) Pasteurella maltophilia (d) Esche-richia coli (With permission from Ref 20)


DISCUSSION

Although tetracyclines have been useful in treating a wide variety of pathogenssince the early 1900s increased resistance has made tetracycline second-line ther-apy until recently with the advent of doxycycline and minocycline Glycylcy-clines appear to be effective in treating a wide range of gram-positive gram-negative and anaerobic bacteria The second generation tetracyclines doxycy-cline and minocycline have increased pharmacokinetic and pharmacodynamicadvantages over tetracycline and other advantages in overall dosing allowingfor their potential once daily dosing as a result of their long PAEs and half-livesand increased activity against common pathogens which maintain their positionin the antibiotic armamentarism as first line agents [27ndash30]

REFERENCES

1 BA Cunha Clinical uses of tetracyclines In RK Blackwood JJ Hlavka JH Bootheds The Tetracyclines Springer-Verlag Berlin 1985393

2 BA Cunha JB Comer M Jonas The tetracyclines Med Clin North Am 198266293

3 NC Klein BA Cunha Tetracyclines Med Clin North Am 199579789ndash8014 N Joshi DQ Miller Doxycycline revisited Arch Intern Med 19971571421ndash

14285 PJ Petersen NV Jacobus WJ Weiss PE Sum RT Testa In vitro and in vivo antibac-

terial activities of a novel glycylcycline the 9-t-butylglycylamido derivative of mi-nocycline (GAR-936) Antimicrob Agents Chemother 199943738ndash744

6 RT Testa PJ Petersen NV Jacobus P Sum VJ Lee FP Tally In vitro and in vivoantibacterial activities of the glycylcyclines a new class of semisynthetic tetracy-clines Antimicrob Agents Chemother 1993372270ndash2277

7 MP Fournet R Zini L DeForges F Lange J Lange JP Tillement Tetracycline anderythromycin distribution in pathological lungs of humans and rat J Pharm Sci 1989781015ndash1019

8 GK McEvoy ed Tetracyclines In American Hospital Formularly Service DrugInformation American Society of Health System Pharmacists Bethesda MD 1997368ndash386

9 BA Cunha The pharmacokinetics of doxycycline Postgrad Med Commun 1979143ndash50

10 BA Cunha Doxycycline Antibiotics Clin 1999321ndash2711 NL Tombs Tissue distribution of GAR-936 a broad spectrum antibiotic in male

rats 39th Interscience Conference on Antimicrobial Agents and Chemotherapy1999 abstr P-413

12 SM Mikels AS Brown L Breden S Compton S Mitelman PJ Petersen WJ WeissIn vivo activities of GAR-936 (GAR) gentamicin (GEN) piperacillin (PIP) alone andin combination in a murine model of Pseudomonas aeruginosa pneumonia 39th Intersci-ence Conference on Antimicrobial Agents and Chemotherapy 1999 abstr P-414


13 G Muralidaharan J Getsy P Mayer I Paty M Micalizzi J Speth B Webster PMojaverian Pharmacokinetics (PK) safety and tolerability of GAR-936 a novelglycylcycline antibiotic in healthy subjects 39th Interscience Conference on Anti-microbial Agents and Chemotherapy 1999 abstr P-416

14 Y Someya A Yamaguchi T Sawai A novel glycylcycline 9-(nn-dimethylglycyl-amido)-6-demethyl-6-deoxytetracycline is neither transported nor recognized by thetransposon TN10-endoced metal-tetracyclineH antiporter Antimicrob AgentsChemother 199539247ndash249

15 WA Craig Pharmacokineticpharmacodynamic parameters Rationale for antibacte-rial dosing of mice and men Clin Infect Dis 1998261ndash12

16 K Totsuka K Shimizu In vitro and in vivo post-antibiotic effects (PAE) of CL331928 (DMG-DMDOT) a new glycylcycline against Staphylococcus aureus 34thInterscience Conference on Antimicrobial Agents and Chemotherapy 1994179abstr F114

17 WA Craig SC Ebert Killing and regrowth of bacteria in vitro A review Scand JInfect Dis Suppl 19917463ndash70

18 O Cars I Odenholt-Tornquist The post-antibiotic sub-MIC effect in vitro and invivo J Antimicrob Chemother 199331159ndash166

19 WA Craig S Gudmundsson Post-antibiotic effect In V Lorian ed Antibiotics inLaboratory Medicine 4th ed Williams and Wilkins Baltimore 1996296ndash329

20 BA Cunha P Domenico CB Cunha Pharmacodynamics of doxycycline Clin Mi-crob Infect Dis 20006270ndash273

21 ML van Ogtrop D Andes TJ Stamstad B Conklin WJ Weiss WA Craig O VesgaIn vivo pharmacodynamic activities of two glycylcyclines (GAR-936 and WAY152288) against various gram-positive and gram-negative bacteria AntimicrobialAgents Chemother 200044943ndash949

22 HW Boucher CB Wennersten RC Moellering GM Eliopoulos In vitro activity ofGAR-936 against gram-positive bacteria 39th Interscience Conference on Antimi-crobial Agents and Chemotherapy 1999 abstr P-406

23 E Mahalingam L Trepeski S Pongporter J De Azavedo DE Lo BN KreiswirthIn vitro activity of new glycylcycline Gar 936 against methicillin-resistant and-susceptible Staphylococcus aureus isolated in North America 39th InterscienceConference on Antimicrobial Agents and Chemotherapy 1999 abstr P-408

24 A Lefort L Massias A Saleh-Mghir M LaFaurie L Garry C Carbon B FantinPharmacodynamics of GAR-936 (GAR) in experimental endocarditis due to VANA-type Enterococcus faecium 40th Interscience Conference on AntimicrobialAgents and Chemotherapy 2000 abstr P-2256

25 M Hedberg CE Nord In vitro activity of anaerobic bacteria to GAR-936 a newglycylcycline 39th Interscience Conference on Antimicrobial Agents and Chemo-therapy 1999 abstr P-411

26 GE Kenny FD Cartwright The susceptibility of human mycoplasmas to a new gly-cylcycline GAR 936 39th Interscience Conference on Antimicrobial Agents andChemotherapy 1999 abstr P-412

27 VX Nguyen DE Nix S Gillikin JJ Schentag Effect of oral antacid administrationon the pharmacokinetics of intravenous doxycycline Antimicrob Agents Chemother198933434ndash436


28 HW Boucher CB Wennersten GM Eliopoulis In vitro activities of the glycyclineGAR-936 against gram positive bacteria Antimicrob Agents Chemother 2000442225ndash2229

29 KW Shea Y Ueno F Abumustafa SMH Qadri BA Cunha Doxycycline activityagainst streptococcus pneumoniae Chest 19951071775ndash1776

30 BA Cunha Doxycycline revisited Intern Med 19971065ndash69

12

Pharmacodynamics of Antivirals

George L Drusano Sandra L Preston

and Peter J Piliero


1 INTRODUCTION

Although pharmacodynamic relationships for antibacterial agents have beensought and identified for a long period of time it has been a general feeling thatthe treatment of viral and fungal infections was different in kind A corollary tothis is that delineation of pharmacodynamic relationships would be much moredifficult

In reality the exact principles that govern the delineation of pharmaco-kineticpharmacodynamic (PKPD) relationships for antibacterials also governantifungals and as will be shown here antivirals

The first issue as always in the development of dynamics relationships isto decide upon an endpoint Most of the data for this chapter have been derivedfrom studies of the human immunodeficiency virus (HIV) Type 1 Consequentlythis chapter addresses the in vitro and in vivo data for this virus Other virusesshould behave in a similar fashion (as is seen with cytomegalovirus) An excep-tion to the rule is hepatitis C virus (among others) because of our inability toobtain more than one round of viral replication in vitro

With HIV-1 there are a number of endpoints that may be chosen Some are

259

260 Drusano et al

1 Survivorship2 Change in CD4 count consequent to therapy3 Change in viral load consequent to therapy4 Change in the hazard of emergence of resistance consequent to therapy5 Durability of maintaining the viral load below detectable levels

Survivorship is as always the ultimate test of an intervention With the adventof potent HIV chemotherapy this disease process has been converted from arelatively rapid killer to one that will likely take its toll over a number of decadesConsequently survivorship may not be the best endpoint to examine for clinicalstudies because of the long lead times In this chapter we concentrate on end-points 2ndash5 for clinical studies

In anti-HIV chemotherapy as in antibacterial chemotherapy one can gener-ate pharmacodynamic relationships from in vitro approaches as well as fromclinical studies One difference between these areas is the ease of employinganimal systems for the generation of dynamic relationships For antibacterialsthere are many examples of the generation of dynamic relationships for differentdrug classes [1ndash3] For HIV the cost and ethical implications of using simianmodels has prevented much work in this area The McCune model and its variantsare attractive but again because of the cost of maintaining the system little hasbeen done to develop dynamic relationships for HIV in animal systems Otherretroviruses have been employed [45] but it is difficult to draw inferences forhuman chemotherapy of HIV from a different pathogen with different pathoge-netic properties

Consequently in this chapter in vitro but not animal model systems arealso reviewed to add insight gleaned from these systems

2 IN VITRO SYSTEMS

Although one might consider determination of viral EC50 or EC90 to be a pharma-codynamic system measurement this chapter takes the view that this is simplya static measure of drug potency It will play (see below) an important role indetermining the final shape of the pharmacodynamic relationship when added toother measures Alone it is merely a measure of compound potency

For in vitro systems to be true pharmacodynamic systems there must bethe possibility of changing drug concentrations over time to examine the effectfor a pathogen of known susceptibility to the drug employed The only systemwith peer-reviewed publication for HIV-1 is shown in Fig 1 In addition it isimportant to factor in the effect of protein binding as only the free drug is virolog-ically active


FIGURE 1 Schematic diagram of the in vitro pharmacodynamic model sys-tem One of the two systems enclosed within a single incubator housed in abiological safety cabinet is shown Cells are grown and samples are removedfrom the extracapillary compartment of HF bioreactors Constant infusionoral or intravenous bolus doses are introduced through the dosing ports inthe diluent reservoir absorption compartment or central reservoir respec-tively Exposure of cells to fluctuating concentrations of D4T is affected byprogrammed dilution of drug within the central reservoir while the volumeof the central compartment is maintained constant by elimination The meanpore diameter of the HF capillaries (10 kDa) would prevent HIV or HIV-infectedcells from exiting the bioreactor and from circulating through the tubing (Fur-ther information is available upon request)

262 Drusano et al

3 PROTEIN BINDING

The effect of protein binding has been examined in greatest detail for its effecton the anti-HIV potency of a drug by this laboratory Bilello et al [6] examinedan HIV-1 protease inhibitor (A-80987) and determined the effect of protein bind-ing on free-drug concentration on drug uptake into infected cells and finallyon the virological effect of the drug This set of experiments has a lsquolsquotransitivelogicrsquorsquo organization

In the first experiment (Fig 2) increasing concentrations of the bindingprotein α1 acid glycoprotein were introduced into the test system and the con-centration of unbound drug was determined As can be seen increasing bindingprotein concentration leads to monotonically decreasing free-drug concentrations

In Fig 3 the amount of drug that penetrates infected CEM cells is shownas a function of the free fraction of the drug Increased external free-drug concen-tration results in an increased amount of intracellular drug (stop oil experiments)in a linear function Finally (Fig 4) the amount of intracellular drug is relatedto the decrement of p24 output as indexed through an inhibitory sigmoid-Emaxmodel It is clear through this series of experiments that only the free-drug con-centration can induce a decrease in viral production

These data make it clear that it is important to interpret drug concentrationsin the hollow fiber system as representing the external free-drug concentrationsnecessary to induce the desired antiviral effect

FIGURE 2 Relationship between increasing Prime1 acid glycoprotein concentra-tion and A-80987 free drug concentration


FIGURE 3 Relationship between increasing A-80987 free fraction and intracel-lular A-80987

FIGURE 4 Relationship (inhibitory sigmoid Emax) between intracellular A-80987 and HIV viral output from infected PBMCs

264 Drusano et al

The most obvious place to initiate evaluation of anti-HIV agents is withthe nucleoside analogs and with the HIV-1 protease inhibitors The first publishedhollow fiber evaluation of an antiviral was the nucleoside analog stavudine (d4T)Bilello et al [7] examined issues of dose finding and schedule dependency forthis drug

It is important to examine the development of stavudine in order to placeits evaluation in the hollow fiber system in the proper perspective The initialclinical evaluation of stavudine was initiated at a total daily dose of 2 mgkg perday The initial schedule chosen was every 8 h Whereas the initial dose had aneffect dose escalation to 12 mgkg per day produced no change in effect butmuch higher rates of drug-related neuropathy [8]

After this the dose was de-escalated to 4 mgkg per day and the schedulewas lengthened to every 12 h Further dose de-escalation was taken from thispoint on a 12 h schedule and nine dosing cohorts were eventually examinedThis process took approximately 2 years

The hollow fiber evaluation took approximately 3 months The identifieddose was ultimately the dose chosen by the phase III trial and has stood the testof time and usage It is at least to our knowledge the first prospective identifica-tion of a drug dose and schedule from an in vitro test system with clinical valida-tion The outcome of the experiments is illustrated in Fig 5 This evaluationmakes clear that for nucleoside analogs the pharmacodynamically linked variableis AUCEC90 With matching AUCs (Fig 5) there was no difference in outcomebetween the exposure being given in a continuous infusion mode and half theexposure every 12 h (data not shown) The reason is likely the phosphorylationof the parent compound into the virologically active form of the molecule (thetriphosphate for stavudine or diphosphate for prephosphorylated compoundssuch as tenofovir) It is clear from Figs 6 and 7 that there is full effect at 05mgkg every 12 h irrespective of starting challenge At half the exposure (025mgkg q12h) there is a hint of loss of control late in the experiment Finally at0125 mgkg q12h there is no discernible antiretroviral effect

HIV-1 protease inhibitors have also been examined in this system The firstto be examined was the early Abbott inhibitor A-77003 This drug was a proof-of-principle agent and was administered intravenously as a continuous infusionConsequently the hollow fiber evaluation was performed as a continuous infu-sion When protein binding was taken into account the concentrations requiredfor effect were above those tolerable clinically Consequently the drug was pre-dicted to fail Indeed an extensive phase III evaluation came to this conclusion[910]

The next protease inhibitor evaluated was amprenavir (141W94) Here[11] time EC90 was demonstrated to be the pharmacodynamically linked vari-able Dosing intervals of q12h and q8h with matching AUCs were compared to


FIGURE 5 The concentrationndashtime profile for stavudine in the hollow fiberunit is displayed for a 1 mg(kg sdot day) dose administered either as a continu-ous infusion or as 05 mgkg administered every 12 h The AUCs developedare identical The ability to suppress viral replication was identical for theregimens

continuous infusion of the same AUC Continuous infusion provided the mostrobust viral control followed closely by q8h dosing Every 12 h dosing lost asignificant fraction of the control of viral turnover This outcome was to be ex-pected as HIV-1 protease inhibitors are reversible inhibitors freely cross cellmembranes without the requirement for energy or a transporter and do not re-quire activation (phosphorylation) for effect as the nucleoside analogs do

A third protease inhibitor was examined the potent once-daily PI BMS232632 [12] In addition to the hollow fiber unit evaluation the technique ofMonte Carlo simulation was also brought to bear on the outcome of these experi-ments

In Fig 8 it is clear that time EC90 is the pharmacodynamically linkedvariable In the one instance complete control of viral replication in vitro isachieved with a continuous infusion regimen at 4 EC50 (about EC90ndash95) Thissame daily AUC administered as a bolus allows breakthrough growth Four timesthis daily AUC administered as a bolus regains control of the viral replicationIndeed this latter part of the experiment demonstrates that free-drug concentra-

266 Drusano et al

FIGURE 6 Panel a displays the increase in p24 output over time in a hollowfiber tube that was an untreated control () in a treated (05 mgkg q12h)tube where 11000 cells were chronically HIV-infected at start () or where1100 cells were HIV-infected at start () Panel b shows numbers of HIV DNAcopies in treated and untreated hollow fiber units


FIGURE 7 Panel a displays the increase in p24 output over time in a hollowfiber tube that was an untreated control () in a treated (025 mgkg q12h)tube where 11000 cells were chronically HIV-infected at start () or where1100 cells were HIV-infected at start () Panel b shows p24 output in atreated hollow fiber unit (0125 mgkg q12h []) and untreated control ()

268 Drusano et al

FIGURE 8 Effect of BMS 232632 on HIV replication Three infected hollow fiberunits were treated with BMS 232632 One tube was treated with a concentra-tion of four times the EC50 as a continuous infusion This produced a 24 hAUC of 4 24 EC50 The second tube received the same 24 h AUC butwas given in a peak-and-valley mode once daily The third tube received anexposure calculated a priori to provide a time EC90 that would give essen-tially the same suppression as the continuous infusion of 4 EC50

tions need to exceed the EC90 for approximately 80ndash85 of a dosing interval inorder to maintain control of the HIV turnover This served as the therapeutictarget for further evaluation

The sponsor provided pharmacokinetic data for BMS 232632 administeredto normal volunteers at doses of 400 mg and 600 mg orally once daily at steadystate Population pharmacokinetic analysis was performed and the mean parame-ter vector and covariance matrix were employed to perform Monte Carlo simula-tion The ability to attain the therapeutic target was assessed accounting for thepopulation variability in the handling of the drug This is demonstrated in Fig9 The viral isolate susceptibilities to BMS 232632 are displayed as EC50 valuesHowever an internal calculation corrects for the difference between EC50 andEC90 as well as for the protein binding of the drug The fractional target attainmentis for free drug being greater than the nominal EC90 for 85 of the dosing interval


FIGURE 9 A Monte Carlo simulation of 1000 subjects performed three timeswas employed to estimate the fraction of these subjects whose concentra-tionndashtime curve would produce maximal viral suppression on the basis ofthe data presented in Fig 2 The evaluation was performed for doses of ()400 mg and () 600 mg of BMS 232632 administered once daily by mouth() Forty-three isolates from a clinical trial of BMS 232632 were tested by theVirologics Phenosense assay

(the therapeutic target determined in Fig 8) As can be seen as the viral isolatesbecome less and less susceptible to BMS 232632 the greater the difference be-tween the 400 mg and 600 mg doses The sponsors also determined the viralsusceptibility to 43 clinical isolates from a phase III clinical trial of the drug inpatients who were HIV-treatment naive As can be seen all had EC50 valuesbelow 2 nM Consequently this allows us to take an expectation over the distribu-tion of measured EC50 values to determine the fraction of patients who will attaina maximal response to the drug under the assumptions that the viral susceptibilitydistribution is correct for naive patients and that the kinetics in normal volunteersand its distribution are a fair representation of the kinetics of the drug in infectedbut treatment-naive patients When this calculation is performed approximately69 of subjects taking the 400 mg dose will obtain a complete response whereasslightly greater than 74 of patients taking 600 mg will obtain a complete re-sponse

270 Drusano et al

A clear lesson learned regarding the chemotherapy of HIV is that combina-tion chemotherapy is more effective than monotherapy However little has beendone to determine optimal combination dosing regimens An initial effort to de-termine the interaction of antivirals in a fully parametric system was publishedby Drusano et al [13] The Greco model was fit to the data from an in vitroexamination of 141W94 (amprenavir an HIV-1 protease inhibitor) plus 1592U89(abacavir a nucleoside analog) This interaction is displayed in Figs 10ndash12 Fig-ure 10 displays the full effect surface from these two agents The effects of proteinbinding are taken into account as the effects are developed in the presence of

FIGURE 10 1592U89 and 141W94 combination study with albumin (40 mgmL) and α1 acid glycoprotein (1 mgmL) A three-dimensional response sur-face representing the interaction from the in vitro matrix is displayed Percentinhibition data from a 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide assay with HIV-1IIIB and MT-2 cells is displayed


FIGURE 11 Synergy plot drawn from the data displayed in Figure 10 Plottedat the 95 confidence level

physiological amounts of the binding proteins human albumin and human α1acid glycoprotein The drug interaction can be seen by subtracting the theoreticaladditive surface and the synergy surface is seen in Fig 11 It is important tonote that there in synergy across all concentrations of the agents As a modelwas fit to the data the weighted residuals are displayed in Fig 12 to demonstratethat the actual regression process was unbiased The actual degree of interactionis given by estimation of the interaction parameter α which is 1144 The 95confidence bound about the interaction parameter is from 0534 to 1754 As thisboundary does not cross zero the synergy is significant at the 005 level

This fully parametric analysis was employed in combination with MonteCarlo simulation to examine whether the drugs at the doses used clinically would

272 Drusano et al

FIGURE 12 Weighted residual plot from the fully parametric analysis The re-siduals are scattered about the zero line without bias

be synergistic and whether the dosing interval affected the outcome [14] Theanswer to both these questions was found to be yes The data are displayed inChapter 14

4 CLINICAL STUDIES OF ANTIVIRAL

PHARMACODYNAMICS

As noted previously the first decision required for determining a pharmacody-namics relationship in the clinic is the choice of an endpoint In this section thechange in CD4 counts the change from baseline viral load prevention of resis-tance and durability of maintenance of viral loads below detectability are theendpoints examined Where possible they are examined for both nucleoside ana-logs and HIV-1 protease inhibitors both alone and in combination

Use of nucleoside analogs as monotherapy occurred mostly at a time whenviral copy number determinations were not freely available Consequently virtu-ally all the available studies employed change in CD4 cell count or p24 as thedynamic endpoint


One of the first studies in this regard examined dideoxyinosine (ddI) usein a naive patient population [15] No concentrationndasheffect response was foundfor CD4 cells but a clear relationship was discerned between the number of CD4

cells present at baseline and the number of cells that returned with the initiationof ddI therapy (Fig 13) In addition this study found a relationship between ddIexposure (as indexed to the area under the plasma concentrationndashtime curveAUC) and the fall in p24 both within patients and across the population (Fig14) The finding reported in the study just cited was also found for zidovudine[16]

More recently Fletcher et al [17] examined the relationship between ddIAUC and fall in viral load in children (Fig 15) This study is flawed by the factthat the relationships were developed in patients receiving combination chemo-therapy without any effort being made to account for the interaction betweendrugs

With regard to emergence of resistance there was one important studylargely ignored that demonstrated the importance of viral susceptibility for effectKozal et al [18] examined patients switching from zidovudine to didanosine(ddI) Over half of such patients developed a mutation at codon 74 by week 24of ddI therapy that is known to confer ddI resistance The effect on the number

FIGURE 13 Linear regression between the number of CD4-positive T-lympho-cytes during therapy with dideoxyinosine and the baseline CD4 count

274 Drusano et al

FIGURE 14 Relation between the suppression of p24 antigen and the steady-state area under the plasma concentrationndashtime curve of dideoxyinosine

FIGURE 15 Didanosine AUC versus baseline to week 24 changes in plasmaHIV RNA levels The solid line represents the line of best fit as determinedwith linear regression the equation for this line is y 1337 (347 AUC)r 2 051 p 003


FIGURE 16 Mean CD4 T-cell changes before the appearance of the HIV-1 re-

verse transcriptase mutation at codon 74 and CD4 T-cell changes after the

appearance of the mutation in 38 patients switched from zidovudine to dida-nosine

of CD4 cells is demonstrated in Fig 16 At the time of appearance of the mutationthe CD4 count dips below the baseline number of CD4 cells indicating that theincrease in EC90 attendant to the mutation drives a loss of virological effect

There is considerably more information relating exposure to effect for theHIV-1 protease inhibitors Because of the time at which they were studied virtu-ally all of the data link some measure of exposure to the change from baselinein the viral load Early publications examining the relationship between indinavirexposure and the CD4 count were published by Stein and Drusano [1920] Inthe first of these publications it was demonstrated that the return of CD4 countwas related to the baseline CD4 count as with nucleoside analogs In the secondit was demonstrated that the return of CD4 cells had another component attachedto it The model was expanded to include the decline in viral load The returnof CD4 cell count was better explained by the larger model of baseline CD4 countplus viral load change as the independent variables than either alone

The first published paper examining the relationship between drug exposureand viral load decline studied high dose saquinavir Schapiro et al [21] demon-strated a relationship between the saquinavir AUC and the change in viral load

276 Drusano et al

FIGURE 17 Drug levels at week 4 (AUC to 24 h) plotted against the decreasein plasma HIV RNA levels at week 4 for each patient in whom pharmacokinet-ics were studied The line represents best fit and was determined using theleast squares algorithm r 0801 expressed as the Pearson correlation coef-ficient () Patients receiving 3600 mg of saquinavir per day () patients re-ceiving 7200 mg of saquinavir per day

A problem with this analysis is that the form of the function is not specifiedConsequently interpretation is difficult

Shortly thereafter Stein et al published a small phase III study of indinavir[22] This was the first lsquolsquohigh dosersquorsquo indinavir study (2400 mgday) and was thefirst to demonstrate robust viral suppression with this drug In addition the au-thors examined the relationship between indinavir exposure and both CD4 cellreturn and viral load decline These relationships are shown in Fig 18 One should

FIGURE 18 Modeling of data using a sigmoid Emax relationship and inhomoge-neous differential equations (a) The relationship of baseline CD4 lymphocytecount to the average CD4 lymphocyte count obtained over 24 weeks of ther-apy (b) The relationship of the inhibition of HIV generation to total drug expo-sure (AUC) (c) The relationship of the inhibition of HIV generation to Cmin

serum concentration


278 Drusano et al

not draw the conclusion that HIV suppression is linked to AUC (as this has aslightly better r2) In this study the drug was administered on a fixed dose andschedule maximizing the co-linearity (ie one could not make the AUC risewithout also increasing the Cmin) The in vitro studies simulated different dosesand schedules minimizing the co-linearity and definitively showed that time EC90 is the dynamically linked variable This is correlated with Cmin The compari-son of the in vitro and in vivo results also raises the issue of the importance ofthe EC90

Drusano et al demonstrated for both indinavir [23] and amprenavir [24]that normalizing the measure of drug exposure to the EC50 (or EC90) of a particularpatientrsquos isolate decreased the variance and increased the r2 This makes senseas the amount of exposure needed to suppress a sensitive isolate will on firstprinciples be less than that needed to suppress a resistant isolate (see above forthe case of ddI and CD4 cells)

An issue that is not addressed by these studies is the duration of HIV sup-pression and the closely linked issue of suppression of emergence of resistanceThe first study to examine this issue was that of Kempf et al [25] who showedthat obtaining a viral load below the detectability limit of the assay was importantto the duration of control of the infection Shortly thereafter Drusano et al [26]examined this issue both for protease inhibitor monotherapy with indinavir andfor the first time for combination therapy that included indinavir The results formonotherapy are displayed in Fig 19 It is clear that patients who attain viralloads that are below the detectability of the assay have the lowest hazard of losingcontrol of the infection or emergence of resistance The time to loss of controlof infection is clearly related to the nadir viral load attained

Of greater interest is the situation with combination chemotherapy as this isthe clinical norm This study also examined the influence of different combinationregimens on the hazard of loss of control of the viral infection Combinationregimens of zidovudine-indinavir zidovudine-didanosine-indinavir and zidovud-ine-lamivudine-indinavir were examined and compared to their monotherapyarms in a stratified analysis The results are displayed in Table 1 Only the regi-men of zidovudine-lamivudine-indinavir remained significantly different frommonotherapy after adjustment for the fall in viral copy number

This result caused the in vitro investigation of this regimen [27] A fullyparametric analysis demonstrated that the interaction among all three drugs waskey to the effect obtained in the clinical studies with this regimen This is shownin Table 2 where the αrsquos represent the interaction parameters There are threetwo-drug interaction parameters and one for the interaction of all three com-pounds Indinavir plus zidovudine interact in an additive manner as the value isclose to zero and the 95 confidence interval overlaps zero The same is trueof indinavir plus lamivudine Zidovudine plus lamivudine has a value that ispositive and a 95 confidence interval that does not overlap zero indicating a


FIGURE 19 KaplanndashMeier estimate of probability that patientrsquos isolate is notresistant to therapy (lack of sustained increase of 075 log10 copiesmL ofHIV-1 RNA from patientrsquos minimum level) at a given study day stratified byminimum HIV-1 RNA achieved Plot shows that probability of remaining sus-ceptible is largest for patients who achieve undetectable level of HIV-1 RNAVertical slashes on plot indicate censoring events

TABLE 1 Effect of Combination Therapy Versus IndinavirMonotherapy Before and After Adjusting for the Effect of the MinimumLevel of HIV-1 RNA

Combination P before Hazard ratiob

therapy adjustment Coefficienta (95 CI) P

IDVAZT 264 035 052 0705 (02541957) 497IDVAZTddI 002 136 090 0258 (00441514) 105IDVAZT3TC 001 168 080 0186 (00390893) 016

a Estimate SE b Hazard ratio is vs IDV monotherapy group in the same study

280 Drusano et al

TABLE 2 In Vitro Assessment of Drug Interactionof AZT-3TC-Indinavir

95 ConfidenceParametera Estimate interval

Econ 9899 9780ndash1002IC50IND 14690 12830ndash16560m IND 1711 1393ndash2030IC50AZT 1184 10820ndash12860mAZT 1289 5576ndash2020IC503TC 10290 1018ndash1041m3TC 6875 369ndash1006

αINDAZT 00001301 06191ndash06194αIND3TC 06881 005189ndash1428αAZT3TC 09692 09417ndash09966αINDAZT3TC 894 3434ndash1445

a Econ effect seen in the absence of drug (percent) IC50 concen-tration of drug necessary to reduce HIV-1 turnover by half whenused alone (nM) m slope parameter corresponding to the rateof rise of effect with increasing drug concentration Prime interac-tion parameter

degree of synergistic interaction that is statistically significant Finally the magni-tude of the synergistic interaction for the three-drug term is very large (and sig-nificant) It may be this exceptionally strong synergistic interaction that explainsthe superb results seen with this particular three-drug combination

It is of interest that lamivudine not only plays a key role in the regimenbut is also its Achillesrsquo heel Holder et al [28] demonstrated that when the tripleregimen fails in about 70 of cases the failure is due to an M184V mutationthat produces high level resistance to lamivudine So that although lamivudineappears to be a key part of this therapeutic regimen and its synergy it also hasthe lowest genetic barrier to resistance If there is some nonadherence to theregimen enough rounds of viral replication may occur to allow the point mutant(M184V) to be amplified in the total population When this clone becomes domi-nant in the population lamivudine will lose most of its contribution to the regi-men Most of the synergy is also lost and the result is viral rebound As Holderand colleagues demonstrated this occurs most of the time with mutation solelyaffecting the low genetic barrier drug

It is obvious then that optimal chemotherapeutic regimens for HIV (andother viral pathogens) are likely to require explicit modeling of the interactionof the drugs in the regimens


Burger et al [29] also examined this combination In a multivariate logisticregression with attaining a viral load below the limit of assay detectability as theendpoint they demonstrated that baseline viral load indinavir trough concentra-tions and prior HIV-1 protease inhibitor use influenced the probability of at-taining this endpoint

All of the above has related to HIV Other viruses can also have a pharma-codynamic evaluation elucidated For CMV there is a clear-cut dynamic relation-ship [30] that has been set forth in Chapter 14

In summary viruses follow the same laws of physics as bacterial pathogens(and fungal pathogens) It is important to delineate relationships both in vitroand in vivo between different measures of drug exposure and the endpoint thatis deemed important The in vitro investigations allow delineation of the truedynamically linked variable in a more straightforward manner The in vivo inves-tigations are important for validation The future is in generating exposurendashresponse relationships for combinations of agents In this way optimal therapyregimens can be generated to provide the greatest benefit for patients infectedwith viral pathogens

REFERENCES

1 B Vogelman S Gudmundsson J Leggett J Turnidge S Ebert WA Craig Correla-tion of antimicrobial parameters with therapeutic efficacy in an animal modelJ Infect Dis 1988 158831ndash847

2 GL Drusano DE Johnson M Rosen HC Standiford Pharmacodynamics of a fluor-oquinolone antimicrobial in a neutropenic rat model of Pseudomonas sepsis Antimi-crob Agents Chemother 1993 37483ndash490

3 A Louie P Kaw W Liu N Jumbe MH Miller GL Drusano Pharmacodynamics ofdaptomycin in a murine thigh model of Staphylococcus aureus infection AntimicrobAgents Chemother 2001 45845ndash851

4 JA Bilello JL Eiseman HC Standiford GL Drusano Impact of dosing scheduleupon suppression of a retrovirus in a murine model of AIDS encephalopathy Anti-microb Agents Chemother 1994 38628ndash631

5 JA Bilello JJ Kort C MacAuley TN Fredrickson RA Yetter JL Eiseman ZDVdelays but does not prevent the transmission of MAIDS by LP-BM MuLV-infectedmacrophage-monocytes J Acquir Immune Defic Syndr 1992 5571ndash576

6 JA Bilello PA Bilello K Stellrecht J Leonard D Norrbeck DJ Kempf T RobbinsGL Drusano The uptake and anti-HIV activity of A 80987 an inhibitor of the HIV-1 protease is reduced by human serum 1-acid glycoprotein Antimicrob Agents Che-mother 1996 401491ndash1497

7 JA Bilello G Bauer MN Dudley GA Cole GL Drusano The effect of 2prime3prime-di-deoxy-2prime3prime-didehydrothymidine (D4T) in an in vitro hollow fiber pharmacodynamicmodel system correlates with results of dose ranging clinical studies AntimicrobAgents Chemother 1994 381386ndash1391

8 MJ Browne KH Mayer SB Chafee MN Dudley MR Posner SM Steinberg KK

282 Drusano et al

Graham SM Geletko SH Zinner SL Denman et al J Infect Dis 1993 16721ndash29

9 JA Bilello PA Bilello JJ Kort MN Dudley J Leonard GL Drusano Efficacy ofconstant infusion of A 77003 an inhibitor of the HIV protease in limiting acuteHIV-1 infection in vitro Antimicrob Agents Chemother 1995 392523ndash2527

10 M Reedijk CA Boucher T van Bommel DD Ho TB Tzeng D Sereni P VeyssierS Jurruaans R Grannemann A Hsu et al Safety pharmacokinetics and antiviralactivity of A77002 a C2 symmetry-based human immunodeficiency virus proteaseinhibitor Antimicrob Agents Chemother 1995 391559ndash1564

11 GL Drusano Antiviral therapy of HIV and cytomegalovirus Abstracts of the 37thInterscience Conference on Antimicrobial Agents and Chemotherapy Abstract S-121 Sept 28ndashOct 1 1997 Toronto ON Canada

12 GL Drusano JA Bilello SL Preston E OrsquoMara S Kaul S Schnittman R EcholsHollow fiber unit evaluation of a new human immunodeficiency virus (HIV)-1 prote-ase inhibitor BMS232632 for determination of the linked pharmacodynamic vari-able J Infect Dis 2000 1831126ndash1129

13 GL Drusano DZ DrsquoArgenio W Symonds PA Bilello J McDowell B Sadler ABye JA Bilello Nucleoside analog 1592U89 and human immunodeficiency virusprotease inhibitor 141W94 are synergistic in vitro Antimicrob Agents Chemother1998 422153ndash2159

14 GL Drusano DZ DrsquoArgenio SL Preston C Barone W Symonds S LaFon M Rog-ers W Prince A Bye JA Bilello Use of drug effect interaction modeling with MonteCarlo simulation to examine the impact of dosing interval on the projected antiviralactivity of the combination of abacavir and amprenavir Antimicrob Agents Chemo-ther 2000 441655ndash1659

15 GL Drusano GJ Yuen JS Lambert M Seidlin R Dolin FT Valentine Quantitativerelationships between dideoxyinosine exposure and surrogate markers of responsein a phase I trial Ann Intern Med 1992 116562ndash566

16 GL Drusano FM Balis SR Gitterman PA Pizzo Quantitative relationships betweenzidovudine exposure and efficacy and toxicity Antimicrob Agents Chemother 199481726ndash1731

17 CV Fletcher RC Brundage RP Remmel LM Page D Weller NR Calles C SimonMW Kline Pharmacologic characteristics of indinavir didanosine and stavudine inhuman immunodeficiency virus-infected children receiving combination chemother-apy Antimicrob Agents Chemother 2000 441029ndash1034

18 MJ Kozal K Kroodsma MA Winters RW Shafer B Efron DA Katzenstein TCMerigan Didanosin resistance in HIV-infected patients switched from zidovudineto didanosine monotherapy Ann Intern Med 1994 121263ndash268

19 DS Stein GL Drusano Modeling of the change in CD4 lymphocyte cell counts inpatients before and after administration of the human immunodeficiency virus prote-ase inhibitor indinavir Antimicrob Agents Chemother 1997 41449ndash453

20 GL Drusano DS Stein Mathematical modeling of the interrelationship of CD4 lym-phocyte count and viral load changes induced by the protease inhibitor indinavirAntimicrob Agents Chemother 1998 42358ndash361

21 JM Schapiro MA Winters F Stewart B Efron J Norris MJ Kozal TC Merigan


The effect of high-dose saquinavir on viral load and CD4 T-cell counts in HIV-infected patients Ann Intern Med 1996 1241039ndash1050

22 DS Stein DG Fish JA Bilello J Chodakewitz E Emini C Hildebrand SL PrestonGL Martineau GL Drusano A 24 week open label phase I evaluation of the HIVprotease inhibitor MK-639 AIDS 1996 10485ndash492

23 GL Drusano This volume Chapter 1424 GL Drusano BM Sadler J Millard WT Symonds M Tisdale C Rawls A Bye

and the 141W94 International Product Development Team Pharmacodynamics of141W94 as determined by short term change in HIV RNA Influence of viral isolatebaseline EC50 Abstract A-16 37th Interscience Conference on Antimicrobial Agentsand Chemotherapy Sept 28ndashOct 1 1997 Toronto ON Canada

25 DJ Kempf RA Rode Y Xu E Sun ME Heath-Chiozzi J Valdes AJ Japour SDanner C Boucher A Molla JM Leonard The duration of viral suppression duringprotease inhibitor therapy for HIV-1 infection is predicted by plasma HIV-1 RNAat the nadir AIDS 1998 12F9ndashF14

26 GL Drusano JA Bilello DS Stein M Nessly A Meibohm EA Emini P DeutschJ Condra J Chodakewitz DJ Holder Factors influencing the emergence of resistanceto indinavir Role of virologic immunologic and pharmacologic variables J InfectDis 1998 178360ndash367

27 S Snyder DZ DrsquoArgenio O Weislow JA Bilello GL Drusano The triple combina-tion indinavir-zidovundine-lamivudine is highly synergistic Antimicrob AgentsChemother 2000 441051ndash1058

28 DJ Holder JH Condra WA Schleif J Chodakewitz EA Emini Virologic failureduring combination therapy with crixivan and RT inhibitors is often associated withexpression of resistance-associated mutations in RT only Conf Retroviruses Oppor-tun Infect 1999 Jan 31ndashFeb 46160 (abstr 492)

29 DM Burger RMW Hoetelmans PWH Hugen et al Low plasma concentrations ofindinavir are related to virological treatment failure in HIV-1-infected patients onindinavir containing triple therapy Antiviral Ther 1998 3215ndash220

30 GL Drusano F Aweeka J Gambertoglio M Jacobson M Polis HC Lane C Eaton SMartin-Munley Relationship between foscarnet exposure baseline cytomegalovirusblood culture and the time to progression of cytomegalovirus retinitis in HIV-positive patients AIDS 1996 101113ndash1119

13

Antifungal Pharmacodynamics

Michael E Klepser

University of Iowa Iowa City Iowa

Russell E Lewis

University of Houston College of Pharmacy Houston Texas

1 INTRODUCTION

Over the last two decades advances in the characterization of antibacterial phar-macodynamics have greatly improved therapeutic strategies in the use of antimi-crobial therapy Progress in our understanding of antifungal pharmacodynamicshowever remains severely limited Prospective clinical trials for serious fungalinfections are difficult to complete and rarely evaluate multiple treatment strate-gies [1] Moreover the high mortality associated with systemic fungal diseaselessens the likelihood that dose-ranging studies for antifungal agents will be pur-sued Perhaps more surprising however is the profound lack of in vitro andanimal data describing concentrationndasheffect relationships for antifungals Thislack of even fundamental knowledge regarding the pharmacodynamics of antifun-gals can even be appreciated in the clinical literature where optimal dosing strate-gies for amphotericin B remain largely undefined despite over 40 years of clinicaluse [23]

With the emergence of the AIDS epidemic widespread use of broad-spec-trum antibacterial therapy and a growing population of immunocompromisedpatients the spectrum of nosocomial pathogens has changed dramatically [4]Fungi are now the fourth most commonly isolated nosocomial bloodstream patho-gens in US hospitals and have been identified as an independent risk factor for

285


in-hospital death [56] This staggering increase in the frequency of fungal infec-tions coupled with the introduction of new antifungal agents has made the identi-fication of optimal antifungal treatment strategies even more critical Althoughstill in its infancy the study of antifungal pharmacodynamics is already providingimportant new information of the activity dosing and use of antifungals fortreatment of candidiasis and cryptococcosis

2 PROBLEMS WITH DESCRIBING ANTIFUNGAL

PHARMACODYNAMICS

21 In Vitro Data

One of the primary factors that has hindered the study of antifungal pharmaco-dynamics over the last two decades was the lack of standardized methods forperforming in vitro susceptibility testing of fungi Prior to the 1980s fungi wererelatively infrequent pathogens and amphotericin B was the sole systemic antifun-gal therapy available thus susceptibility testing of fungi was impractical andremained largely undeveloped With the frequency of fungal infections increasingand the number of new antifungal agents growing the development of standard-ized antifungal susceptibility testing methods has become a priority Problemswith inter- and intralaboratory reproducibility however hindered early effortsto develop antifungal susceptibility testing into a clinically useful tool One studyevaluating the interlaboratory reproducibility of antifungal susceptibility testingnoted a 512-fold variation in MIC values among collaborating laboratories thatused the same published but unstandardized methodology [7]

In the late 1990s the National Committee for Clinical Laboratory Standards(NCCLS) approved standardized methods for in vitro antifungal susceptibilitytesting and susceptibility breakpoints for Candida species [89] This step notonly enabled the quantitative description of antifungal resistance but also pro-vided the groundwork for performing in vitro pharmacodynamic work with anti-fungals

22 Animal Models

Historically animal (mostly rodent) models have been the preferred method oftesting antifungal efficacy Much of this deference to the use of animal modelswas a result of not having standardized in vitro susceptibility testing methodsfor fungi Moreover many fungal pathogens exhibit different morphology andgrowth characteristics in vivo from those of growth in culture media Candidaalbicans for example generally does not form the hyphal structures in culturemedia that are seen with in vivo tissue invasion [10ndash12] Published data fromanimal models however also suffer the limitation of unstandardized methodol-ogy and poor reproducibility [13] Selection of the endpoint for evaluation of


study results in vivo ie survival versus mycology can profoundly influenceinterpretation of data Although survival is often the desired endpoint in vivoanimal studies are generally not conducted over a sufficient length of time toprovide an adequate opportunity for relapse of infection once therapy is discon-tinued Additionally it is reasonable to theorize that if fungi are detectable inbody tissues or fluids at the end of a treatment cycle these viable organisms mayproliferate and cause relapse after antifungal therapy is withdrawn Thereforecounts of viable fungi often provide the best information regarding in vivo anti-fungal efficacy This can be problematic however for extrapolating pharmacody-namic parameters predictive of in vivo antifungal efficacy Many antifungals pos-sess fungistatic activity thus the actual differences in counts of viable fungi invivo may be too small to delineate pharmacodynamic relationships [1314]

23 Clinical Data

Using clinical data to determine dosendashresponse relationships for antifungals canbe especially difficult The multiplicity of non-drug factors that play a part inthe development of severe fungal infections (ie previous chemotherapy intra-vascular catheters etc) profoundly influence the overall effectiveness of antifun-gal therapy in the individual patient Fungal infections are also a diagnostic chal-lenge and serial blood cultures are generally insensitive markers for diagnosisand response to antifungal therapy [15] Moreover monitoring of serum drugconcentration for antifungals is rarely performed thus comparison of differentclinical studies is unfeasible

Distinct diagnostic criteria compared to other systemic fungal infectionsgenerally make cryptococcal meningitis andor oropharyngeal candidiasis bestsuited to the extrapolation of outcome data with respect to antifungal therapyCryptococcal meningitis however may be preferred for outcome measurementbecause the mortality associated with oropharyngeal candidiasis is comparativelylower than that of other systemic infections

3 PHARMACODYNAMICS

31 Amphotericin B

Despite the availability of better-tolerated agents the broad spectrum of activityand potency of amphotericin B have maintained its role as the drug of choicefor deep-seated mycoses Amphotericin B is thought to act primarily by bindingto ergosterol in the fungal cell membrane resulting in intercalation of the mem-brane and leakage of intracellular contents [3] Amphotericin B has also beenshown to induce oxidative damage to the cell membrane and to stimulate hostimmune responses [16] which may contribute to the overall elimination of fungalpathogens from the infected host


Even with nearly 40 years of clinical use relatively few pharmacodynamicdata exist for this agent This lack of data is surprising considering the numeroustoxicities associated with amphotericin B therapy The recent introduction of lipidformulations of amphotericin B which have considerably different pharmacoki-netic characteristics than those of the standard formulations have further compli-cated issues regarding the optimal dosing of this agent

311 In Vitro Data

In vitro data describing the pharmacodynamics of amphotericin B are relativelyscarce Although the NCCLS has proposed standardized methods for microbrothsusceptibility testing of amphotericin B these methods are still not well suitedfor detecting amphotericin B resistance and are still under active investigation[891718]

Standardized methods for performing time-kill studies with amphotericinB against Candida and Cryptococcus spp however have been published [19]These methods have been used to evaluate concentrationndasheffect relationships ofamphotericin B against Candida albicans [20] and Cryptococcus neoformans[21] A representative graph from these studies is presented in Fig 1 Against

FIGURE 1 Representative graph of amphotericin B time-kill activity againstCandida and Cryptococcus species (From Refs 20 and 21)


both fungal species amphotericin B at concentrations above the MIC producedrapid fungicidal activity As concentrations of amphotericin B in the growth me-dia increased both the rate and extent of antifungal activity improved This im-provement in activity continued up to the maximal concentrations tested 16ndash32times the MIC of the test isolates This concentration-dependent relationship ofkilling has also been reported by other investigators [22]

The postantifungal effect (PAE) of amphotericin B has also been evaluatedfor both Candida and Cryptococcus species [23] Amphotericin B produces aprolonged PAE ranging from 05 to 106 h against Candida species and from 28to 104 h against C neoformans Additionally the authors noted that the PAEexerted by amphotericin B was dependent on the duration of exposure and theconcentrations of drug tested

According to these data it appears that amphotericin B exhibits concentra-tion-dependent antifungal activity in vitro However it is important to gaugein vitro results against clinically achievable drug concentrations Examining thesigmoidal dosendashresponse curves constructed from the time-kill data representa-tive of fully susceptible Candida and Cryptococcus spp (Fig 2) one notes thatthe transitional portion of the dose response curve for amphotericin B againstthese isolates (in vitro) occurred over a concentration range of 025ndash2 times the

FIGURE 2 Representative sigmoidal dosendashresponse curves of amphotericinB and fluconazole against Candida and Cryptococcus spp (From Refs20 and 21)


MIC (02ndash20 microgmL) This transitional portion represents a relatively broadrange of serum concentrations that can be achieved with clinically used dosagesof amphotericin B of 025ndash15 mgkg q24h [3] Therefore these data predictover a range of clinically achieved concentrations that amphotericin B would beexpected to exhibit concentration-dependent pharmacodynamics

312 In Vivo Data

Currently no studies animal or human specifically designed to evaluate the phar-macodynamic characteristics of amphotericin B have been published Howeversome animal and clinical studies do exist that have evaluated multiple doses ofamphotericin B under identical or similar testing conditions and can therefore becarefully evaluated for dosendashresponse relationships George et al [24] used arabbit model of aspergillosis to evaluate the efficacy of amphotericin B adminis-tered at 05 and 15 mg(kg sdot day) These investigators employed both clinical(survival) and mycological (tissue burden) endpoints to evaluate the efficacy ofthe two dosing regimens They reported that although there was no differencebetween the two amphotericin B regimens with respect to survival (both regimensyielded 100 survival) amphotericin B at 15 mg(kg sdot day) resulted in fewerpositive cultures and reduced fungal tissue burdens compared with the lower doseregimen

Pharmacodynamic data from controlled human studies are lacking There-fore we are left to evaluate data from multiple studies in order to construct apicture of pharmacodynamic relationships As mentioned one of the best patientpopulations available for comparison among studies are patients with cryptococ-cal meningitis Some of the primary reasons that this patient population lendsitself to interstudy comparisons are the defined criteria used to diagnose and de-fine disease the ability to detect the pathogen relatively reliably and the relativelystandard endpoints used for clinical and microbiological outcomes The majorproblem with extrapolating pharmacodynamic relationships from studies ofcryptococcal meningitis however is the lack of data describing amphotericin Bconcentrations at the site of infection (cerebrospinal fluid) This could somewhatskew the dosendashresponse relationships observed because distribution of amphoter-icin B into the CSF is generally poor (10) [3] Several studies evaluatingamphotericin B for the treatment of cryptococcal meningitis are presented inTable 1 Examination of these studies reveals a trend of improved outcomes withincreasing doses of amphotericin B It is interesting to note that mortality associ-ated with cryptococcal meningitis decreased from 14 at a dose of 03 mgkgper day to 29 at a dose of 10 mgkg per day [25ndash27]

313 Summary

In vitro and in vivo data both reveal improved fungicidal activity with amphoteri-cin B as concentrations or doses of the drug are increased Therefore in light of


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the long half-life and PAE associated with the drug it appears that the optimaldosing strategy for this agent would be to administer amphotericin B at relativelyhigh doses (ie 08 mgkg per day) at intervals of no less than every 24 h

32 Azole Pharmacodynamics

Fluconazole has been the most widely studied of the azole antifungals A syn-thetic triazole antifungal fluconazole exhibits fungistatic activity against a varietyof Candida species and C neoformans

321 In Vitro Data

Similar to amphotericin B limited data are available regarding the pharmacody-namic characteristics of fluconazole Klepser et al [2021] examined the fungi-static activity produced by fluconazole against C albicans and C neoformansusing time-kill methods According to their data the fungistatic activity producedby fluconazole was maximized at concentrations of 2ndash4 times the MIC of thetest isolates (Figs 2 and 3) Similar findings have been reported by other investi-gators [22] Additionally using an in vitro dynamic model of infection these

FIGURE 3 Representative graph of fluconazole time-kill activity against Can-dida and Cryptococcus spp (From Refs 20 and 21)


investigators compared the activity of two fluconazole dosing regimens 200 mgand 400 mg administered every 24 h against C albicans [28] No differenceswith respect to the rate or extent of activity produced by the two fluconazoleregimens were noted It is important to note that the fluconazole MICs for thetest isolates were 025 and 05 microgmL Given the long half-life of fluconazoleapproximately 20ndash50 h drug concentrations in the model for both regimenswould be expected to remain above 10 times the MIC for each regimen andisolate tested

Voriconazole is an investigational triazole antifungal with an enhancedspectrum of activity against Candida Cryptococcus and Aspergillus species Us-ing time-kill methods Klepser et al [29] examined the in vitro activity of vorico-nazole against C albicans C glabrata C tropicalis and C neoformans overconcentrations ranging from 0125 to 16 times the MIC of test isolates AgainstC albicans and C neoformans voriconazole produced fungistatic activity thatwas maximized at concentrations between 1 and 4 times the MIC

In vitro fluconazole produces a short PAE against Candida species [30ndash33] Against C albicans fluconazole produces a PAE of 05 h The durationof the PAE is not increased with higher concentrations or by increasing the dura-tion of exposure Some investigators have noted that the PAE induced by fluco-nazole may increase if human serum is added to the in vitro system [30] Thisobservation led the investigators to suggest that the PAE induced by fluconazolemay be significantly longer in vivo than in vitro

322 In Vivo Data

Using a murine model of deep-seated C albicans infection Louie et al [34]evaluated the pharmacodynamic characteristics of fluconazole In this study theinvestigators determined the range of concentrations over which response in themodel was noted to change from minimal to maximal (transition portion ofthe dosendashresponse curve) Then dose-fractionation studies were performed toevaluate the relationship between pharmacodynamic parameters such as peakMIC and AUCMIC ratios and time above the MIC and outcome at concentra-tions near the transition portion of the dosendashresponse curve Upon analysis theauthors determined that a concentration-associated parameter the AUCMIC ra-tio was best correlated with positive results Although these in vivo data mayappear to contradict previous in vitro findings some limitations of this studywarrant cautious interpretation of this study First the doses of fluconazole usedin the study produced serum concentrations in the model that ranged from approx-imately 4 to 8 microgmL well below clinically observed concentrations even withlow-dose 200 mg daily fluconazole Second viable colony counts of fungi inkidney homogenate served as the endpoint for the study and the authors statethat fungal densities were similar for groups that received the same daily doseof fluconazole However according to their data there does not appear to be a


difference among fungal densities in any of the dosing groups suggesting poorsensitivity in the model Finally the authors specifically studied the transitionportion of the dosendashresponse curve By definition the measured response shouldincrease as exposure to increased amounts of drug increase This portion of thecurve was noted to occur over a narrow range of concentrations If one were toexamine these data in the context of clinically achievable concentrations (Fig2) it would be apparent that this transition portion of the dosendashresponse curvewould be easily surpassed Therefore one might conclude that even though theactivity noted in the transition portion of the curve may correlate with the AUCMIC ratio under clinical conditions activity would be dictated according to theconcentration relationships of the flat portion or non-concentration-dependentportion of the curve against fully susceptible fungi (Fig 2)

323 Clinical Studies

Clinical trials designed specifically to study the pharmacodynamic properties offluconazole have not been completed However Witt et al [35] examined theactivity of fluconazole at doses ranging from 400 to 2000 mg in patients withcryptococcal meningitis A total of 12 15 20 and 10 patients received intrave-nous fluconazole at doses of 400 1200 1600 and 2000 mg respectively Theauthors reported that no association was observed between the dose of flucona-zole administered and outcome

324 Summary

Although the fungistatic activity of fluconazole may improve as drug concentra-tions increase to 2ndash4 times the MIC of a given fungus fluconazole appears toexhibit non-concentration-dependent characteristics over the range of concentra-tions yielded by clinically used doses Therefore it is not likely that the activityproduced by fluconazole will be improved via the administration of larger thannormal doses ie 400 mg daily unless the fungal pathogens exhibit relativelyhigh MICs (32ndash64 microgmL) to fluconazole

33 Flucytosine Pharmacodynamics

331 In Vitro Data

Flucytosine is a fluorinated pyrimidine that possesses antifungal activity againsta variety of fungal species including Candida and Cryptococcus In order to exertits antifungal activity flucytosine must be converted to fluorouracil by cytosinedeaminase located inside the fungal cell The use of flucytosine is frequentlylimited secondary to its narrow therapeutic index four times daily dosing intervaland rapid emergence of resistance if single-drug therapy is attempted

As with the other antifungal agents discussed few data currently exist re-


garding the pharmacodynamic characteristics of flucytosine Lewis et al [36]examined the influence of drug concentrations on the antifungal activity ex-pressed by flucytosine using time-kill methods Several Candida species and Cneoformans were tested at multiples of their flucytosine MICs ranging from 025to 64 times the MIC Against isolates of C albicans C glabrata C kruseiand C tropicalis flucytosine exhibited fungistatic activity that improved as theconcentration of flucytosine increased up to 16 times the MIC In contrast againstC neoformans there did not appear to be an identifiable relationship betweenconcentration and increased antifungal activity once flucytosine concentrationsexceeded the MIC of the isolate It should be noted that even at 16 times theMIC of the test isolates these concentrations are well within the range of clini-cally observed concentrations In fact steady-state serum and cerebrospinal fluidconcentrations in humans average 50ndash100 microgmL [2637ndash39] The MICs of theisolates used by Lewis et al ranged from 006 to 4 microgmL therefore clinicallyachievable concentrations would represent levels approximately 50ndash1600 timesthe MICs of these isolates As a result even though flucytosine does exhibitconcentration-dependent activity in vitro it is likely that concentration-indepen-dent activity would be observed clinically

A measurable PAE has been reported for flucytosine [2336] In generalflucytosine produces an in vitro PAE ranging from 05 to approximately 4 h Theduration of the PAE is prolonged as the concentration of drug tested increases[36]

332 In Vivo Data

Using a murine model of disseminated candidiasis Andes and colleagues exam-ined the pharmacodynamic characteristics of flucytosine [40] In their model theinvestigators determined that minimal concentration-dependent activity wasnoted They determined that the time that concentrations remained above theMIC of the pathogen and the AUCMIC ratio held the best correlation with ob-served effect It is important to note however that the half-life of flucytosine intheir model was approximately 035ndash045 h significantly less than the 6 h half-life reported for humans

333 Summary

Flucytosine exhibits non-concentration-dependent fungistatic activity over arange of clinically achievable concentrations Therefore given the relatively longhalf-life of this agent its narrow therapeutic window and correlation betweentime above the MIC and activity it appears reasonable to advocate the use oflow dose 100 mg(kg sdot day) regimens administered at extended intervals every8ndash12 h This strategy would appear to provide optimal activity while minimizingpatient exposure to the drug


34 Antifungal Combinations

With increasing reports of antifungal resistance and a limited number of therapeu-tic agents the use of combination antifungal regimens to offset toxicity and im-prove efficacy has become common clinical strategy Amphotericin Bndashflucyto-sine combination therapy has been proven to be an efficacious combination inthe treatment of cryptococcal meningitis [2638] Some clinical data are availableto suggest that azole-flucytosine combinations may be useful in the treatment offungal peritonitis [41]

One of the most controversial topics in antifungal therapeutics howeverconcerns azole-amphotericin B combinations Considering the pharmacology ofthese two antifungals this combination should result in antagonism Theoreti-cally the inhibition of ergosterol synthesis by fluconazole should result in de-creased binding and antagonism of amphotericin B activity Results from manyin vitro studies have supported this theory of azole-amphotericin B antagonismyet the results of in vivo rodent and rabbit studies have been unequivocal [42]Unlike many of the other azole antifungals fluconazole exhibits reversible bind-ing to the target enzyme 14α-demethylase and is relatively hydrophilic [43]Based of these differences and data from in vivo models several investigatorshave predicted that antagonism between amphotericin B and fluconazole wouldnot occur in vivo [4244ndash48]

If fluconazole does antagonize the activity of amphotericin B against yeasttwo important pharmacodynamic questions need to be answered (1) What isthe time course (rate) for the development of antagonismmdashis pre-exposure tofluconazole necessary (2) If antagonism does develop to what extent will overallantifungal activity be lost This second question is critical because it may explainwhy many in vivo models have failed to show increased mortality with an ampho-tericin B-fluconazole combination

Recently studies have been published examining these questions [2849]De novo ergosterol biosynthesis and incorporation into the fungal membrane inCandida species have been reported to occur over a period of approximately 6h [50] Considering the pharmacodynamics of each antifungal (Figs 1ndash3) it ap-pears that some amount of pre-exposure to fluconazole would be necessary toantagonize the activity of amphotericin B Ernst and coworkers noted that thefungicidal activity of amphotericin B is dramatically decreased when isolates arepre-exposed to fluconazole for more than 8 h The activity of amphotericin Bagainst yeast exposed to fluconazole for at least 8 h was indistinguishable fromthat of fluconazole alone and was fungistatic (2 log10 decrease from startinginoculum) A limitation of these studies however was the static nature of drugsin the test conditions

To test the likelihood of fluconazole antagonizing the fungicidal activityof amphotericin B over a range of dynamic concentrations Lewis et al [28]


developed an in vitro pharmacodynamic infection model of candidemia In thismodel the investigators simulated the human pharmacokinetic profile of five anti-fungal regimens plus control (1) fluconazole 200 mg q24h (2) fluconazole 400mg q24h (3) amphotericin B 1 mgkg per day (4) amphotericin B plus flucon-azole (400 mg) simultaneously and (5) amphotericin B administered 8 h afterthe fluconazole bolus Similar to previous time-kill studies amphotericin B ad-ministered 8 h after a fluconazole bolus drastically reduced amphotericin B fungi-cidal activity (Fig 4)

Data from these pharmacodynamic studies have several implications Firstthe time course and order of administration for amphotericin B and fluconazoleare important for the development of antagonism Therefore traditional methodsfor screening antifungal antagonism that rely on static concentration of antifun-gals administered simultaneously (ie checkerboard testing) may not necessarilydetect antagonism Especially notable the fungistatic activity of fluconazoleseems to persist despite the loss of amphotericin B fungicidal activity (Fig 4)If this antifungal activity persists in vivo it is unlikely that studies using mortalityas the principal endpoint would be able to detect antagonism of amphotericin BThis is also true clinically as fluconazole has been shown to be as efficaciousas moderate doses of amphotericin B for the treatment of systemic candidiasisin neutropenic and non-neutropenic patients [5152]

FIGURE 4 Representative graph of amphotericin B-fluconazole antifungalcombination activity tested in an in vitro pharmacodynamic infection modelagainst Candida albicans (From Ref 28)


4 CONCLUSIONS

Clearly the study of antifungal pharmacodynamics is still in its infancy Never-theless early in vitro and in vivo work has provided some important new informa-tion on the activity dosing and use of antifungals for the treatment of candidiasisand cryptococcosis The next challenge will be to bring pharmacodynamic datafrom benchtop and animal studies to the bedside Other systemic mycoses criti-cally need pharmacodynamic study Aspergillosis for example has become amajor cause of mortality among oncology patients yet no clinical trials existdescribing the optimal therapy for these fungal infections Future progress in thefield of antifungal pharmacodynamics will hopefully optimize our ability to treatmycoses despite a limited armamentarium

REFERENCES

1 JE Edwards Jr GP Bodey RA Bowden T Buchner BE de Pauw SG Filler MAGhannoum M Glauser R Herbrecht CA Kauffman S Kohno P Martino F Meu-nier T Mori MA Pfaller JH Rex TR Rogers RH Rubin J Solomkin C ViscoliTJ Walsh M White International Conference for the Development of a Consensuson the Management and Prevention of Severe Candidal Infections Clin Infect Dis19972543ndash59

2 MP Nagata CA Gentry EM Hampton Is there a therapeutic or pharmacokineticrationale for amphotericin B dosing in systemic Candida infections Ann Pharma-cother 199630811ndash818

3 HA Gallis RH DrewWW Pickard Amphotericin B 30 years of clinical experienceRev Infect Dis 199012308ndash329

4 SN Banerjee TG Emori DH Culver RP Gaynes WR Jarvis T Horan JR EdwardsJ Tolson T Henderson WJ Martone Secular trends in nosocomial primary blood-stream infections in the United States 1980ndash1989 National Nosocomial InfectionsSurveillance System Am J Med 19919186Sndash89S

5 D Pittet N Li RF Woolson RP Wenzel Microbiological factors influencing theoutcome of nosocomial bloodstream infections A 6-year validated population-based model Clin Infect Dis 1997241068ndash1078

6 MA Pfaller RN Jones GV Doern HS Sader RJ Hollis SA Messer Internationalsurveillance of bloodstream infections due to Candida species Frequency of occur-rence and antifungal susceptibilities of isolates collected in 1997 in the United StatesCanada and South America for the SENTRY Program The SENTRY ParticipantGroup J Clin Microbiol 1998361886ndash1889

7 DL Calhoun GD Roberts JN Galgiani JE Bennett DS Feingold J Jorgensen GSKobayashi S Shadomy Results of a survey of antifungal susceptibility tests in theUnited States and interlaboratory comparison of broth dilution testing of flucytosineand amphotericin B J Clin Microbiol 198623298ndash301

8 JH Rex MA Pfaller JN Galgiani MS Bartlett A Espinel-Ingroff MA GhannoumM Lancaster FC Odds MG Rinaldi TJ Walsh AL Barry Development of interpre-tive breakpoints for antifungal susceptibility testing Conceptual framework and


analysis of in vitrondashin vivo correlation data for fluconazole itraconazole and Can-dida infections Subcommittee on Antifungal Susceptibility Testing of the NationalCommittee for Clinical Laboratory Standards Clin Infect Dis 199724235ndash247

9 National Committee for Clinical Laboratory Standards 1997 Reference method forbroth dilution antifungal susceptibility testing of yeasts approved standard Vil-lanova PA NCCLS

10 RB Ashman A Fulurija TA Robertson JM Papadimitriou Rapid destruction ofskeletal muscle fibers by mycelial growth forms of Candida albicans Exp Mol Pathol199562109ndash117

11 JC Parker Jr JJ McCloskey KA Knauer Pathobiologic features of human candidia-sis A common deep mycosis of the brain heart and kidney in the altered host AmJ Clin Pathol 197665991ndash1000

12 TJ WalshWG Merz Pathologic features in the human alimentary tract associatedwith invasiveness of Candida tropicalis Am J Clin Pathol 198685498ndash502

13 DM Dixon In vivo models evaluating antifungal agents Methods Find Exp ClinPharmacol 19879729ndash738

14 ME Klepser RE Lewis MA Pfaller Therapy of Candida infections Susceptibilitytesting resistance and therapeutic options Ann Pharmacother 1998321353ndash1361

15 LA Mermel DG Maki Detection of bacteremia in adults Consequences of culturingan inadequate volume of blood Ann Intern Med 1993119270ndash272

16 G Medoff Controversial areas in antifungal chemotherapy Short-course and combi-nation therapy with amphotericin B Rev Infect Dis 19879403ndash407

17 JH Rex MA Pfaller M Lancaster FC Odds A Bolmstrom MG Rinaldi Qualitycontrol guidelines for National Committee for Clinical Laboratory StandardsmdashRec-ommended broth macrodilution testing of ketoconazole and itraconazole J Clin Mi-crobiol 199634816ndash817

18 JH Rex MA Pfaller MG Rinaldi A Polak JN Galgiani Antifungal susceptibilitytesting Clin Microbiol Rev 19936367ndash381

19 ME Klepser EJ Ernst RE Lewis ME Ernst MA Pfaller Influence of test conditionson antifungal time-kill curve results Proposal for standardized methods AntimicrobAgents Chemother 1998421207ndash1212

20 ME Klepser EJ Wolfe RN Jones CH Nightingale MA Pfaller Antifungal pharma-codynamic characteristics of fluconazole and amphotericin B tested against Candidaalbicans Antimicrob Agents Chemother 1997411392ndash1395

21 ME Klepser EJ Wolfe MA Pfaller Antifungal pharmacodynamic characteristicsof fluconazole and amphotericin B against Cryptococcus neoformans J AntimicrobChemother 199841397ndash401

22 F Meunier C Lambert P Van der Auwera In-vitro activity of SCH39304 in compar-ison with amphotericin B and fluconazole J Antimicrob Chemother 199025227ndash236

23 JD Turnidge S Gudmundsson B Vogelman WA Craig The postantibiotic effectof antifungal agents against common pathogenic yeasts J Antimicrob Chemother19943483ndash92

24 D George D Kordick P Miniter TF Patterson VT Andriole Combination therapyin experimental invasive aspergillosis J Infect Dis 1993168692ndash698

25 F de Lalla G Pellizzer A Vaglia V Manfrin M Franzetti P Fabris C Stecca Am-photericin B as primary therapy for cryptococcosis in patients with AIDS Reliability


of relatively high doses administered over a relatively short period Clin Infect Dis199520263ndash266

26 CM van der Horst MS Saag GA Cloud RJ Hamill JR Graybill JD Sobel PCJohnson CU Tuazon T Kerkering BL Moskovitz WG Powderly WE DismukesTreatment of cryptococcal meningitis associated with the acquired immunodefi-ciency syndrome N Engl J Med 199733715ndash21

27 MS Saag GA Cloud JR Graybill JD Sobel CU Tuazon PC Johnson WJ FesselBL Moskovitz B Wiesinger D Cosmatos L Riser C Thomas R Hafner WE Dis-mukes A comparison of itraconazole versus fluconazole as maintenance therapy forAIDS-associated cryptococcal meningitis National Institute of Allergy and Infec-tious Diseases Mycoses Study Group Clin Infect Dis 199928291ndash296

28 RE Lewis BC Lund ME Klepser EJ Ernst MA Pfaller Assessment of antifungalactivities of fluconazole and amphotericin B administered alone and in combinationagainst Candida albicans by using a dynamic in vitro mycotic infection model Anti-microb Agents Chemother 1998421382ndash1386

29 ME Klepser D Malone RE Lewis EJ Ernst MA Pfaller Evaluation of voriconazolepharmacodynamic characteristics using time-kill methodology Antimicrob AgentsChemother 2000441917ndash1920

30 F Minguez JE Lima MT Garcia J Prieto Influence of human serum on the postanti-fungal effect of four antifungal agents on Candida albicans Chemotherapy 199642273ndash279

31 F Minguez MT Garcia JE Lima MT Llorente J Prieto Postantifungal effect andeffects of low concentrations of amphotericin B and fluconazole on previouslytreated Candida albicans Scand J Infect Dis 199628503ndash506

32 F Minguez ML Chiu JE Lima R Nique J Prieto Activity of fluconazole Postanti-fungal effect effects of low concentrations and of pretreatment on the susceptibilityof Candida albicans to leucocytes J Antimicrob Chemother 19943493ndash100

33 EJ Ernst ME Klepser MA Pfaller Postantifungal effect of echinocaudin azole andpolyene agents against Candida albicans and Cryptococcus neoformans AntimicrobAgents Chemother 2000441108ndash1111

34 A Louie GL Drusano P Banerjee QF Liu W Liu P Kaw M Shayegani H TaberMH Miller Pharmacodynamics of fluconazole in a murine model of systemic candi-diasis Antimicrob Agents Chemother 1998421105ndash1109

35 MD Witt RJ Lewis RA Larsen EN Milefchik MA Leal RH Haubrich JA RichieJE Edwards Jr MA Ghannoum Identification of patients with acute AIDS-associ-ated cryptococcal meningitis who can be effectively treated with fluconazole Therole of antifungal susceptibility testing Clin Infect Dis 199622322ndash328

36 RE Lewis ME Klepser MA Pfaller In vitro pharmacodynamic characteristics offlucytosine determined by time kill methods Dicy Microbiol Infect Dis 200036101ndash105

37 JE Bennett Flucytosine Ann Intern Med 197786319ndash32138 JE Bennett WE Dismukes RJ Duma G Medoff MA Sande H Gallis J Leonard

BT Fields M Bradshaw H Haywood ZA McGee TR Cate CG Cobbs JF WarnerDW Alling A comparison of amphotericin B alone and combined with flucytosinein the treatment of cryptoccal meningitis N Engl J Med 1979301126ndash131

39 P Francis TJ Walsh Evolving role of flucytosine in immunocompromised patients


New insights into safety pharmacokinetics and antifungal therapy Clin Infect Dis1992151003ndash1018

40 DA Andes M van Ogtrop In vivo characterization of the pharmacodynamics offlucytosine in a neutropenic murine disseminated candidiasis model AntimicrobAgents Chemother 200044938ndash942

41 G Barbaro G Barbarini G Di Lorenzo Fluconazole vs itraconazole-flucytosine as-sociation in the treatment of esophageal candidiasis in AIDS patients A double-blind multicenter placebo-controlled study The Candida Esophagitis MulticenterItalian Study (CEMIS) Group Chest 19961101507ndash1514

42 AM Sugar Use of amphotericin B with azole antifungal drugs What are we doingAntimicrob Agents Chemother 1995391907ndash1912

43 SM Grant SP Clissold Fluconazole A review of its pharmacodynamic and pharma-cokinetic properties and therapeutic potential in superficial and systemic mycosesDrugs 199039877ndash916

44 M Scheven F Schwegler Antagonistic interactions between azoles and amphoteri-cin B with yeasts depend on azole lipophilia for special test conditions in vitroAntimicrob Agents Chemother 1995391779ndash1783

45 M Scheven C Scheven Quantitative screening for fluconazole-amphotericin B an-tagonism in several Candida albicans strains by a comparative agar diffusion assayMycoses 199639111ndash114

46 M Scheven L Senf Quantitative determination of fluconazole-amphotericin B an-tagonism to Candida albicans by agar diffusion Mycoses 199437205ndash207

47 AM Sugar Interactions of amphotericin B and SCH 39304 in the treatment of experi-mental murine candidiasis Lack of antagonism of a polyene-azole combinationAntimicrob Agents Chemother 1991351669ndash1671

48 AM Sugar CA Hitchcock PF Troke M Picard Combination therapy of murineinvasive candidiasis with fluconazole and amphotericin B Antimicrob Agents Che-mother 199539598ndash601

49 EJ Ernst ME Klepser MA Pfuller In vitro interaction of fluconazole and amphoteri-cin B administered sequentially against candida albicans effect of concentration andexposure time Diag Microbiol Infect Dis 199832205ndash210

50 SL Kelly J Rowe PF Watson Molecular genetic studies on the mode of action ofazole antifungal agents Biochem Soc Trans 199119796ndash798

51 EJ Anaissie RO Darouiche D Abi-Said O Uzun J Mera LO Gentry T WilliamsDP Kontoyiannis CL Karl GP Bodey Management of invasive candidal infectionsResults of a prospective randomized multicenter study of fluconazole versus am-photericin B and review of the literature Clin Infect Dis 199623964ndash972

52 JH Rex JE Bennett AM Sugar PG Pappas CM van der Horst JE Edwards RGWashburn WM Scheld AW Karchmer AP Dine A randomized trial comparingfluconazole with amphotericin B for the treatment of candidemia in patients withoutneutropenia Candidemia Study Group and the National Institute N Engl J Med19943311325ndash1330

53 MS Saag WG Powderly GA Cloud P Robinson MH Grieco PK Sharkey SEThompson AM Sugar CU Tuazon JF Fisher N Hyslop JM Jacobson R HafnerWE Dismukes Comparison of amphotericin B with fluconazole in the treatment ofacute AIDS associated cryptococcal meningitis N Engl J Med 199232683ndash89

14

Human Pharmacodynamics ofAnti-Infectives Determination fromClinical Trial Data

George L Drusano


1 INTRODUCTION

Determination of the relationship between drug exposure and response or be-tween drug exposure and toxicity is key to achieving the ultimate aim of chemo-therapy obtaining the maximal probability of a good therapeutic response whileengendering the smallest possible probability of toxicity

In the area of anti-infective chemotherapy there is a single difference fromother areas of clinical pharmacological investigation In other areas we deal withreceptors for the drug that are human in origin There are true between-patientdifferences in receptor affinity for the drug that are currently not measurableThis unmeasured variance leads to difficulty in generating pharmacodynamic re-lationships

In anti-infectives however we are dealing with an external invaderWhether we are dealing with bacteria viruses or fungi we can with few excep-tions (eg hepatitis C virus) grow the offending pathogen and obtain a measureof the drugrsquos potency for that particular pathogen These measures have differentnames depending on the pathogen [eg minimal inhibitory concentration (MIC)

303

304 Drusano

effective concentration that reduces growth by half (EC50) minimal fungicidal(static) concentration (MFC)] These measures of pathogen sensitivity to the drugcan then be used to normalize the drug exposure in the patient relative to theinvading pathogen This markedly reduces the observed variability and improvesthe ability to define a relationship between exposure and response

2 DETERMINANTS OF A PHARMACODYNAMIC

RELATIONSHIP FOR ANTI-INFECTIVES

21 Endpoints

In order to determine a relationship between exposure and response or betweenexposure and toxicity the first step is to identify an endpoint Such endpointsdiffer according to what is being studied Endpoints may be continuous in nature[eg change in glomerular filtration rate (GFR) after drug exposure] dichoto-mous or polytomous (eg success vs failure survival vs death a four-pointpain scale) or time to an event (time to death time to relapse time to viralclearance) As will be discussed later the endpoint chosen will in many waysdetermine a good deal of the rest of the analysis

The first and most important step in defining a pharmacodynamic relation-ship is to obtain solid response endpoint data If all other steps are performedwell but the endpoint data are poorly defined then the relationship will be poorat best and possibly misleading

22 Pathogen Identification and Susceptibility

Determination

If one is attempting to construct an effect relationship (ie between drug expo-sure and response) it is imperative that the offending pathogen be isolated andidentified and that the susceptibility of that specific pathogen to the drug beingused for therapy be measured This is straightforwardly performed in many clini-cal antibacterial trials because pathogen identification and MIC determination areintegral parts of making a clinical study case both clinically and microbiologicallyevaluable for the Food and Drug Administration

Amazingly little has been done regarding the EC50 of viruses and theirinfluence on outcome in clinical trials of antiviral chemotherapy It has only beenrecently with the availability of commercial homologous recombination assaysor rapid sequencing assays for HIV that determination of drug susceptibility hasbecome a part of the clinical trial arena Nonetheless EC50 has an important roleto play as demonstrated by the data in Figs 1andash1f In this evaluation the HIVprotease inhibitor indinavir was administered as a single agent Plasma concentra-tions were measured by HPLC and EC50 values were determined for indinavirby the ACTGDOD consensus assay A sigmoidal Emax effect model was fit to


FIGURE 1 Pharmacodynamics of indinavir Emax model of plasma copy num-ber change For part explanations see text

306 Drusano

FIGURE 1 Continued


FIGURE 1 Continued

308 Drusano

the data with area under the plasma concentrationndashtime curve (AUC) peak con-centration and trough concentration each as the independent variable (Figs 1andash1c) In Figs 1dndash1f the drug exposures were normalized to the EC50 of that pa-tientrsquos virus [1] It can be seen that the normalization improves the fit of themodel to the data It should be noted that the normalization transforms severalpoints from well off the best-fit curve to an area where the fit improves This isbecause of the added information gained from treating a very susceptible viralstrain

It should also be noted that because the drug was administered at essentiallythe same dose and schedule in all the patients there is significant co-linearityThat is one cannot make the peak rise without also raising the trough and withoutincreasing the AUC Therefore one should not draw the inference from thesedata that the AUCEC50 ratio is the pharmacodynamically linked variable forindinavir Indeed from other sources of data the troughEC50 ratio or (perhapspreferably) the time EC95 is the linked variable for protease inhibitors [2]Clearly normalization to a measure of potency for the viral isolate to indinavirimproves the relationship between exposure and response

Much the same is true for any type of pathogen Our ability to grow theorganism and identify its sensitivity to the therapeutic agent is key to our abilityto formulate an exposurendashresponse relationship

However the measure of sensitivity of the pathogen to the drug althoughimportant is not a sufficient condition for the development of a dynamics rela-tionship In order to have the highest probability of attaining a robust dynamicsrelationship obtaining a good estimate of drug exposure for the individual patientis also critical This was seen in a neutropenic rat model of fluoroquinolone phar-macodynamics [3] Two stable mutants of a parental strain of Pseudomonas aeru-ginosa (MICs to the test fluoroquinolone of 1 4 and 8 mgL) were derivedTherapy with the same dose of drug produced a clear difference in response byMIC (80 mgkg once daily as therapy with survivorships of 70 15 and 0for the groups challenged with MICs of 1 4 and 8 mgL respectively) Howeverwhen the dose was altered (20 mgkg once daily for the MIC of 10 mgL chal-lenge strain) so that the peakMIC ratio and AUCMIC ratio were the same asthose seen for the challenge group with an MIC of 40 treated with 80 mgkgonce daily the survivorship curves were identical Because isogenic mutants wereemployed this demonstrates that both pieces of information (drug exposure plusa measure of drug susceptibility) are necessary for the best pharmacodynamicrelationships to be developed

23 Drug Exposure in Clinical Trial Patients

Amazingly few pharmacodynamic relationships have been derived in the anti-infective arena Part of the reason for this is that patients being treated for infec-


tions are often quite ill and unwilling or unable to undergo the rigors of a tradi-tional pharmacokinetic evaluation Often dose has been employed as a surrogatefor actual exposure estimates This has proven to be a failed strategy Dose isa poor measure of exposure There are true between-patient differences in thepharmacokinetic parameter values such as clearance and volume of distributionSuch true differences (but unmeasured when dose is used as a measure of expo-sure) translate into large differences in peak concentration trough concentrationand AUC in a population of patients receiving the same dose It should not besurprising that dose is a particularly poor measure of drug exposure and a poorexposure variable to employ in developing pharmacodynamic relationships

Figure 2 demonstrates the inadequacy of examining just dose as a measureof drug exposure This is the marginal density plot for clearance for levofloxacinThis drug was studied in 272 patients enrolled in the first study to prospectivelydevelop a relationship between exposure and response [4] This was done in amulticenter study that included 22 centers in the United States In the study proto-col patients with serum creatinine values in excess of 20 mgdL were excludedNonetheless by inspection the range of clearance exceeded tenfold This alsoindicates that the range of AUC for a fixed dose would exceed tenfold Obviouslyany attempt to link exposure to outcome employing dose as the measure of expo-sure would be doomed to failure

Over the past decade a number of mathematical techniques have foundtheir way into the toolbox of the kineticist or clinician wishing to construct suchrelationships The first is optimal sampling theory This technique allows identi-

FIGURE 2 Approximate marginal density for clearance of levofloxacin

310 Drusano

fication of sample times that are laden with lsquolsquoinformationrsquorsquo The definition oflsquolsquoinformationrsquorsquo is dependent upon the measure that is defined by the user Forinstance the most commonly employed measure is the determinant of the inverseFisher information matrix This is referred to as D-optimality It has several prop-erties that are desirable The answers obtained are independent of how the systemis parameterized and are also independent of units This measure also has theremarkable property of replicativeness That is if one defines a four-parametersystem there will be exactly four optimal sampling times If the investigatorwishes to make the sampling scheme more robust to errors D-optimality willtell the investigator to repeat one of the optimal sampling times This is becauseD-optimality is deterministic and is based upon the (incorrect) assumption thatthere is only one true parameter vector without true between-patient variabilityMost other measures of information content (eg C-optimality A-optimality)also suffer from being deterministic Publications by DrsquoArgenio [5] and Tod andRocchisani [6] extended optimal sampling into the stochastic framework and al-lowed true between-subject variability in the parameter values This allows theinvestigator to increase the number of samples and to have increasing amountsof information in the sampling scheme for patients whose values are more re-moved from the mean values

Traditional (deterministic) optimal sampling has been well validated Fur-ther it is possible to employ traditional optimal sampling and still obtain sam-pling schedule designs robust for a large portion of the population

One problem with optimal sampling strategy is that it assumes that theanswer is already known that is that one knows the true mean parameter vectorfor the model system This obviously places limitations on the use of optimalsampling strategy in the early phases of drug development when little is knownregarding the lsquolsquotruersquorsquo model to be employed for a specific drug and less is knownregarding the true mean parameter vector Nonetheless with only a little informa-tion regarding these issues optimal sampling has been employed successfully

There was no validation of this technique in patients until a series of studieswere published by Drusano and coworkers [7ndash10] In what was to our knowl-edge the first clinical validation of optimal sampling theory the drug ceftazidimewas examined in young patients with cystic fibrosis receiving a single dose [7]With the use of a Bayesian estimator the optimal sampling subset of the fullsampling set produced precise and unbiased estimates of the important pharmaco-kinetic parameter values

This group then examined the drug piperacillin in a population of septicneutropenic cancer patients [8] Whereas the study with ceftazidime was per-formed with a single dose of drug the study with piperacillin examined twoissues (1) whether optimal sampling would provide precise and unbiased esti-mates of parameter values in the steady-state situation and (2) whether obtaining


duplicate samples obtained at the specified sample times would improve the preci-sion of parameter estimation

The results demonstrated that optimal sampling would as expected pro-vide reasonably precise and unbiased parameter estimates Further this studyalso showed that resampling at the designated optimal times did not improve theprecision of parameter estimation The latter result was a bit of a surprise andflew in the face of the then-accepted theory regarding optimal sampling Optimalsampling theory assumes that the mean parameter vector is known without errorFurther true between-patient variance is not incorporated into the optimal sam-pling time calculation Given these limitations it is not surprising that when que-ried regarding the next most optimal time to obtain a sample after the originaloptimal times have all been obtained the theory forces one of the optimal timesto be repeated (property of replication) This strategy may improve the precisionfor the mean patient but in the clinical situation where one is trying to constructa population model (part of the creation of a pharmacodynamic model) it isimportant to recognize that true between-patient variance exists for the parametervalues

If an investigator is to limit the number of plasma samples obtained to anoptimal sampling set it is important to know how robust optimal sampling iswith regard to errors in nominal parameter values This group also addressed thisissue [10] Theophylline has been demonstrated to have its clearance altered bysmoking cigarettes The degree of this alteration has been on the order of a 50increase in the mean clearance of the population It was felt that by studying apopulation of smokers as well as a population of nonsmokers and employingoptimal sampling strategies for both smokers and nonsmokers they could examinehow badly optimal sampling sets performed when systematic errors on the orderof 50 (either high or low) were introduced into the nominal value for clearanceThis study demonstrated that errors of this magnitude did not introduce significantbias or imprecision into the overall estimation of theophylline clearance Furtherbecause this study was performed in two stages after the first stage they embed-ded a sampling set that was calculated by employing the patientrsquos initial parame-ter values estimated from the full sample set obtained during the first stage Theydemonstrated that the patientrsquos own optimal samples provide excellent precisionand minimal bias for the second stage of the study (patient by patient) Such afinding is important in that it means that toxic drugs can be adequately controlledwith minimal sample acquisition if patients are to be dosed over a relatively longperiod of time (as is the case in antiretroviral chemotherapy) Likewise obtaininginformation about the patientrsquos parameter values for effect control with limitedsample acquisition also becomes possible in the routine clinical situation

Others have recognized the importance of optimal sampling theory in guid-ing the acquisition of plasma samples in the clinical trial setting for the develop-

312 Drusano

TABLE 1 Precision () of Kinetic Parameters of Theophylline asDetermined from Different Optimal Sampling Strategies Relative to ThoseDetermined from the Full Sampling Strategya

Vc Vss Varea SCl T12β

Correct7 220 126 130 297 299Wrong7 166 101 104 356 398Patientrsquos7 228 134 130 298 366Patientrsquos4 260 220 228 299 377

a Correct7 represents the 7 sample times derived from the lsquolsquocorrectrsquorsquo prior populationWrong7 represents the seven sample times derived from the lsquolsquowrongrsquorsquo prior populationPatientrsquos7 and Patientrsquos4 represent the seven and four sample times derived from thepatientrsquos own prior parameter values

ment of exposurendashresponse relationships Forrestrsquos group [11] developed an opti-mal sampling strategy for ciprofloxacin that is useful in the environment ofseriously ill hospitalized patients with lower respiratory tract infections Fletcherand colleagues [12] adapted optimal sampling strategy to the AIDS arena for thedevelopment of concentration-controlled trials

The second technique is population pharmacokinetic modeling Credit forthe initial development of this technique reflects to Sheiner Beal and colleagues[13ndash15] After the initial development of the NONMEM system other groupsdeveloped population modeling programsmdashMallet (NPML) [16] Schumitzky etal (NPEM) [17] the PPHARM system [18] Davidian and Gallant [19] Lind-strom and Bates [20] and Forrest et al [21] among others Population modelingallows the development of a mean parameter vector for the model without requir-ing that every patient have a robust sampling set It also provides an estimate ofthe covariance matrix allowing construction of parameter distributions and alsoallowing Monte Carlo simulation which has recently been shown to be useful inevaluation of doses and schedules Of course the important issue with populationmodeling is that the data must be well timed The looseness of execution oftenassociated with performing population PK modeling is not an excuse for poortiming of sample collection Such poor attention to detail can severely impactupon the estimates rendering them either biased or imprecise Nonetheless itshould be recognized that the ability to perform population modeling has resultedin nothing short of a revolution in our ability to obtain information about drugdisposition in ill target patients The data presented in Fig 2 are from an analysisemploying NPEM [4] Ill patients with community-acquired infections were stud-ied with each patient having an optimal sampling set of seven plasma determina-tions each guided by stochastic design theory


Once population modeling has been performed it is then useful to performmaximal a posteriori probability (MAP) Bayesian estimation This allows pointestimates of the model parameters to be obtained for all the patients in the popula-tion Measures of drug exposure [peak concentration trough concentration areaunder the plasma concentrationndashtime curve (AUC)] can be calculated and thennormalized to the potency parameter (eg peakMIC ratio AUCMIC ratiotime MIC) It is now possible to examine the relationship between exposureand response andor toxicity

In the study cited above a parameter vector was calculated for each patientby Bayesian estimation The plasma drug concentrations were then simulated forthe specific times they were obtained and a predicted versus observed plot wasproduced Figure 3 displays this analysis The best-fit line was

Observed 1001 predicted 00054 r2 0966 p 0001

Once robust estimates of parameter values are obtained for each patientit is straightforward to attempt to link measures of exposure (peak concentrationMIC or AUCMIC ratio time MIC etc) to outcomes For continuous outcomevariables (eg viral copy number CD4 counts) continuous functions such as atraditional sigmoidal Emax effect function would be a natural choice (see Figs1andash1f)

However clinical trials frequently have either dichotomous outcome vari-ables (eg successfailure eradicationpersistence) or have time-to-event end-

FIGURE 3 Scatterplot and least squares line for the entire population

314 Drusano

points (eg time to death time to opportunistic infection time to lesion changein CMV retinitis) For dichotomous outcome variables logistic regression analy-sis is a natural choice For the prospective study examining levofloxacin citedabove we had an analysis plan that tested 13 covariates univariately [422]Model building then ensued from the covariates that significantly altered theprobability of a good clinical or microbiological outcome (separate sets of analy-ses) The final models for clinical and microbiological outcomes are displayedgraphically in Figs 4 and 5 respectively

It should also be noted that small boxes on the probability curve denoteindependent variable lsquolsquobreakpointsrsquorsquo These are arrived at through classificationand regression tree (CART) analysis These merely indicate that patients whoseindependent variable (here peak concentrationMIC ratio) has a value equal toor greater than the breakpoint value have a significantly higher probability ofobtaining a good outcome CART is a useful adjunctive technique in pharmaco-dynamic analyses but should probably be seen as an exploratory tool and onefor rational setting of breakpoints Logistic regression should be seen as the pri-mary tool for analysis with dichotomous endpoints

In addition to modeling successfailure logistic regression can also be em-ployed to model the probability of occurrence of toxicity An example can beseen in the analysis of aminoglycoside-related nephrotoxicity published by Rybaket al [23] These authors performed a prospective randomized double-blind trialin which patients received their aminoglycoside either once daily or twice daily

FIGURE 4 Logistic regression relationship between levofloxacin peakMIC ra-tio and the probability of a good clinical outcome


FIGURE 5 Logistic regression relationship between levofloxacin peakMIC ra-tio and the probability of organism eradication

In the final model the schedule of administration the daily AUC of aminoglyco-side and the concurrent use of vancomycin all independently influenced the prob-ability of occurrence of aminoglycoside-related nephrotoxicity

Sometimes as with the therapy of cytomegalovirus retinitis the endpointexamined is the time to an event here the time to CMV lesion progression Inthis circumstance after having performed the Bayesian estimation the measuresof exposure may be employed as covariates in a Cox proportional hazards modelanalysis This semiparametric approach is a useful way to approach such analysesFor those instances where fuller knowledge of the shape of the hazard functionis available fully parametric analyses (eg Weibull analysis) can be performed

In an analysis of the use of foscarnet for the therapy of cytomegalovirusretinitis Drusano et al [24] performed a population pharmacokinetic analysisfollowed by Bayesian estimation The exposures then became part of the pharma-codynamic analysis Five covariates were examined (1) baseline CD4 count (2)peak CD4 count during therapy (3) whether or not the patient had a baselineblood culture positive for CMV (4) the peak concentration achieved and (5) theAUC achieved Trough concentrations were not considered because they wouldgenerally be below the level of assay detection In fact all five covariates signifi-cantly shifted the hazard function With model building only AUC and the base-line CMV blood culture status remained in the final model Figure 7 demonstratesthe exposure response from the final Cox model

Monte Carlo simulation has recently been demonstrated to be useful forthe evaluation of doses and schedules for anti-infective agents This technique

316 Drusano

FIGURE 6 Probability of aminoglycoside toxicity (a) Twice daily dosingmdashvancomycin use and daily AUC (b) Once daily dosingmdashvancomycin use anddaily AUC


FIGURE 7 Exposure response of final Cox model (a) Baseline cytomegalovi-rus blood culture negative (b) baseline cytomegalovirus blood culture posi-tive () Lowest AUC observed in the population ( ) 20th 50th and80th percentiles respectively of AUC in the population

was first applied for this purpose by Drusano at a meeting of the FDA Anti-Infective Drug Products Advisory Committee [25] Two applications are demon-strated here The first is for dose adequacy and for preclinical MIC breakpointdetermination The second is for the evaluation of the dosing schedule

To evaluate the adequacy of a 500 mg dose of the fluoroquinolone lev-ofloxacin Drusano and Craig collaborated for the following analysis The meanparameter vector and covariance matrix from the levofloxacin study cited earlierwere employed to create a 10000 subject Monte Carlo simulation The AUCdistribution for a 500 mg IV dose for these subjects was generated The data

318 Drusano

FIGURE 8 Levofloxacin 10000-subject Monte Carlo simulation Pneumococ-cal target attainment with a 500 mg qid dose () 1 Log drop target () stasistarget () MIC distribution

from the levofloxacin TRUST (Tracking Resistance in the United States Today)study was employed for the MIC distribution for Streptococcus pneumoniae Thekey for this analysis was to set a lsquolsquotarget goalrsquorsquo Craigrsquos mouse thigh modelallowed setting the AUCMIC target goal of 270 (total drug) associated withstasis and 345 (total drug) for a drop in the CFU of one log10 unit (associatedwith the shutoff of bacteremia) for levofloxacin [26] Each of the 10000 AUCsin the distribution were divided by the MIC range from 0125 to 40 (in a twofolddilution series) The resultant values were compared to the target goals and thefrequency with which the target was achieved was ascertained The outcome ofthis analysis is displayed in Fig 8

It is obvious that the goal attainment rate is 100 for both targets until anMIC of 05 mgL is reached At 10 mgL both target attainments decline but

TABLE 2 Levofloxacin 10000-Subject Monte Carlo Simulation TargetAttainment Over a 4296 Isolate Database of Streptococcus pneumoniae

Target 1 Log drop (345 AUCMIC ratio) Stasis (27 AUCMIC ratio)Attainment 947 02 978 01

Source PK parameters from Ref 33 isolate MICs from the 1998ndash1999 TRUST studytarget attainment data from Ref 26


FIGURE 9 One randomly chosen concentration time profile resulting fromMonte Carlo simulations of different dosage schedules for abacavir and am-prenavir with 500 subjects (andashd) Concentrationndashtime profiles (a c) () Am-prenavir 800 mg g8h () abacavir 300 mg q12h (b d) () Amprenavir 1200mg q12h () abacavir 300 mg q12h (e f) Effectndashtime profiles for a specificpatient

320 Drusano

FIGURE 10 Average percent of maximal antiretroviral effect for combinationtherapy with abacavir plus amprenavir Amprenavir schedule of administra-tion was 800 mg (q8h) or 1200 mg (q12h) Abacavir schedule was 300 mg(q12h) in both groups Results weere calculated from a Greco interactionmodel Drug concentrations were from Monte Carlo simulation

both are in excess of 90 Only after this do we see a large decline in targetattainment

It is possible to remove the variability in the MIC by performing an expecta-tion over the MIC distribution In essence we can multiply the target attainmentrate by the fraction of the strains of pneumococcus represented at each levofloxa-cin MIC value This gives us an estimate of the target attainment rate in a clinicaltrial subject to the assumptions that the MIC distribution is representative of thatseen in clinical trials and that the AUC distribution is likewise representative ofa clinical trial The target attainment rates are shown in Table 2

This example demonstrates that a 500 mg dose of levofloxacin would likelybe adequate for pneumococcal infections given the distribution of the AUCs forthe drug and the distribution of the MICs This has been demonstrated in clinicaltrials of levofloxacin in community-acquired pneumonia [2227]

It is also possible to examine schedule with this technique Drusano et al[28] examined the combination of abcavir plus amprenavir for HIV The interac-tion of the two agents was quantitated in the presence of human binding proteinsin vitro using the Greco interaction equation [29] Population pharmacokineticmodels were then derived from clinical trial data for both drugs Monte Carlosimulations were derived of the effectndashtime curves for 500 subjects In the simu-lations doses of 300 mg of abacavir every 12 h (q12h) plus 800 mg of amprenavir


every 8 h (q8h) were simulated as well as doses of abacavir 300 mg every 12h plus 1200 mg of amprenavir every 12 h In Figs 9a and 9b the mean concentra-tionndashtime profiles are shown for the various simulations for 500 subjects Figures9c and 9d show one subject selected from the population Figures 9e and 9f showthe effect vs time curves derived from that specific patient at steady state forthe different schedules of administration

The effectndashtime curves can be integrated over a 24 h steady-state interval

FIGURE 11 Frequency histograms for average percent of maximal effect forabacaviramprenavir combination therapy In (a) abacavir was 300 mg (q12h)and amprenavir dose was 800 mg (q8h) In (b) abacavir was 300 mg (q12h)and amprenavir was 1200 mg (q12h) Values were determined as in Fig-ure 10

322 Drusano

and divided by the interval length (24 h) An average percent of maximal effectresults from the calculation These are plotted in Fig 10 for the two schedulesof administration for all 500 simulated subjects

It is obvious from inspection that the schedule of administration that ismore fractionated for the protease inhibitor (amprenavir q8h) is providing greatereffects This can clearly be seen in the frequency histograms presented in Fig11 Irrespective of how one tests the differences between regimens (frequency90 maximal effect frequency 70 maximal effect difference betweenmean percent maximal effects) the more fractionated regimen is always statisti-cally significantly superior

Consequently Monte Carlo simulation can be used for both dose and regi-men evaluations and can also set preclinical and after the human pharmacody-namic trials have been performed clinical MIC breakpoints It is obvious thatthere are other uses for this flexible and powerful technique for the clinical arena(eg drug penetration to the site of infection [30])

Each of the foregoing examples leads to a simple paradigm It is straightfor-ward to attempt to define pharmacodynamic relationships in the clinical trial set-ting The approach is set forth in Table 3 Once such relationships are definedparticularly when relationships are available for both efficacy and toxicity as isthe case for aminoglycoside antibiotics [2331] the true goal of anti-infectivechemotherapy (maximal effect with minimal toxicity) can be sought by usingstochastic control techniques [32] As part of the approach it is clear that plasmaconcentration determination is a requirement It should be brought home force-

TABLE 3 Paradigm for the Development of ExposurendashResponseRelationships for Anti-Infective Agents

1 Decide on an endpoint2 Make potency measurements on pathogens from trials (MIC EC50 etc)3 Obtain exposure estimates for patients from those trials

a Stochastic design for sampling schemeb Population pharmacokinetic modelingc MAP-Bayesian estimation for individual-patient exposure esti-

mates4 Decide on an endpoint analysis (the following are examples only)

a Sigmoidal Emax analysis for a continuous variableb Logistic regression for dichotomouspolytomous outcomesc Cox proportional hazards modeling (or a variant) for time-to-event

datad Classification and regression tree analysis for breakpoint determi-

nation5 Stochastic control when effectndashtoxicity relationships are available


fully to pharmaceutical sponsors that the paradigm has shifted We can success-fully seek and generate such relationships In the lsquolsquoold daysrsquorsquo the necessity forconcentration determination was the death knell for the development of a com-pound Now it is clear that for the patientrsquos sake we can achieve maximal proba-bility of a good clinical and microbiological outcome coupled with the minimalprobability of toxicity and (perhaps) emergence of resistance by measuring drugconcentrations in plasma This should be the new (and positive) way of differenti-ating drugs We should prefer those that provide us the possibility of having arational basis for producing the best patient outcomes Again third party payorsalso need to understand that drugs with such relationships developed should havepriority on clinical pathways because they provide maximal probability of re-sponse with minimal probability of toxicity

REFERENCES

1 GL Drusano Antiviral therapy of HIV and cytomegalovirus In Symposium 130-A S-121 37th Interscience Conference on Antimicrobial Agents and ChemotherapyToronto Canada Sept 28ndashOct 1 1997 Am Soc Microbiol Washington DC

2 GL Drusano JA Bilello SL Preston E OrsquoMara S Kaul S Schittman R EcholsHollow fiber unit evaluation of BMS232632 a new HIV-1 protease inhibitor forthe linked pharmacodynamic variable In Session 171 Presentation 1662 40th In-terscience Conference on Antimicrobial Agents and Chemotherapy Toronto Can-ada Sept 17ndash20 2000 Am Soc Microbiol Washington DC

3 GL Drusano DE Johnson M Rosen HC Standiford Pharmacodynamics of a fluor-oquinolone antimicrobial in a neutropenic rat model of Pseudomonas sepsis Antimi-crob Agents Chemother 199337483ndash490

4 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornseif V ReichlJ Natarajan FA Wong M Corrado Levofloxacin population pharmacokinetics inhospitalized patients with serious community-acquired infection and creation of ademographics model for prediction of individual drug clearance Antimicrob AgentsChemother 199842631ndash639

5 DZ DrsquoArgenio Incorporating prior parameter uncertainty in the design of samplingschedules for pharmacokinetic parameter estimation experiments Math Biosci 199099105ndash118

6 M Tod J-M Rocchisani Implementation of OSPOP an algorithm for the estimationof optimal sampling times in pharmacokinetics by the ED EID and API criteriaComput Methods Programs Biomed 19965013ndash22

7 GL Drusano A Forrest MJ Snyder MD Reed JL Blumer An evaluation of optimalsampling strategy and adaptive study design Clin Pharmacol Ther 198844232ndash238

8 GL Drusano A Forrest KI Plaisance JC Wade A prospective evaluation of optimalsampling theory in the determination of the steady state pharmacokinetics of pipera-cillin in febrile neutropenic cancer patients Clin Pharmacol Ther 198945635ndash641

9 GJ Yuen GL Drusano A Forrest KI Plaisance ES Caplan Prospective use of opti-

324 Drusano

mal sampling theory Steady-state ciprofloxacin pharmacokinetics in critically illtrauma patients Clin Pharmacol Ther 198946(4)451ndash457

10 GL Drusano A Forrest JG Yuen KI Plaisance Optimal sampling theory Effectof error in a nominal parameter value on bias and precision of parameter estimationJ Clin Pharmacol 199434967ndash974

11 AD Kashuba CH Ballow A Forrest Development and evaluation of a Bayesianpharmacokinetic estimator and optimal sparse sampling strategies for ceftazidimeAntimicrob Agents Chemother 1996401860ndash1865

12 SE Noormohamed WK Henry FS Rhames HH Balfour Jr CV Fletcher Strategiesfor control of zidovudine concentrations in serum Antimicrob Agents Chemother1995392792ndash2797

13 TH Grasela LB Sheiner Population pharmacokinetics of procainamide from routineclinical data Clin Pharmacokinet 19849545ndash554

14 SL Beal LB Sheiner Estimating population kinetics CRC Crit Rev Bioeng 19828195ndash222

15 LB Sheiner B Rosenberg VV Marathe Estimation of population characteristics ofpharmacokinetic parameters from routine clinical data J Pharmacokinet Biopharm19775445ndash479

16 A Mallet A maximum likelihood estimation method for random coefficient regres-sion models Biometrika 198673645ndash656

17 A Schumitzky R Jelliffe M Van Guilder NPEM2 A program for pharmacokineticpopulation analysis Clin Pharmacol Ther 199455163

18 F Mentre R Gomeni A two step iterative algorithm for estimation in non-linearmixed effects models with an evaluation in population pharmacokinetics J Bio-pharm Stat 19955141ndash158

19 M Davidian AR Gallant Smooth nonparametric maximum likelihood estimationfor population pharmacokinetics with application to quinidine J Pharmacokinet Bi-opharm 199220529ndash556

20 M Lindstrom D Bates Nonlinear mixed effects models for repeated measures dataBiometrics 199046673ndash687

21 A Forrest CH Ballow DE Nix MC Birmingham JJ Schentag Development of apopulation pharmacokinetic model and optimal sampling strategies for intravenousciprofloxacin Antimicrob Agents Chemother 1993371065ndash1072

22 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornseif V Reichl JNatarajan M Corrado Prospective development of pharmacodynamic relationshipsbetween measures of levofloxacin exposure and measures of patient outcome Anew paradigm for early clinical trials J Am Med Assoc 1998279125ndash129

23 MJ Rybak BJ Abate SL Kang MJ Ruffing SA Lerner GL Drusano Prospectiveevaluation of the effect of an aminoglycoside dosing regimen on rates of observednephrotoxicity and ototoxicity Antimicrob Agents Chemother 1999431549ndash1555

24 GL Drusano F Aweeka J Gambertoglio M Jacobson M Polis HC Lane C Eaton SMartin-Munley Relationship between foscarnet exposure baseline cytomegalovirusblood culture and the time to progression of cytomegalovirus retinitis in HIV-posi-tive patients AIDS 1996101113ndash1119

25 GL Drusano SL Preston C Hardalo R Hare C Banfield D Andes WA Craig Useof pre-clinical data for the choice of a phase IIIII dose for SCH27899 with applica-


tion to identification of a pre-clinical MIC breakpoint Antimicrob Agents Chemo-ther 20014513ndash22

26 Craig WA Personal communication27 TM File Jr J Segreti L Dunbar R Player R Kohler RR Williams C Kojak A

Rubin A multicenter randomized study comparing the efficacy and safety of intra-venous andor oral levofloxacin versus ceftriaxone andor cefuroxim axetil in treat-ment of adults with community-acquired pneumonia Antimicrob Agents Chemother1997411965ndash1972

28 GL Drusano DZ DrsquoArgenio SL Preston C Barone W Symonds S LaFon M Rog-ers W Prince A Bye JA Bilello Use of drug effect interaction modeling with MonteCarlo simulation to examine the impact of dosing interval on the projected antiviralactivity of the combination of abacavir and amprenavir Antimicrob Agents Chemo-ther 2000441655ndash1659

29 GL Drusano DZ DrsquoArgenio W Symonds PA Bilello J McDowell B Sadler ABye JA Bilello Nucleoside analog 1592U89 and human immunodeficiency virusprotease inhibitor 141W94 are synergistic in vitro Antimicrob Agents Chemother1998422153ndash2159

30 GL Drusano SL Preston M Van Guilder D North M Gombert M Oefelein LBoccumini B Weisinger M Corrado J Kahn A population pharmacokinetic analy-sis of the prostate penetration of levofloxacin Antimicrob Agents Chemother 2000442046ndash2051

31 AD Kashuba AN Nafziger GL Drusano JS Bertino Jr Optimizing aminoglycosidetherapy for nosocomial pneumonia caused by gram-negative bacteria AntimicrobAgents Chemother 199943623ndash629

32 A Schumitzky Applications of stochastic control theory to optimal design of dosageregimens In DZ DrsquoArgenio ed Advanced Methods of Pharmacokinetic and Phar-macodynamic Systems Analysis Plenum New York 1991137ndash152

33 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornseif V ReichlJ Natarajan FA Wong M Corrado Levofloxacin population pharmacokinetics andcreation of a demographic model for prediction of individual drug clearance in pa-tients with serious community-acquired infection Antimicrob Agents Chemother1998421098ndash1104

15

Antibacterial Resistance

Philip D Lister

Creighton University School of Medicine Omaha Nebraska

1 INTRODUCTION

Bacterial resistance to antibiotics is an inescapable response to the overuse ofantimicrobial agents in the environment It can arise from the selection of resistantsubpopulations among susceptible strains or from the increased prevalence ofstrains expressing intrinsic or acquired resistance The impact of antibiotic resis-tance on patients and society is overwhelming as infections caused by resistantpathogens are associated with higher rates of morbidity and mortality than infec-tions caused by pathogens [12] Furthermore microbial drug resistance has beenprojected to add between $100 million and $30 billion annually to health carecosts [3] Recognition of the factors associated with increasing resistance is essen-tial for the development of new strategies to slow the evolution and progressionof antimicrobial resistance among bacteria

The heavy use and abuse of some antimicrobial agents have been directlylinked to some of the more serious resistance problems challenging clinicianstoday [45] Perhaps the best example is the association between excessive ceftaz-idime use and the development and spread of multidrug resistance among gram-negative bacteria [67] With a diminishment in the number of novel agents enter-ing the clinical arena the judicious use of currently available antibiotics becomesessential if we are to preserve their clinical efficacy Drug control strategies must

327

328 Lister

not only involve tighter regulation on the use of antibiotics but also must involvea more scientific approach to dosing strategies The field of antimicrobial pharma-codynamics strives to establish relationships between the pharmacokinetics of adrug and the effective treatment of infections However just as important are therelationships between pharmacokinetics and selection of resistance during ther-apy This chapter will review some of the most critical resistance problems facingclinicians today the problems encountered with accurate detection of resistancein the clinical laboratory and the role of pharmacodynamic strategies in slowingthe emergence of resistance among bacteria

2 EMERGING RESISTANCE PROBLEMS IN

INFECTIOUS DISEASES


211 Enterococcus Species

Among the numerous species of enterococci E faecalis and E faecium are thetwo most important clinically In addition to causing serious hospital-acquiredinfections such as bacteremia and endocarditis [8] E faecalis and E faeciumare two of the most intrinsically resistant bacteria challenging clinicians today[9] These two pathogens naturally exhibit low-level resistance to the β-lactamsaminoglycosides clindamycin trimethoprim-sulfamethoxazole and other classesof antimicrobial agents When bacterial killing is required for clinical cure thecombination of a cell-wall-active agent (penicillin or vancomycin) with an amino-glycoside provides one of the true examples of antimicrobial synergy

Unfortunately E faecalis and E faecium have not been satisfied with theirintrinsic resistance and have been acquiring high-level resistance to these antibi-otics as well [9] Once high-level resistance to either the β-lactams or aminogly-cosides is acquired synergistic killing is lost therapy of serious infections iscompromised and the search for more effective antimicrobial agents or alterna-tive combinations becomes essential

212 Staphylococcus aureus

In contrast to the impressive intrinsic resistance displayed by the enterococci Saureus represents a prototype bacterium for rapid acquisition and developmentof resistance in response to antibiotics in the environment Soon after penicillinbecame available for clinical use Spink and Ferris [10] reported their isolationof a penicillin-resistant S aureus that produced a penicillin-inactivating enzymePenicillinases in S aureus are usually encoded on a plasmid and can spread easilyamong the genus Currently 70ndash90 of staphylococci are resistant to penicillinand the aminopenicillins [11] To circumvent this problem penicillinase-resistantpenicillins such as methicillin and oxacillin were synthesized and introduced inthe early 1960s However the first methicillin-resistant S aureus (MRSA) was


discovered as early as 1961 [12] Resistance to this class of penicillins is notmediated through enzymatic inactivation but rather is mediated through the ex-pression of a low-affinity penicillin binding protein PBP2prime [13] By the late1980s 25 of all staphylococcal isolates in the United States were resistant tomethicillin [14] with prevalence rates varying markedly between geographicalareas and between individual institutions

Strains of MRSAs are frequently resistant to multiple antibiotics includingthe penicillins cephalosporins cephamycins carbapenems β-lactamasendashinhibi-tor combinations aminoglycosides macrolides tetracyclines and sulfonamides[11] The glycopeptides vancomycin and teicoplanin remain the preferred therapyfor MRSA infections However strains of S aureus exhibiting intermediate resis-tance to both vancomycin and teicoplanin have been reported [1516]

213 Streptococcus pneumoniae

Like S aureus S pneumoniae has shown a remarkable ability to evolve anddevelop resistance to antibiotics in the environment In contrast to S aureushowever the rate of resistance development is much slower among pneumococciClinical isolates of S pneumoniae remained susceptible to penicillin until the1960s when the first intermediate-resistant isolates were reported in Boston in1965 [17] Thereafter pneumococcal susceptibility to penicillin gradually de-creased with reports of intermediate resistance increasing globally and the firstreports of high-level resistance and clinical failures with penicillin appearing inthe literature [18ndash20] In the United States the prevalence of penicillin-nonsus-ceptible pneumococci steadily increased through the 1980s and 1990s with 40of pneumococci across the country having now lost their susceptibility to penicil-lin [21]

The mechanism of penicillin resistance among S pneumoniae involves theacquisition of lsquolsquoresistantrsquorsquo PBP gene segments from other streptococci in theenvironment [2223] As susceptibility to penicillin decreases a concurrent de-crease in susceptibility to other penicillins cephalosporins cephamycins andcarbapenems is also observed [24] In addition penicillin-resistant pneumococciare increasing their resistance to other classes of antimicrobial agents includingthe macrolides tetracyclines chloramphenicol and trimethoprim-sulfamethoxa-zole [24] The increased prevalence of multidrug-resistant pneumococci presentsa therapeutic dilemma for the treatment to serious pneumococcal infections

A summary of emerging resistance problems among gram-positive bacteriais provided in Table 1


221 Haemophilus influenzae

It is now well established that ampicillin resistance among H influenzae strainscan result from mutations affecting the PBP targets decreased permeability

330 Lister

TABLE 1 Emerging Resistance Problems Among Gram-Positive Pathogens

Bacterial species Class of antibiotics Molecular mechanisms

Enterococcus faecium High-level β-lactam Low-affinity PBPsEnterococcus faecalis β-lactamase inactivation

Vancomycin Production of alteredcell-wall precursorsthat vancomycinbinds with lower af-finity

Aminoglycosides Inactivating enzymesStaphylococcus aureus Penicillin and Penicillinase

aminopenicillinsAll β-lactams Acquired low-affinity

PBP2primeVancomycin Increased targets in

outer cell wallStreptococcus pneu- β-Lactams Acquired lsquolsquoresistantrsquorsquo

moniae PBP gene segments

through the outer membrane andor acquisition of plasmid-mediated β-lacta-mases [25] Of these three resistance mechanisms production of β-lactamaseaccounts for 90 of ampicillin resistance among H influenzae globally withTEM-1 being the most common enzyme produced [25] On average 30ndash37 ofH influenzae strains from every region of the United States produce β-lactamase[21] Although β-lactamase-producing strains remain susceptible to the β-lacta-masendashinhibitor combinations and extended-spectrum cephalosporins permeabil-ity andor PBP changes can provide resistance to these agents as well [2126]In addition to β-lactam resistance H influenzae are developing impressive resis-tance to the macrolide antibiotics with 54 of over 3000 isolates exhibitingresistance in a 1998 national survey [21]

222 Escherichia coli and Klebsiella pneumoniae

Strains of E coli and K pneumoniae have shown a remarkable ability to evolveand adapt to the threat of antibiotics in the environment especially to the β-lactam class Resistance to β-lactams among these two species is mediated pri-marily through the production of β-lactamases Whereas both species encode forβ-lactamase on their chromosome plasmid-encoded β-lactamases are the pre-dominant mechanism of resistance with TEM-1 and SHV-1 being the most com-mon enzymes produced [6] TEM-1 and SHV-1 are considered broad-spectrumβ-lactamases and account for the majority of E coli and K pneumoniae resistanceto the penicillins and early narrow-spectrum cephalosporins Two approaches


used to circumvent β-lactamase-mediated resistance have been to develop lsquolsquoen-zyme-resistantrsquorsquo cephalosporins and to combine an inhibitor of β-lactamases withenzyme-labile penicillins E coli and K pneumoniae have evolved impressivelyin response to these two approaches and have rapidly developed resistance tothe extended-spectrum lsquolsquothird generationrsquorsquo cephalosporins and the β-lactamasendashinhibitor combinations

In response to the β-lactamasendashinhibitor combinations E coli and K pneu-moniae have mutated to either increase production of their plasmid-encoded β-lactamases [2728] or decrease the sensitivity of their β-lactamases to inhibitionby clavulanate tazobactam and sulbactam [2930] In response to the extended-spectrum cephalosporins (ceftazidime cefotaxime ceftriaxone) E coli and Kpneumoniae have evolved through two distinct pathways They have either mu-tated the active sites of their TEM-1 or SHV-1 enzymes to increase the hydrolysisof these drugs or they have acquired plasmid-encoded AmpC cephalosporinasesthat originated from the chromosomal AmpCs of other species (see Sections223 and 224) and are inherently capable of providing resistance to the ex-tended-spectrum cephalosporins The mutated variants of TEM-1 and SHV-1have been termed extended-spectrum β-lactamases (ESBLs) because they haveextended the resistance potential of E coli and K pneumoniae to include theextended-spectrum cephalosporins and aztreonam Unfortunately many strainsproducing these enzymes appear falsely susceptible to the newer cephalosporinsand aztreonam in routine susceptibility tests [3132] and the scope of this resis-tance problem remains largely unknown Although most ESBL-producing bacte-ria are also resistant to ampicillin-sulbactam amoxicillin-clavulanate and ticar-cillin-clavulanate many remain susceptible to piperacillin-tazobactam Similarlymany ESBL-producing E coli and K pneumoniae remain susceptible to thelsquolsquofourth generationrsquorsquo cephalosporin cefepime However the clinical relevance ofpiperacillin-tazobactam and cefepime susceptibility has not been established andthere are concerns of false susceptibility similar to that observed with the ex-tended-spectrum cephalosporins and aztreonam Further complicating therapy ofESBL-producing bacteria is that these enzymes are encoded on plasmids that alsocarry genes encoding resistance to other classes of antimicrobial agents [3334]Therefore it is not uncommon to find ESBL-producing bacteria that are alsoresistant to the aminoglycosides trimethoprim-sulfamethoxazole chlorampheni-col and the tetracyclines The most reliably active agents for treatment of ESBL-producing E coli and K pneumoniae are the carbapenems cephamycins andfluoroquinolones However strains of K pneumoniae resistant to all availableantimicrobial agents have been isolated [35] and may signal the beginning of theprophesied lsquolsquopost-antibiotic erarsquorsquo

223 Other Enterobacteriaceae

Although the predominant ESBL-producing bacteria are E coli and K pneumon-iae ESBLs have also been observed in species of Enterobacter Citrobacter

332 Lister

Proteus Salmonella Serratia and other species of Klebsiella [63637] How-ever it is an inducible chromosomal AmpC cephalosporinase that is most com-monly involved in the development of β-lactam resistance among some of thesespecies [38] Since the 1980s Enterobacteriaceae possessing inducible AmpCcephalosporinases have played an important role in the emergence of resistance

TABLE 2 Emerging Resistance Problems Among Gram-NegativePathogens

Bacterial species Antibiotic class Molecular mechanisms

H influenzae Penicillins TEM-1 β-lactamasePBP affinity decreaseOuter membrane per-

meabilityK pneumoniae E coli Penicillins and narrow- TEM-1 and SHV-1

spectrum cephalo- β-lactamasessporins

β-Lactamasendashinhibitor Inhibitor-resistantcombinations β-lactamases or

hyperproductionof TEM-1SHV-1

Penicillins cephalospo- Extended-spectrumrins inhibitorndashpenicil- β-lactamaseslin combinationsand monobactams

Cephamycins Plasmid-encodedAmpC β-lactamase

E cloacae E aerogen- Penicillins cephalospo- Chromosomal AmpCese S marcescens rins inhibitorndashpenicil- β-lactamaseC freundii lin combinations

cephamycins andmonobactams

Carbapenems CarbapenemasesAmpC outer mem-

brane porinsP aeruginosa Antipseudomonal peni- Plasmid-encoded

cillins β-lactamasesPenicillins cephalospo- Chromosomal AmpC

rins inhibitorndashpenicil- β-lactamaselin combinationsand monobactams

Multiple drug classes Outer membrane porinsAntibiotic efflux pumps


to the antipseudomonal penicillins extended-spectrum cephalosporins and az-treonam Studies have shown that the rapid development of resistance to virtuallyall β-lactams is associated with mutant subpopulations that constitutively producehigh levels of their AmpC cephalosporinase [39] With one mutant cell beingpresent among every 106ndash107 viable cells high-level β-lactam resistance has beenshown to emerge in anywhere from 19 to 80 of patients infected with isolatespossessing an inducible AmpC cephalosporinase [6] Although the emergence ofAmpC-mediated resistance has been observed during therapy with many of theavailable β-lactam antibiotics there appears to be a particular association withextended-spectrum cephalosporins most particularly ceftazidime [2] In contrastto the β-lactam class of drugs carbapenem resistance is very rare among membersof the Enterobacteriaceae When resistance is observed it is due to either theproduction of a β-lactamase capable of hydrolyzing carbapenem antibiotics (car-bapenemase) or a reduction in the accumulation of drug in the periplasmic space[384041]

224 Pseudomonas aeruginosa

Among the gram-negative bacterial species P aeruginosa is one of the mosttalented in terms of rapidly developing resistance to antibiotics Similar to theEnterobacteriaceae the mechanisms responsible for β-lactam resistance amongP aeruginosa often involve the production of β-lactamases both plasmid- andchromosomally encoded and the emergence of high-level β-lactam resistanceduring therapy is also primarily due to the selection of mutants that overproducetheir chromosomal AmpC cephalosporinase [42] In contrast to the Enterobacteri-aceae however rates of imipenem resistance among P aeruginosa can exceed50 [43] The β-lactams and carbapenem antibiotics are not the only classes thatare impacted by P aeruginosarsquos ability to rapidly develop resistance Due to itsability to rapidly alter outer membrane permeability P aeruginosa can developresistance during the course of therapy with virtually any antibiotic

A summary of emerging resistance problems among gram-negative bacteriais provided in Table 2

3 DETECTION OF RESISTANCE IN THE

CLINICAL LABORATORY

31 Clinical Relevance of Susceptibility Tests

The susceptibility of a bacterial pathogen to antibiotics is an important therapeuticguide for clinicians However antimicrobial susceptibility is just one piece of acomplex puzzle determining clinical outcome Just as important are the pharma-cokinetics of the antibiotic at the site of infection the physical and chemicalenvironment at the site of infection the pharmacodynamic interactions between

334 Lister

the antibiotic and target bacterium the immune status of the host the need forsurgical intervention and the emergence of resistance during therapy The inter-play between some or all of these factors can lead to a clinical failure even whenthe bacterium exhibits susceptibility to an antibiotic in vitro Conversely clinicalsuccess can sometimes be achieved in cases when the bacterial pathogen exhibitsresistance in vitro This is especially true when interpretive breakpoints for resis-tance are based upon serum pharmacokinetics yet the antimicrobial agents areknown to concentrate to much higher levels at the site of infection

There have been relatively few studies investigating the predictive valueof susceptibility tests and comparison of their data is difficult due to differencesin susceptibility tests used definitions of sensitivity or resistance microorgan-isms studied and criteria used for clinical success or failure [44] Despite theseproblems general conclusions can be reached The correlation between in vitrosusceptibility and favorable clinical outcome is moderately good at best mostlydue to the multitude of other factors that can significantly affect therapeutic suc-cess Although the correlation between in vitro resistance and clinical failure ismuch stronger the relationship is not 100 [44] This is not surprising howeverconsidering that in vitro susceptibility assays are performed in the absence ofhost defense mechanisms and that interpretive breakpoints based on serum phar-macokinetics are inappropriate for drugs that concentrate to higher levels in tis-sues or urine Despite these imperfections there is a clinical relevance to thedata obtained from in vitro susceptibility assays especially when antimicrobialresistance is accurately detected Unfortunately most efforts directed toward im-proving susceptibility testing to date have focused on achieving a reproducibledetection of susceptibility As the threat of antimicrobial resistance continues toincrease in our environment it becomes essential that scientists shift the focusof susceptibility assays from the reproducible detection of susceptibility to theaccurate detection of old and newly evolving resistance mechanisms There arethree general approaches that can be used to detect resistance in clinical isolates(1) detection of resistance genes (2) detection of gene products that mediateresistance and (3) detection of phenotypic resistance through routine susceptibil-ity assays These three approaches will be reviewed in this chapter

32 Methods for the Detection of Resistance Genes

One advantage of using genetic techniques to detect resistance is that they cangive the clinician therapeutic guidance faster than routine culture and susceptibil-ity methods In addition genetic techniques can sometimes be more accurate thansusceptibility tests in defining the resistance mechanisms of a bacterium Forexample oxacillin resistance among strains of S aureus can be mediated eitherthrough the production of PBP2prime or through the hyperproduction of a penicillinase[45] It is not always possible to differentiate between these two resistance mecha-


nisms based on routine susceptibility data yet the therapeutic implications areimportant [46] A genetically based assay that accurately detects mecA in staphy-lococci could provide important therapeutic information to the clinician and pre-vent the unnecessary use of vancomycin in some cases It is important to notehowever that these genetically based assays are useful only when they detect aresistance gene in a bacterial isolate A negative result is of little use to the clini-cian because other unrelated resistance mechanisms may be operative in thebacterium Therefore a negative result should never be used as a confirmationof susceptibility

Two approaches are currently used to detect resistance genes (1) directdetection of genes with DNA probes and (2) amplification of genes using poly-merase chain reaction (PCR) methodology Variations of these methodologiesare currently being developed to target resistance genes in numerous bacterialspecies However this section will only briefly review those genetic methods thattarget aminoglycoside and glycopeptide resistance in enterococci and β-lactamresistance mediated through mecA and β-lactamases (see Table 3)

321 Glycopeptide and Aminoglycoside Resistance

To date there have been four different genotypes of vancomycin resistance char-acterized vanA vanB vanC and vanD [5] Of these vanA and vanB are the mostimportant clinically providing high-level vancomycin resistance among strainsof E faecalis and E faecium Gene probes and PCR assays have been designedto detect the vanA vanB vanC genotypes [4748] and PCR assays are currentlybeing evaluated for the identification of vancomycin-resistant enterococci in fecalsamples [49]

Owing to the diversity of aminoglycoside-modifying enzymes amonggram-negative bacteria it is very difficult to find a consensus sequence that wouldallow for detection of multiple genes using one DNA probe or PCR primer setIn contrast the genes encoding aminoglycoside-modifying enzymes in gram-pos-itive bacteria are much more conserved To date most efforts have focused ondetecting the genes encoding ANT(6) and AAC(6prime)-APH(2Prime) which providehigh-level aminoglycoside resistance to enterococci [50]

322 β-Lactam Resistance

Among gram-positive bacteria most attention has focused on the genetic detec-tion of mecA-mediated resistance in isolated colonies of staphylococci or bloodculture [5152] These assays can require as few as 500 staphylococci to obtaina positive reaction using a PCR enzyme-linked colorimetric assay

The enzymes that are responsible for β-lactam resistance among gram-neg-ative bacteria are diverse not only in their function but also in their genetic codeDNA probes and PCR primer sets have already been prepared for several plas-mid-encoded β-lactamases [5354] and DNA probes have been used to detect

336 Lister

TABLE 3 Assays to Detect Antimicrobial Resistance Genes

Bacterial species Resistance mechanism Genes targeted

Enterococcus faecium Vancomycin resistance vanAEnterococcus fae- through VanA VanB vanBcalis Enterococcus or VanC vanCspp

High-level aminoglyco- ant(6prime)side through inactivat- aac(6prime)-aph(2Prime)ing enzymes ANT(6)or AAC(6prime)-APH(2Prime)

Staphylococcus aureus β-Lactam resistance mecAthrough production oflow-affinity PBP2prime

Escherichia coli Resistance to penicillins blaTEM

Klebsiella pneumoniae and early cephalospo- blaSHV

rins through TEM-1or SHV-1 β-lactamase

Haemophilus influenzae Resistance to penicillins blaTEM

through production blaROB

of TEM-1 or ROB-1β-lactamases

Enterobacteriaceae Resistance to penicil- Oligonucleotide typinglins cephalosporins to detect nucleotideinhibitorndashpenicillin changes at criticalcombinations and basepairs of blaTEM

monobactams and blaSHV

through productionof ESBLs

the blaTEM gene (TEM-1) in urethral exudates urine and spinal fluid [55ndash57]However because of the diversity of β-lactamase produced by gram-negativebacteria samples may need to be tested with multiple probes or PCR primer setsFor example to accurately detect β-lactamase-mediated resistance in E coli andK pneumoniae probes or primer sets for TEM-1 SHV-1 and a multitude ofother β-lactamases may need to be tested on a single isolate Similarly to accu-rately detect β-lactamase-mediated resistance among H influenzae probes orprimer sets for TEM-1 or ROB-1 should be evaluated even though TEM-1 isby far the most predominant enzyme Even with the development of probes toevery known β-lactamase gene this genetically based approach would not detectresistance mediated by porin or PBP changes and thus a negative result shouldbe used as a confirmation of susceptibility


The ever-evolving family of ESBLs presents an additional problem forgenetically-based assays Because these enzymes have evolved over time throughpoint mutations within the active sites of TEM-1 and SHV-1 [3337] probes andprimer sets designed for blaTEM and blaSHV will detect an ESBL gene Howeverthese probes cannot differentiate an ESBL-encoding gene from a gene encodingTEM-1 or SHV-1 Considering the therapeutic implications associated withESBLs it is critical that any genetically-based assays demonstrate this differentia-tion capability Mabilat and Courvalin [58] described an oligonucleotide-basedhybridization method that can differentiate β-lactamase genes based on nucleo-tide changes at critical basepairs However because of the logistics and labor-intensive nature of this technology oligonucleotide typing presents a greater ben-efit for epidemiological studies at this time As technologies continue to evolvethe direct detection of ESBLs may become a reality in the clinical laboratory

33 Methods For Detection of Gene Products That

Mediate Resistance

Similar to the genetically-based methods discussed above methods that directlydetect the active products of resistance genes can provide valuable therapeuticinformation to the clinician faster than routine culture and susceptibility methodsA positive result from these tests can guide the clinician by inferring resistanceto those drugs known to be affected by the expressed resistance mechanismHowever similar to genetically based methods a negative result does not ensurethe lack of resistance as other mechanisms of resistance may be operative Asummary of the methods used for direct detection of β-lactamases and chloram-phenicol acetyltransferase (CAT) is provided in Table 4

331 Direct Detection of β-Lactamases

There are three direct β-lactamase detection assays used clinically (1) iodometricassay (2) acidometric assay and (3) chromogenic assay The iodometric andacidometric assays are based upon the colorimetric detection of penicillinoic acidproduced from β-lactamase hydrolysis of penicillin [5960] In the acidometricassay hydrolysis of citrate-buffered penicillin causes a decrease in pH and acolor change of phenol red to yellow In the iodometric assay penicillinoic acidreduces iron and prevents the interaction of iron with starch Without the hydroly-sis of penicillin to penicillinoic acid iron and starch interact and the mediumturns bluish purple In the chromogenic assay the chromogenic cephalosporinnitrocefin can either be incorporated into liquid suspension or impregnated intosterile paper disks Hydrolysis of nitrocefin results in an electron shift and a redproduct [61] The disk chromogenic assay commercially known as the cefinasedisk is the most common method used for β-lactamase detection in clinical labo-ratories

338 Lister

TA

BLE

4A

ssay

sto

Det

ect

Gen

eP

rod

uct

sth

atM

edia

teA

nti

mic

rob

ial

Res

ista

nce

Bac

teri

alsp

ecie

sR

esis

tan

cem

ech

anis

mM

eth

od

olo

gy

Hae

mo

ph

ilus

infl

uen

zae

Mo

ra-

Pla

smid

-en

cod

edβ-

lact

amas

eA

cid

om

etri

cd

etec

tio

no

fp

enic

illin

oic

acid

xella

cata

rrh

alis

E

nte

roco

ccu

sfr

om

β-la

ctam

ase

hyd

roly

sis

of

pen

icil-

spp

N

eiss

eria

go

no

rrh

eae

lin

Dec

reas

ein

pH

chan

ges

ph

eno

lre

dS

tap

hyl

oco

ccu

sau

reu

sto

yello

w

Iod

om

etri

cd

etec

tio

no

fp

enic

illin

oic

acid

w

hic

hre

du

ces

iro

nan

dp

reve

nts

inte

r-ac

tio

nw

ith

star

ch

Lack

of

iro

nndashs

tarc

hin

tera

ctio

np

reve

nts

colo

rch

ang

eC

hro

mo

gen

icn

itro

cefi

n

insu

spen

sio

no

rim

pre

gn

ated

into

pap

erd

isks

is

hy-

dro

lyze

db

yβ-

lact

amas

ean

del

ectr

on

shif

tre

sult

sin

red

colo

rp

rod

uct

H

aem

op

hilu

sin

flu

enza

eS

trep

to-

Ch

lora

mp

hen

ico

lac

etyl

tran

sfer

ase

Ace

tyl

coen

zym

eA

isco

nve

rted

toh

ydro

-co

ccu

sp

neu

mo

nia

eS

alm

o-

(CA

T)

gen

sulfi

de

coen

zym

eA

du

rin

gth

ere

-n

ella

spp

ac

tio

no

fC

AT

wit

hch

lora

mp

hen

ico

lFr

eesu

lfh

ydry

lo

fco

enzy

me

Ais

de-

tect

edb

ya

colo

rch

ang

ein

the

reac

-ti

on

med

ium


The direct detection of β-lactamase in species of staphylococci entero-cocci H influenzae Neisseria gonorrhea and Moraxella catarrhalis indicatesresistance to all penicillin antibiotics but does not indicate resistance to all thecephalosporins Furthermore a negative reaction does not rule out penicillin re-sistance because some strains are resistant to penicillins through altered PBPsandor decreased permeability [62] Another consideration is the inducibility ofpenicillinase production in staphylococci Unlike the constitutive productionof β-lactamase by H influenzae most staphylococci produce detectable levelsof their penicillinase only after induction with a β-lactam drug [63] Thereforeany negative results from uninduced cultures of staphylococci must be repeatedwith cultures grown in the presence of subinhibitory concentrations of a β-lactamantibiotic

332 Detection of CAT

The majority of chloramphenicol resistance is mediated through the enzymechloramphenicol acetyltransferase (CAT) [64] A rapid method for detectingCAT in cultures of H influenzae has been described [65] and is available com-mercially as a kit This assay has also been used to detect the production of CATin S pneumoniae [66] and Salmonella spp [67] In this assay acetyl coenzymeA is converted to hydrogen sulfide coenzyme A during the reaction of CAT withchloramphenicol The free sulfhydryl of coenzyme A then reacts with 55prime-dithi-obis(2-nitrobenzoic acid) and a resultant color change from pale yellow to deepyellow is observed Similar to β-lactamase detection assays a negative resultdoes not ensure that the bacterium is susceptible to chloramphenicol

34 Susceptibility Methods for Phenotypic Detection

of Resistance

Assays that evaluate the growth of bacteria on or in antibiotic-containing mediahave been the standard approach to susceptibility testing for decades Theseassays can be either qualitative quantitative or semiquantitative Qualitativeassays provide susceptible intermediate and resistance phenotypes but do notprovide information on how susceptible or resistant a bacterium is to antibioticsIn contrast quantitative assays provide a minimum inhibitory concentration(MIC) and thus provide information on the degree of susceptibility or resistanceSemiquantitative assays fall between the qualitative and quantitative methodsmdashthey evaluate susceptibility using a short range of antibiotic concentrationsaround an important lsquolsquobreakpointrsquorsquo concentration or a single concentration thatcan differentiate between mechanisms of resistance

341 In Vivo Correlation of Susceptibility Assays

The primary criticism of any in vitro susceptibility test is that the in vivo environ-ment is not simulated and that the assays are artificially evaluating antimicrobial

340 Lister

susceptibility The four most important criticisms focus on the growth of bacteriain nutrient-rich media the size of starting inocula the absence of serum proteinsthat bind antibiotics and the lack of pharmacodynamic correlations

Growth In or On Artificial Media When the interactions of an antibioticwith a bacterium are evaluated in vitro the bacteria are usually growing logarith-mically in or on nutrient-rich media This is in contrast to the slow growth orstasis governed by the nutrient-deprived environment of most infections There-fore cells that are growing rapidly in vitro may appear more susceptible togrowth-phase-dependent antibiotics such as β-lactam antibiotics than theywould actually be in the infected patient In addition other factors such as pHosmolarity and levels of gas exchange which can play an important role in theinteractions of antibiotics with bacteria may also be misrepresented in broth cul-tures and in the growing bacterial colony [68]

Inoculum Size Because the load of bacteria can vary substantially frompatient to patient and between different sites of infection it is difficult to definea clinically relevant inoculum Therefore scientists have chosen to focus theirattention on standardizing inocula so that the reproducibility of data is maximized[6970] Unfortunately accurate reproducibility is best achieved through reducingthe inoculum size to provide a more uniform bacterial population By reducingthe inoculum size one also increases the apparent activity of some antibiotics[6871] and limits the ability to detect resistant subpopulations that may causetherapeutic failure [72 73]

Regardless of the inoculum size selected however the methods used toachieve the standard inoculum are also important Standard inocula can be ob-tained either by growing the bacteria in broth to a desired turbidity or throughthe suspension of colonies from agar cultures to achieve a desired turbidity Ithas been suggested that inocula be prepared from at least four or five coloniesto ensure that all phenotypes are present [74] However others have suggestedthat five colonies is less than adequate to represent the clinical situation [75]

Protein Binding The degree of protein binding can vary widely betweenand within the different classes of antibiotics For example just among the β-lactam antibiotics percent protein binding can range from 5 to 90 [76]Although the degree of protein binding has been shown to influence the move-ment of antibiotics from the bloodstream to extravascular spaces [77] the impactof protein binding on antibacterial activity remains unsettled The interaction be-tween antibiotics and serum proteins is a dynamic reversible process with drugmolecules constantly binding and unbinding at a rapid rate [78] In studies withciprofloxacin and ofloxacin which exhibit 20ndash40 binding to serum proteinsthe addition of 40ndash50 serum to MIC assays did not affect the potency of thesefluoroquinolones [79ndash82] In studies with trovafloxacin which exhibits 70 pro-


tein binding intra- and interspecies variations have been observed ranging fromno effect at all to significant reductions in potency in the presence of serumproteins [83] Just as conflicting are data from studies with cefonicid which ishighly protein-bound and exhibits a significant decrease in in vitro potency inthe presence of serum proteins [84] Although protein binding has been used toexplain clinical failures with cefonicid when the pathogens are susceptible invitro [85] the efficacy of cefonicid against K pneumoniae was actually enhancedby the administration of excess serum albumin to mice [86] The best explanationfor this observation is that the binding of cefonicid to the excess serum albuminextended its elimination half-life and increased the time for which concentrationsremained above the MIC

Pharmacodynamic Correlations One of the major drawbacks of mostsusceptibility assays is that they evaluate only the inhibition of visible growthWith the exception of broth dilution methodologies susceptibility assays cannotdifferentiate bacteriostasis from bactericidal killing and this differentiation canbe critical for successful treatment of some serious infections Another drawbackof in vitro susceptibility assays is that they evaluate antibioticndashbacterium interac-tions using static concentrations over limited periods of time In contrast duringthe treatment of an infection bacteria are exposed to constantly changing concen-trations of antibiotic over several days Therefore important pharmacodynamicinteractions may be missed during routine susceptibility assays especially theoutgrowth of slow-growing mutant subpopulations that could result in therapeuticfailure in the patient

Whereas it is obvious that in vitro susceptibility assays are far from perfectand fall far short of mimicking the in vivo environment the data obtained fromthem can be useful if their limitations are recognized and more attention is di-rected toward their accurate detection of antibacterial resistance The salient as-pects of currently used quantitative semi quantitative and qualitative assays willbe reviewed

342 Quantitative Assays

The four quantitative assays used in research and clinical laboratories are themacrobroth dilution assay microbroth dilution assay agar dilution assay and E-test These assays provide MIC data that can be directly compared to achievabledrug concentrations in the patient especially at the site of infection Thereforequantitative assays provide clinicians with the type of information that allowsthem to optimize therapeutic strategies to not only help the patient but also slowthe emergence of resistance

Macrobroth Dilution The macrobroth dilution assay is the most conser-vative of the quantitative assays and is considered the gold standard against whichall susceptibility tests are evaluated One advantage of macrobroth dilution assays

342 Lister

is that they allow evaluation of bacterial killing as well as evaluation of the MICHowever because of the laboriousness of this method its use in clinical labora-tories is not practical To perform a macrobroth dilution assay twofold serialdilutions of antibiotic are prepared in 1ndash3 mL of broth media and each tube isinoculated with 5 105 colony-forming units (CFU) per milliliter of the testbacterial culture [87] Therefore the total number of bacteria inoculated into eachtube ranges from 5 105 to 1 106 CFU Although the macrobroth dilutionassay is the most reliable for detecting resistance among clinical isolates thestarting inoculum is often insufficient to evaluate the presence of mutant subpopu-lations that could cause therapeutic failures

Microbroth Dilution Because of the laboriousness of macrobroth dilutionassays a microbroth dilution version was developed This assay is performed insmall plastic trays with individual wells containing 50ndash100 microL of media andtwofold serial dilutions of antibiotic Although microdilution assays also use start-ing inocula of approximately 105 CFUmL the total number of bacteria inocu-lated into each well is only 104 CFU Considering the established relationshipbetween inoculum size and susceptibility it is not surprising that bacteria appearto be more susceptible in microdilution assays than in macrodilution assays Fur-thermore with an even lower starting inoculum microdilution assays are evenless likely to detect resistant subpopulations that could cause therapeutic failure

Agar Dilution The basic principle of the agar dilution assay is similarto that of broth dilution methods with the exception that drug is incorporatedinto an agar medium and bacteria are inoculated on top of the agar One majorlimitation of agar dilution assays is that they can only evaluate the inhibition ofvisible growth and cannot distinguish bacteriostasis from bacterial killing Likemicrobroth dilution assays agar dilution assays are hindered by a starting inocu-lum of only 104 CFU which make bacteria appear more susceptible than withmacrobroth methods [88ndash90] Furthermore the detection of resistant subpopula-tions is even less likely with agar dilution assays than with either of the brothdilution methods The reason for this is that one resistant cell will grow into onlya single colony on an agar plate and this single mutant colony would likely beignored In contrast one mutant cell in a broth culture would eventually growto turbidity and would not likely be ignored

E-test The E-test is a quantitative assay that represents a marriage be-tween agar dilution and the disk diffusion assay discussed in Section 345 Inthis test a standard inoculum of bacteria (05 McFarland turbidity) is used tocreate a confluent lawn of growth on an agar plate Onto this lawn of bacteriaare placed plastic-coated strips impregnated with a continuous and exponentialgradient of antibiotic concentrations As antibiotic diffuses from the E-test stripinto the agar medium it interacts with the lawn culture of bacteria In either the


absence of drug or presence of subinhibitory drug concentrations confluentgrowth is observed However near the E-test strips an elliptical zone of inhibitionis observed reflecting the increasing concentrations of antibiotic along the stripThe MIC is obtained from the point of intersection between the ellipse of growthinhibition and the antibiotic gradient on the E-test strip Unlike in the other dilu-tion-based methods the E-test inoculum is not measured by the number of viablebacteria per milliliter or spot However the density of the inoculum still playsa critical role in the data obtained If the inoculum is too light an unwantedadvantage is provided to the drug diffusing from the E-test strip thus resultingin larger zones of inhibition and lower MICs Even with the strictest of standardprocedures bacteria appear more susceptible with E-test methodology than withbroth dilution assays

Problems Associated with Interpretive Breakpoints Once an MIC is ob-tained from a quantitative assay the next step is to determine the susceptibleintermediate or resistant phenotype of the bacterium These phenotypes are deter-mined by comparing MICs to established interpretive breakpoints When drugpharmacokinetics are considered during the establishment of interpretive criteriabreakpoints are based on the serum pharmacokinetics of the drug There are twocritical flaws with this approach The first problem is that some are known toconcentrate to much higher levels in tissues and urine than their peak concentra-tions in serum Therefore establishing susceptible breakpoints based on the se-rum pharmacokinetics of a drug that concentrates outside the bloodstream mayincorrectly classify bacteria as nonsusceptible when in fact therapeutic levelswould be achieved at the site of infection The second problem is just the converseof the first in that drug access to the central nervous system and other restrictedareas is often limited and only a fraction of serum antibiotic is able to penetrateTherefore establishing breakpoints based on serum pharmacokinetics may actu-ally overestimate the activity of some drugs for treatment of infections in inacces-sible sites This is best illustrated by the problems encountered with establishinginterpretive breakpoints for cephalosporins in the treatment of pneumococcalmeningitis As susceptibility to cephalosporins began to decrease with increasingproblems of penicillin resistance clinical failures with cephalosporins in the treat-ment of meningitis were observed despite in vitro susceptibility based on theestablished interpretive breakpoints Therefore it was apparent that the originalinterpretive breakpoints for cephalosporins did not relate to pneumococcal infec-tions within the central nervous system and re-evaluation was necessary

Even when interpretive breakpoints do correlate with the pharmacokineticsof a drug problems with detection of resistance can occur The best example toillustrate this problem is the dilemma of detecting ESBL-mediated resistanceamong E coli and K pneumoniae As previously discussed the MICs of ceftazi-dime and cefotaxime against ESBL-producing E coli and K pneumoniae are

344 Lister

often in the susceptible or intermediate range [91] Therefore current interpretivecriteria do not reliably identify ESBL-producing bacteria as resistant and theresults can be detrimental to the infected patient To address this problem newassays and screening criteria are being developed to increase the detection ofESBL-mediated resistance in the clinical laboratory However similar problemswill likely occur with other antibiotics and bacterial species as mechanisms ofantimicrobial resistance continue to evolve and spread in the environment

343 Automated Susceptibility Systems

There are a number of semiautomated or fully automated systems available forsusceptibility testing in clinical laboratories Most of these involve the automatedmonitoring of bacterial growth in drug-free and drug-containing wells Data areanalyzed using algorithms that are then converted into quantitative results Toevaluate multiple drugs and drug classes on a single susceptibility card thesesystems are limited in the number of drug concentrations that can be testedTherefore susceptibility is measured with just a narrow range of drug concentra-tions around a critical lsquolsquobreakpointrsquorsquo concentration and semiquantitative data areobtained The major drawback of these systems is that they rely on the assumptionthat the breakpoint concentrations selected have clinical relevance Thereforeany errors in selecting the appropriate lsquolsquobreakpointrsquorsquo concentrations can providepotentially harmful susceptibility data for patients Another disadvantage ofbreakpoint assays is they have no way of detecting declining susceptibility in theenvironment Only when susceptibility crosses the critical breakpoint is resistancedetected This creates a problem for the detection of those resistance mechanismsthat fail to increase MICs above the susceptible breakpoint ie ESBLs Onefinal problem associated with automated susceptibility systems is that they aredesigned to provide rapid results within hours rather than the usual overnightincubations associated with routine dilution assays This rapid approach has beenshown to be unreliable in detecting resistance to some antibiotics and researchcontinues to solve these problems

344 Resistance Screening Assays

Similar to the automated susceptibility systems resistance screening assays aresemiquantitative assays that evaluate growth or lack of growth at a single drugconcentration In contrast to automated systems however the concentrations cho-sen for these assays are not lsquolsquobreakpointrsquorsquo concentrations but rather concentra-tions that allow for differentiation between resistance mechanisms intrinsic andhigh-level resistance and truly susceptible and falsely susceptible populationsA summary of the screening assays for mecA-mediated methicillin resistanceamong staphylococci vancomycin and high-level aminoglycoside resistanceamong enterococci and ESBL-mediated resistance among E coli K pneumon-iae and K oxytoca is provided in Table 5


TA

BLE

5S

cree

nin

gA

ssay

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Det

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Dif

fere

nti

ate

Mec

han

ism

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fA

nti

mic

rob

ial

Res

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nce

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teri

alsp

ecie

sR

esis

tan

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ech

anis

mM

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od

olo

gy

Sta

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ylo

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us

spp

β-

Lact

amre

sist

ance

du

eto

low

Ino

cula

te10

4C

FUs

po

to

nto

surf

ace

of

agar

sup

ple

-af

fin

ity

PB

P2prime

fro

mm

ecA

men

ted

wit

h4

sod

ium

chlo

rid

ean

d6

microgm

Lo

fo

xaci

llin

A

ny

gro

wth

afte

r24

ho

fin

cub

atio

nat

37o C

isin

dic

ativ

eo

fm

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-med

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dre

sist

ance

toal

lβ-

lact

ams

En

tero

cocc

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faec

ium

V

anco

myc

inre

sist

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du

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Ino

cula

te10

4C

FUs

po

to

nto

surf

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of

agar

sup

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-E

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sfa

ecal

isV

anB

or

Van

Cm

ente

dw

ith

6microg

mL

of

van

com

ycin

A

ny

gro

wth

afte

r24

ho

fin

cub

atio

nat

37degC

isin

dic

a-ti

veo

fva

nco

myc

inre

sist

ance

H

igh

-lev

elam

ino

gly

cosi

de

re-

Ag

arsc

reen

In

ocu

late

106

CFU

sp

ot

on

tosu

rfac

esi

stan

ceo

fag

arsu

pp

lem

ente

dw

ith

500

microgm

Lo

fg

enta

-m

icin

or

2000

microgm

Lo

fst

rep

tom

ycin

A

ny

gro

wth

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r24

ndash48

ho

fin

cub

atio

nat

37degC

isin

-d

icat

ive

of

hig

h-l

evel

amin

og

lyco

sid

ere

sist

ance

M

icro

dilu

tio

nb

roth

scre

en

Ino

cula

te5

10

5C

FU

mL

into

bro

thco

nta

inin

g50

0microg

mL

of

gen

tam

i-ci

no

r10

00microg

mL

of

stre

pto

myc

in

Pre

sen

ceo

fvi

sib

leg

row

thaf

ter

24h

of

incu

bat

ion

isin

dic

a-ti

veo

fh

igh

-lev

elam

ino

gly

cosi

de

resi

stan

ce

Esc

her

ich

iaco

liE

xten

ded

-sp

ectr

um

β-la

cta-

Ino

cula

te5

10

5C

FUm

Lin

tob

roth

con

tain

ing

1K

leb

siel

lap

neu

mo

nia

em

ases

microgm

Lo

fce

fpo

do

xim

ece

ftaz

idim

ece

fota

xim

eK

leb

siel

lao

xyto

cace

ftri

axo

ne

or

aztr

eon

am

Pre

sen

ceo

fvi

sib

leg

row

thaf

ter

24h

of

incu

bat

ion

issu

gg

esti

veo

fE

SB

Lp

rod

uct

ion

C

on

firm

ato

ryas

say

req

uir

ed

346 Lister

MRSA Screen The therapeutic importance of differentiating between dif-ferent mechanisms of methicillin resistance in staphylococci has been discussedBecause hyperproduction of β-lactamase or alterations in PBPs unrelated to mecAresult in borderline resistance strains expressing these mechanisms of resistanceare typically inhibited by concentrations of oxacillin that do not inhibit the growthof MRSA expressing PBP2prime An agar-based oxacillin screening assay has beendescribed that differentiates mecA-mediated resistance from other mechanismsof oxacillinmethicillin resistance [87] In this assay agar supplemented with 4sodium chloride and 6 microgmL of oxacillin is inoculated with 104 CFU of bacteriaas in agar dilution methodology Growth on this agar screen indicates that theisolate is expressing mecA Although this screening assay is not 100 accurateit does allow for the differentiation of the majority of MRSA and can be usedto guide appropriate therapy

High-Level Aminoglycoside Resistance Screen Enterococci are intrinsi-cally resistant to aminoglycosides with MICs in the range of 8ndash256 microgmL [9]Although intrinsic resistance prevents aminoglycosides from being used as singleagents for serious enterococcal infections it does not interfere with their syner-gistic interactions with cell-wall-active agents In contrast the acquisition ofhigh-level resistance is characterized by much higher MICs and does interferewith synergistic killing Broth- and agar-based screening assays have been devel-oped to detect high-level aminoglycoside resistance among enterococcal isolates[87] Due to cross-resistance the only two aminoglycosides that need to be testedare gentamicin and streptomycin In the agar screening methodology 500 microgmL of gentamicin or 2000 microgmL of streptomycin is incorporated into agar me-dium and 106 CFUspot of enterococcal isolates is inoculated onto the agar sur-face much as in agar dilution methodology After 24ndash48 h of incubation thepresence of more than one colony or hazy growth indicates high-level resistanceto aminoglycosides For broth screening purposes a microdilution theme is usedand high-level aminoglycoside resistance is detected by the visible growth of a5 105 CFUmL inoculum in the presence of 500 microgmL of gentamicin or 1000microgmL of streptomycin

Vancomycin Resistance Screen Because vancomycin resistance can below level with strains expressing the VanB and VanC phenotypes routine suscep-tibility methods may fail to detect these resistance mechanisms [9293] There-fore an agar screening method is used that evaluates the growth of enterococcalisolates on agar containing 6 microgmL of vancomycin [8793] After 24 h of incuba-tion the presence of more than one colony indicates resistance to vancomycin

ESBL Screen Although MICs of extended-spectrum cephalosporins andaztreonam against ESBL-producing bacteria do not always exceed susceptiblebreakpoints they are elevated compared to those of strains lacking ESBLs TheNCCLS recommends a broth-based screening assay for E coli K pneumoniae


and K oxytoca that evaluates the growth of these three species in the presenceof 1 microgmL of cefpodoxime ceftazidime cefotaxime ceftriaxone or aztreonam[94] Visible growth after 16ndash20 h of incubation is suggestive of ESBL produc-tion and this phenotype must then be confirmed Confirmation of ESBL produc-tion involves the measurement ceftazidime-clavulanate and cefotaxime-clavula-nate MICs and comparison with the MICs obtained with each cephalosporinalone A threefold or greater difference confirms the production of an ESBLSimilar assays are being developed for some automated systems and the E-testassay

345 Qualitative Assays

Qualitative assays provide simple lsquolsquoyesrsquorsquo or lsquolsquonorsquorsquo susceptibility informationthrough establishment of susceptible intermediate or resistant phenotypes How-ever quantitative information on the level of susceptibility is not provided

Disk Diffusion Assay The disk diffusion assay is well standardized re-producible and inexpensive and is relatively easy to perform Because of thesequalities the disk diffusion is the most common susceptibility assay used in clini-cal laboratories Although similar in methodology to the E-test disk diffusionassays use paper disks impregnated with a single drug concentration rather thanthe range of antibiotic concentrations associated with E-test strips This differenceresults in circular zones of growth inhibition the size of which ultimately deter-mines the susceptibility phenotype of the bacterium Although the susceptibilityof a bacterium to an antibiotic influences the size of the inhibition zone theinoculum incubation temperature incubation time and media consistency anddepth are all important factors that influence the size of growth inhibition zones

The NCCLS establishes the interpretive breakpoint criteria from which thesizes of growth inhibition zones are translated into susceptible intermediate andresistant phenotypes These breakpoints are established through error-rate-bounded analysis of disk diffusion and MIC data for hundreds of bacterial iso-lates The ultimate goal of any disk diffusion assay is to achieve 100 correlationbetween the phenotypes determined by lsquolsquogold standardrsquorsquo MIC assays and thoseobtained with the disk diffusion assay Because 100 correlation is not oftenattainable it becomes important to limit or prevent any false susceptibility errorswith the disk diffusion assay False susceptibility errors occur when a bacteriumappears susceptible by disk diffusion assay but is found to be truly resistant byMIC methods This type of error has also been referred to as a lsquolsquofatal errorrsquorsquobecause of the life-threatening problems it can present to patients with seriousinfections What level of false susceptibility error rates is acceptable with thedisk diffusion assay has been a controversial subject for many years

Special Disk Diffusion Assays for Detection of ESBLs Similar to prob-lems encountered with dilution-based quantitative assays the disk diffusion assay

348 Lister

TA

BLE

6M

od

ified

Dis

kD

iffu

sio

nA

ssay

sfo

rC

on

firm

atio

no

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SB

L-M

edia

ted

Res

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nce

Det

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on

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eth

od

olo

gy

Inte

rpre

tati

on

Th

ree-

dim

ensi

on

alT

he

test

bac

teri

alis

ola

teis

ino

cula

ted

on

top

of

anIf

the

bac

teri

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ola

tep

rod

uce

san

ES

BL

the

en-

dis

kd

iffu

sio

nag

arp

late

sim

ilar

tost

and

ard

dis

kd

iffu

sio

nzy

me

will

inac

tiva

ted

rug

dif

fusi

ng

fro

mth

eco

m-

assa

ym

eth

od

olo

gy

Inad

dit

ion

aco

nce

ntr

ated

susp

en-

mer

cial

dis

ksth

rou

gh

the

lsquolsquowal

lrsquorsquoo

fb

acte

ria

insi

on

of

the

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ltu

reis

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ted

into

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it

Th

isin

tera

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nw

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sult

ina

tru

n-

slit

pre

par

edin

the

agar

wit

ha

scal

pel

C

om

mer

-ca

ted

zon

eo

fg

row

thin

hib

itio

n

cial

dis

kso

fce

fota

xim

ece

ftaz

idim

ece

ftri

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is unreliable in detecting ESBL-mediated resistance Up to 75 of ESBL-produc-ing strains appear susceptible to cefotaxime and 43 appear susceptible to ceftaz-idime according to disk diffusion methodology [3195] Three modifications ofthe disk diffusion assay have been shown to provide improved detection ofESBLs in clinical isolates (see Table 6)

The three-dimensional assay [32] is performed much like the standard diskdiffusion assay The one exception is that the bacterial inoculum is introducedinto a slit in the agar as well as on top of the agar Commercial disks containingextended-spectrum cephalosporins or aztreonam are placed in close proximity tothe slit in the agar If the inoculated organism produces an ESBL drug is inacti-vated before it can diffuse through the concentrated culture in the agar slit andtruncated zones of inhibition are observed The three-dimensional assay reliablydetects 90 of ESBL-mediated resistance but is currently not suited for routinelaboratory use

The double-disk diffusion assay [31] is based upon the sensitivity of ESBLsto inhibition by clavulanic acid In this assay disks containing extended-spectrumcephalosporins or aztreonam are placed proximate to a disk containing clavulanicacid The inhibition of ESBLs by clavulanic acid results in an enhancement ofthe inhibition zone around commercial ceftazidime cefotaxime ceftriaxone oraztreonam disks Although this assay substantially improves the detection ofESBLs the optimum distance between disks required for detection varies sub-stantially from strain to strain Therefore multiple assays must be run with vary-ing distances between the clavulanate- and drug-containing disks Of even moreconcern are the reports of ESBLs that also exhibit resistance to clavulanate inacti-vation The increased prevalence of these enzymes threatens the future usefulnessof the double-disk assay and any other ESBL detection assays that require clavu-lanate inhibition of the enzymes

The NCCLS has approved a set of disk diffusion screening assays to detectESBLs in E coli K pneumoniae and K oxytoca [94] For initial screeningzones of inhibition around cefpodoxime ceftazidime cefotaxime ceftriaxoneor aztreonam disks are evaluated for diameters that fall below ESBL-indicativebreakpoints ESBL production is then confirmed by a special assay in which 10microg of clavulanate is added to commercial disks of ceftazidime and cefotaximeand a 5 mm increase in the zone of inhibition around the cephalosporin-clavula-nate disks compared to the cephalosporins alone confirms ESBL production

4 PHARMACODYNAMICS AND ANTIMICROBIAL

RESISTANCE

The field of antimicrobial pharmacodynamics strives to establish relationshipsbetween the pharmacokinetics of a drug its interaction with target bacteria andthe effective treatment of infections As previously discussed the emergence of

350 Lister

resistance can result in therapeutic failure when the original isolate is susceptibleby routine assays Therefore the relationship between antimicrobial pharmacody-namics and suppression of resistant subpopulations is also critical for optimizingtherapy of many infections

Resistance to antimicrobial agents can develop through two distinct path-ways (1) acquisition of new genes encoding resistance-mediating proteins and(2) evolutionary mutations in existing genes The impact of optimizing antimicro-bial pharmacodynamics on each of these pathways is very different Althoughthe optimization of antimicrobial pharmacodynamics can slow the evolution ofmost mutation-mediated resistance mechanisms there are many acquired resis-tance mechanisms that cannot be treated with safe doses of antibiotic Thereforethe goal of pharmacodynamics should be to optimize therapy such that mutationalresistance is prevented This section will differentiate acquired from mutationalresistance and discuss the pharmacodynamic parameters that can impact theemergence of mutational resistance during therapy

41 Acquired Versus Mutational Resistance

411 Acquired Resistance

Acquired resistance as the name suggests represents the development of resis-tance in a bacterium through the acquisition of a new gene or set of genes fromother bacteria in the environment Examples of acquired resistance mechanismsinclude plasmid-encoded β-lactamases plasmid-encoded aminoglycoside-inacti-vating enzymes acquired vancomycin resistance and methicillin resistance me-diated through mecA The common theme with these resistance problems is thatthey are passed from bacterium to bacterium on transferable genetic elementsie plasmids or transposons Once a bacterium acquires one of these resistancemechanisms the level of resistance that is expressed usually exceeds the levelsof antibiotic that can be safely achieved in serum or other sites of infection Forexample acquisition of the TEM-1 β-lactamase by an E coli can increase theMIC of ampicillin from 2 to 256 microgmL [96] Similarly acquisition of the genesresponsible for vancomycin resistance can increase the MIC of vancomycinagainst enterococci from 2 to 512 microgmL [97] Although acquisition of new genesusually results in an instantaneous conversion to high-level resistance there areexceptions to this rule It has been well established that penicillin resistanceamong pneumococci is mediated through the acquisition of PBP gene segmentsfrom other resistant streptococci in the environment However development ofhigh-level penicillin resistance does not result from a single transfer event butrather requires multiple steps affecting multiple PBPs

412 Mutational Resistance

In contrast to acquired resistance mutational resistance is mediated through ge-netic changes (point mutations) within a bacteriumrsquos own chromosome These


evolutionary mutations can alter the binding of a drug to its target decrease theaccumulation of a drug at the site of action or disrupt the regulatory genes con-trolling the production of resistance-mediating enzymes Similar to acquired re-sistance mechanisms mutational events can result in instantaneous high-levelresistance or gradual decreases in susceptibility requiring multiple steps toachieve clinically significant resistance Whether high-level resistance is achievedwith a single point mutation or requires multiple mutational events depends uponthe specific drug and the specific target bacterium In fact differences betweendrugs within the same family can be observed This point is best illustrated usingthe example of β-lactam resistance in gram-negative bacteria as mediated throughderepression of the ampC operon

As previously discussed derepression of the ampC results from point muta-tions within a regulatory gene ampD resulting in a loss of the negative controlof ampC transcription [38] Once negative control is lost AmpC production in-creases significantly and the bacteria become resistant to virtually all β-lactamantibiotics Such mutations occur in one out of every 106ndash107 viable bacteriaTherefore one or more highly resistant mutants are likely to be present at thestart of therapy of some infections If these mutants are not eliminated by thehost or killed by the antibiotic they can eventually become the predominantpopulation and lead to therapeutic failure Clinical failure due to the emergenceof this resistance problem has been observed in 19ndash80 of patients infected withbacteria possessing inducible AmpC cephalosporinases [6] Although almost anyβ-lactam antibiotic can select for these mutants there appears to be a particularassociation with the lsquolsquothird generationrsquorsquo cephalosporin ceftazidime [2]

In contrast a single mutational event is not always sufficient to provideresistance to the lsquolsquofourth generationrsquorsquo cephalosporin cefepime especially amongthe Enterobacteriaceae In a 1994 survey of five medical centers in the UnitedStates cefepime was tested against 256 ceftazidime-resistant clinical isolates[98] Among the Enterobacter in this study almost all species were 100 suscep-tible to cefepime The only exception was E cloacae of which 94 of the ceftaz-idime-resistant isolates remained susceptible to cefepime In a separate study100 of Enterobacter strains with proven ampC derepression were susceptibleto cefepime [99] The activity of cefepime against ceftazidime-resistant Entero-bacter spp can be explained by the differing number of mutational steps requiredto achieve resistance with each of these drugs In contrast to the single mutationalevent required for ceftazidime resistance it takes two independent mutationalevents to achieve cefepime resistance among Enterobacter spp [100] First thelevel of AmpC production must be increased through derepression of the ampCoperon and then the penetration of cefepime through the outer membrane mustbe slowed or prevented through a second mutational event Since each of thesemutational steps occurs in one out of every 106ndash107 viable bacteria it is unlikelythat resistance will develop in a ceftazidime-susceptible enterobacter during thecourse of therapy with cefepime This hypothesis is supported by data from a

352 Lister

murine infection model [101] However once an enterobacter mutates to a stateof ampC derepression and bacterial numbers increase above 106 the chances ofselecting the second permeability mutation increases and emergence of cefepimeresistance becomes a real threat

Differences between antibiotics in the number of mutational steps requiredto achieve clinically relevant resistance is not unique to the β-lactam class assimilar observations have been reported with other drug classes as well particu-larly the fluoroquinolones [102] Although appropriate dosing of antibiotics doesnot often impact problems associated with acquired resistance pharmacodynam-ics can play an influential role in slowing the development of mutational resis-tance especially when first step mutations do not result in high-level resistanceTherefore the relationship between antimicrobial pharmacodynamics and theemergence of mutational resistance is essential for developing optimum therapeu-tic strategies

42 Antimicrobial Pharmacodynamics and Emergence

of Resistance

The three best characterized pharmacodynamic parameters influencing the clini-cal efficacy of antibiotics are the time antibiotic concentrations remain above theMIC (T MIC) the ratio of peak concentrations to the MIC (peakMIC) andratio of the area under the concentration curve to the MIC (AUCMIC) Whichpharmacodynamic parameter is most predictive of clinical efficacy depends uponthe class of drugs being used For example with β-lactam antibiotics the T MIC is the pharmacodynamic parameter that most influences clinical outcomewhereas with fluoroquinolones the most important parameter can either be thepeakMIC ratio or the AUCMIC ratio Further complicating therapeutic strate-gies is the observation that the pharmacodynamic parameter that most influencesoverall efficacy may not be the most important parameter impacting the emer-gence of resistance during therapy With the data accumulated to date it appearsthat the peakMIC ratio is the most critical pharmacodynamic parameter dictatingwhether resistance will emerge during therapy with virtually all drug classes

421 PeakMIC Ratio and Emergence of ResistanceDuring Therapy

The likelihood of a bacterium undergoing two mutational events during thecourse of therapy is very small Therefore to prevent the emergence of resistanceduring therapy the pharmacodynamic focus must be on effectively treating boththe original isolate and any first-step mutants present within the initial bacterialpopulation This demands that the concentration of antibiotic at the site of infec-tion exceed the MIC for the first-step mutant Since neither T MIC nor AUCMIC directly addresses this need the peakMIC ratio becomes the pharmacody-


namic parameter that can most impact selection of resistance Indirect evidenceto support this hypothesis has come from studies in an animal model of infection[103] Using a neutropenic rat model of Pseudomonas sepsis Drusano and col-leagues evaluated the effects of dose fractionation of lomefloxacin on survivalAlthough the emergence of resistance was not directly evaluated in this studythe peakMIC ratio was most closely linked to survival when the ratios exceeded101 Their hypothesis to explain these results was that sufficient peakMIC ratiosresulted in suppression of mutant subpopulations thus preventing death due tothe emergence of resistance Similar conclusions were reached in a study of lev-ofloxacin in the treatment of human infections [104]

The best evidence supporting the importance of peakMIC ratios in pre-venting the emergence of resistance comes from studies with in vitro pharmacoki-netic models The flexibility provided by these models allows for the design ofexperiments to specifically address this issue Using a two-compartment pharma-cokinetic model Blaser et al [105] varied the pharmacokinetics of enoxacin andnetilmicin such that bacteria were exposed to varying peakMIC ratios whilemaintaining a constant total drug exposure over 24 h These investigators ob-served the outgrowth of resistant subpopulations when simulated peakMIC ratiosfailed to exceed 81 Similarly Marchbanks et al [106] used a two-compartmentpharmacokinetic model to simulate ciprofloxacin doses of 400 mg TID (peakMIC ratio 41) and 600 mg BID (peakMIC ratio 61) and observed out-growth of resistant subpopulations of P aeruginosa When the same daily doseof 1200 mg was simulated as a single injection the 111 peakMIC ratio pre-vented the outgrowth of resistant subpopulations However in the same studypeakMIC ratios of only 61 were sufficient to prevent the emergence of resistantsubpopulations of S aureus Similarly Lacy et al [107] observed differencesbetween levofloxacin and ciprofloxacin and the emergence of resistance in experi-ments with S pneumoniae Although these investigators simulated peakMICratios of 51 with both fluoroquinolones they observed the emergence of resis-tance only in studies with ciprofloxacin Resistance did not emerge in studieswith levofloxacin despite peakMIC ratios as low as 11 These data suggest thatalthough the peakMIC ratio exerts an important influence on the emergence ofresistance during therapy the minimum peakMIC ratio needed to prevent thisproblem will likely vary depending upon the drug used and the bacterium tar-geted This conclusion is supported with data from my own pharmacodynamicstudies of P aeruginosa

422 Studies with Levofloxacin Against P aeruginosa

A two-compartment pharmacokinetic model was used to simulate the serum phar-macokinetics of a once daily 500 mg dose of levofloxacin and evaluate pharmaco-dynamic interactions against a panel of six P aeruginosa strains Three of thestrains evaluated were fully susceptible to levofloxacin (MIC 05 microgmL) and

354 Lister

three strains were borderline susceptible (MIC 2 microgmL) The peak concentra-tion simulated for the 500 mg dose was 6 microgmL which provided peakMICratios of 131 for the fully susceptible strains and 31 for the borderline-suscepti-ble strains Levofloxacin was dosed into the model at 0 and 24 h The pharmaco-dynamics of levofloxacin against representative susceptible and borderline-sus-ceptible strains of P aeruginosa are shown in Fig 1 In studies with the fullysusceptible strains levofloxacin exhibited rapid and significant bactericidal activ-ity with viable counts falling to undetectable levels (10 CFUmL) within 4ndash12 h after the first dose In studies with the borderline-susceptible strains lev-ofloxacin also exhibited significant bacterial killing However killing was ob-served only over the initial 6ndash8 h with viable counts increasing rapidly thereafterdue to the outgrowth of resistant subpopulations By 24 h viable counts in thelevofloxacin-treated cultures were approaching those in the drug-free control cul-tures The MICs of levofloxacin against the resistant subpopulations ranged from8 to 16 microgmL Therefore emergence of resistance could have been predictedbecause peak concentrations with each dose of levofloxacin never exceeded theMICs for these mutant subpopulations

423 Studies with Ceftazidime and Ticarcillin AgainstP aeruginosa

A two-compartment pharmacokinetic model was used to investigate the relation-ships between mutational frequencies pharmacodynamics and the emergence ofresistance during the treatment of P aeruginosa with ticarcillin and ceftazidimeMutational frequency studies were performed in agar by exposing logarithmicphase cultures of P aeruginosa 164 to ticarcillin and ceftazidime at concentra-tions two- four- and eightfold above their respective MICs The initial MICsfor ticarcillin and ceftazidime against P aeruginosa 164 were 16 microgmL and 2microgmL respectively Analysis of mutational frequencies showed there was oneresistant mutant present among every 106ndash107 parent cells with MICs of ticarcil-lin and ceftazidime against these mutants increasing to 512 microgmL and 32 microgmL respectively Using a two-compartment pharmacokinetic model the kineticsof a 30 g QID dose of ticarcillin and 20 g TID dose of ceftazidime were simu-lated and their pharmacodynamics against P aeruginosa 164 were evaluatedover 24 h With peak concentrations of 260 microgmL for ticarcillin and 140 microgmL for ceftazidime peakMIC ratios were 161 for ticarcillin and 701 for cefta-zidime The observed pharmacodynamic interactions are shown in Fig 2 In stud-ies with ceftazidime viable counts decreased over 3 logs throughout the 24 hexperimental period and no resistant mutants were detected on drug selectionplates The lack of resistance emergence was not unexpected because peak con-centrations achieved with the 20 g dose of ceftazidime were fourfold above the32 microgmL MIC of first-step mutants In contrast outgrowth of a resistant mutantwas observed in studies with ticarcillin despite simulated peakMIC ratios of


FIGURE 1 Pharmacodynamics of (A) levofloxacin against P aeruginosaGB240 and (B) P aeruginosa GB65 in an in vitro pharmacokinetic model Le-vofloxacin MICs were 05 microgmL for P aeruginosa GB240 and 20 microgmL forP aeruginosa GB65 Levofloxacin was dosed at 0 and 24 h and the pharmaco-kinetics of a 500 mg dose were simulated Each datum point represents themean number of viable bacteria per milliliter of culture for duplicate experi-ments Error bars show standard deviations

356 Lister

FIGURE 1 Continued


FIGURE 2 Pharmacodynamics of ticarcillin and ceftazidime against P aerugi-nosa 164 in an in vitro pharmacokinetic model Ticarcillin and ceftazidimeMICs were 16 microgmL and 20 microgmL respectively Ticarcillin was dosed at 06 12 and 18 h and the pharmacokinetics of a 30 g dose were simulatedover each 6 h dose interval Ceftazidime was dosed at 0 8 and 16 h and thepharmacokinetics of a 20 g dose were simulated over each 8 h dose intervalEach datum point represents the mean number of viable bacteria per milliliterof culture for duplicate experiments Error bars show standard deviations

358 Lister

161 against P aeruginosa 164 The outgrowth of these mutant subpopulationscould have been predicted because peak concentrations of ticarcillin never ex-ceeded the MIC against the resistant subpopulation

These pharmacodynamic data and data from other investigators highlightthe importance of the peakMIC ratio in dictating whether emergence of resis-tance will complicate therapy Unfortunately these studies also demonstrate thatthe optimum peakMIC ratio needed to prevent the emergence of resistance variesbetween different drug classes and with different bacteriandashdrug combinationsFurthermore these studies suggest that optimization of therapy cannot just bedirected toward the lsquolsquoparentrsquorsquo isolate Rather optimization of therapy to preventemergence of resistance during therapy requires a greater understanding of thequantitative influence that different resistance mechanisms exert on susceptibilityIn other words the relationship between peak concentrations and the MIC againstfirst-step mutants is more important in preventing the emergence of resistancethan targeting peakMIC ratios based on MICs against the original isolate

5 CONCLUDING REMARKS

Antibacterial resistance is an inevitable consequence of the overuse of antibioticsin the environment The emergence of multidrug resistance among prominentgram-positive and gram-negative pathogens presents serious therapeutic prob-lems not only for clinicians but also for the clinical microbiologists who haveto reliably detect new resistance mechanisms in the laboratory Where assays failto detect resistance new assays are being developed and provide some hope forthe future However the reliable detection of resistance gains nothing for theclinician faced with dwindling therapeutic options How then do we curve theevolution of antibiotic resistance among bacteria and once again take control ofthe ongoing resistance battle

As the number of stronger or truly novel antimicrobial agents achievingFDA approval declines scientists must continue to develop strategies to slow theemergence of resistance in the environment One approach is to control the useof antibiotics and to rotate antibiotics before resistance becomes a problem Thistheory is referred to as antibiotic cycling [108] A further step that can be takenis to pharmacodynamically optimize antimicrobial therapy such that the evolutionof mutational resistance is slowed This requires dosing antibiotics such that peakconcentrations at the site of infection exceed the MICs of the parent and anyresistant subpopulations that might be present Through the combination of judi-cious antimicrobial use and pharmacodynamically based optimization of dosingthe evolution of antibiotic resistance can be slowed and the lsquolsquopostantibiotic erarsquorsquoneed not become a reality


REFERENCES

1 SD Holmberg SL Solomon PA Blake Health and economic impacts of antimicro-bial resistance Rev Infect Dis 198791065ndash1078

2 JW Chow MJ Fine DM Shlaes JP Quinn DC Hooper MP Johnson R RamphalMM Wagener DK Miyashiro VL Yu Enterobacter bacteremia Clinical featuresand emergence of antibiotic resistance during therapy Ann Int Med 1991115585ndash590

3 CE Phelps Bugdrug resistance Sometimes less is more Med Care 198927194ndash203

4 JE McGowan Antimicrobial resistance in hospital organisms and its relations toantibiotic use Rev Infect Dis 198351033ndash1048

5 DCE Speller The clinical impact of antibiotic resistance J Antimicrob Chemother198822583ndash586

6 CC Sanders WE Sanders Jr β-lactam resistance in gram-negative bacteria Globaltrends and clinical impact Clin Infect Dis 199215824ndash839

7 KS Thomson AM Prevan CC Sanders Novel plasmid-mediated β-lactamases inEnterobacteriaceae Emerging problems for new β-lactam antibiotics In JS Rem-ington MN Swartz eds Current Clinical Topics in Infectious Diseases CambridgeMA Blackwell Science 1996151ndash163

8 J Robert C Moellering Enterococcus species Streptococcus bovis and Leuconos-toc species In GL Mandell JE Bennett R Dolin eds Principles and Practice ofInfectious Diseases 4th ed New York Churchill Livingstone 19951826ndash1835

9 GM Eliopoulos Antibiotic resistance in Enterococcus species An update In JSRemington ed Current Clinical Topics in Infectious Diseases Cambridge MABlackwell Science 199621ndash51

10 WW Spink V Ferris Quantitative action of penicillin inhibitor from penicillin-resistant strains of staphylococci Science 1945102221

11 RN Jones EN Kehrberg ME Erwin Prevalence of important pathogens and antimi-crobial activity of parenteral drugs at numerous medical centers in the UnitedStates I Study on the threat of emerging resistances Real or perceived DiagnMicrobiol Infect Dis 199419203ndash215

12 M Barber Methicillin-resistant staphylococci J Clin Pathol 19611438513 BM Hartman A Tomasz Low-affinity penicillin binding protein associated with

beta-lactam resistance in Staphylococcus aureus J Bacteriol 198415851314 RN Jones AL Barry RV Gardiner The prevalence of staphylococcal resistance

to penicillinase-resistant penicillins A retrospective and prospective trial of isolatesfrom 40 medical centers Diagn Microbiol Infect Dis 198912383ndash394

15 K Kiramatsu H Hanaki T Ino K Yabuta T Oguri FC Tenover Methicillin-resis-tant Staphylococcus aureus clinical strain with reduced vancomycin susceptibilityJ Antimicrob Chemother 199740135ndash136

16 CDC Staphylococcus aureus with reduced susceptibility to vancomycinndashUnitedStates 1997 MMWR 199746765ndash766

17 JW Kislak LMB Razavi AK Daly M Finland Susceptibility of pneumococci tonine antibiotics Am J Med Sci 1965250262ndash268

360 Lister

18 PC Applebaum A Bhamjee JN Scragg AJ Hallett AF Bowen RC Cooper Strep-tococcus pneumoniae resistant to penicillin and chloramphenicol Lancet 1977ii995ndash997

19 S Naraqi GP Kirkpatrick S Kabins Relapsing pneumococcal meningitis Isolationof an organism with decreased susceptibility to penicillin G J Pediatr 197485671ndash673

20 VJ Howes RG Mitchell Meningitis due to relatively penicillin-resistant pneumo-coccus Br Med J 19761996

21 JE Mortensen MR Jacobs LM Koeth Susceptibility of Streptococcus pneumoniaeand Haemophilus influenzae in 1997 Results of a hospital-based US study InAbstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemo-therapy Abstr C-19 Washington DC Am Soc Microbiol 1998

22 CG Dowson A Hutchison JA Brannigan RC George D Hansman J Linares ATomasz JM Smith BG Spratt Horizontal transfer of penicillin-binding proteingenes in penicillin-resistant clinical isolates of Streptococcus pneumoniae Proc NatAcad Sci USA 1989868842ndash8846

23 CG Dowson TJ Coffey C Kell RA Whiley Evolution of penicillin resistance inStreptococcus pneumoniae The role of Streptococcus mitis in the formation of alow affinity PBP2B in Streptococcus pneumoniae Mol Microbiol 19939635ndash643

24 PD Lister Multiply-resistant pneumococcus Therapeutic problems in the manage-ment of serious infections Eur J Clin Microbiol Infect Dis 199514(1)18ndash25

25 AL Smith Antibiotic resistance in Haemophilus influenzae In EM Ayoub GHCassell WC Branche TJ Henry eds Microbial Determinants of Virulence andHost Response Washington DC Am Soc Microbiol 1990321ndash343

26 JH Jorgensen GV Doern C Thornsberry Susceptibility of multiply-resistantHaemophilus influenzae to newer antimicrobial agents Diagn Microbiol Infect Dis1988927ndash32

27 CC Sanders JP Iaconis GP Bodey G Samonis Resistance to ticarcillin-potassiumclavulanate among clinical isolates of the family Enterobacteriaceae Role of PSE-1 β-lactamase and high levels of TEM-1 and SHV-1 and problems with false sus-ceptibility in disk diffusion tests Antimicrob Agents Chemother 1988321365ndash1369

28 KS Thomson DA Weber CC Sanders J Eugene W Sanders β-Lactamase produc-tion in members of the family Enterobacteriaceae and resistance of β-lactamndashenzyme inhibitor combinations Antimicrob Agents Chemother 199034622ndash627

29 CJ Thomson SGB Amyes TRC-1 Emergence of a clavulanic acid-resistant TEMβ-lactamase in a clinical strain FEMS Microbiol Lett 199291113ndash118

30 J Blazquez M Baquero R Canton Characterization of a new TEM-type β-lacta-mase resistant to clavulanate sulbactam and tazobactam in a clinical isolate ofEscherichia coli Antimicrob Agents Chemother 1993372059ndash2063

31 V Jarlier M-H Nicolas G Fournier A Philippon Extended broad-spectrum β-lacta-mases conferring transferable resistance to newer β-lactam agents in Enterobacteri-aceae Hospital prevalence and susceptibility patterns Rev Infect Dis 198810867ndash878


32 KS Thomson CC Sanders Detection of extended-spectrum β-lactamases in mem-bers of the family Enterobacteriaceae Comparison of the double-disk and three-dimensional tests Antimicrob Agents Chemother 1992361877ndash1882

33 A Philippon R Labia GA Jacoby Extended-spectrum β-lactamases AntimicrobAgents Chemother 1989331131ndash1136

34 GA Jacoby Genetics of extended-spectrum β-lactamases Eur J Clin MicrobiolInfect Dis 1994(suppl 1)132ndash11

35 PA Bradford C Urban N Mariano SJ Projan JJ Rahal K Bush Imipenem resis-tance in Klebsiella pneumoniae is associated with the combination of ACT-1 aplasmid-mediated AmpC β-lactamase and the loss of an outer membrane proteinAntimicrob Agents Chemother 199741563ndash569

36 GA Jacoby AA Medeiros More extended-spectrum β-lactamases AntimicrobAgents Chemother 1991351697ndash1704

37 SK DuBois MS Marriott SGB Amyes TEM- and SHV-derived extended-spec-trum β-lactamases Relationship between selection structure and function J Anti-microb Chemother 1995357ndash22

38 CC Sanders β-Lactamases of gram-negative bacteria New challenges for newdrugs Clin Infect Dis 1992141089ndash1099

39 CC Sanders Chromosomal cephalosporinases reponsible for multiple resistance tonewer β-lactam antibiotics Ann Rev Microbiol 198741573ndash593

40 Y Yang P Wu DM Livermore Biochemical characterization of a β-lactamasethat hydrolyzes penems and carbapenems from two Serratia marcescens isolatesAntimicrob Agents Chemother 199034755ndash758

41 E-H Lee MH Nicholas MD Kitzkis G Pialou E Collatz L Gutmann Associationof two resistance mechanisms in a clinical isolate of Enterobacter cloacae withhigh-level resistance to imipenem Antimicrob Agents Chemother 1991351093ndash1098

42 WE Sanders CC Sanders Inducible β-lactamases Clinical and epidemiologicalimplications for use of newer cephalosporins Rev Infect Dis 198810830ndash838

43 JP Quinn AE Studemeister CA DiVincenzo SA Lerner Resistance to imipenem inPseudomonas aeruginosa Clinical experience and biochemical mechanisms RevInfect Dis 198810892ndash898

44 WE Sanders CC Sanders Do in vitro antimicrobial susceptibility tests accuratelypredict therapeutic responsiveness in infected patients In V Lorian ed Signifi-cance of Medical Microbiology in the Care of Patients 2nd ed Baltimore MDWilliams and Wilkins 1982325ndash340

45 HF Chambers Methicillin-resistant staphylococci Clin Microbiol Rev 19881173ndash186

46 RM Massanari MA Pfaller DS Wakefield GT Hammons LA McNutt RF Wool-son CH Helms Implications of acquired oxacillin resistance in the managementand control of Staphylococcus aureus infections J Infect Dis 1988158702ndash709

47 S Dutka-Malen S Evers P Courvalin Detection of glycopeptide resistance geno-types and identification to the species level of clinically relevant enterococci byPCR J Clin Microbiol 19953324ndash27

362 Lister

48 N Clark RC Cooksey BC Hill JM Swenson FC Tenover Characterization ofglycopeptide-resistant enterococci from US hospitals Antimicrob Agents Chemo-ther 1993372311ndash2317

49 S Sachiko N Clark D Rimland FS Nolte FC Tenover Detection of vancomycin-resistant enterococci in fecal samples by PCR J Clin Microbiol 1997352325ndash2330

50 JAMvd Klundert JS Vliegenthart PCR detection of genes coding for aminoglyco-side modifying enzymes In DH Persing TF Smith FC Tenover TJ White edsDiagnostic Molecular Microbiology Principles and Applications Washington DCAm Soc Microbiol 1993547ndash552

51 K Ubukata S Nakagami A Nitta A Yamane S Kawakami M Sugiura A KonnoRapid detection of the mecA gene in methicillin-resistant staphylococci by enzy-matic detection of polymerase chain reaction products J Clin Microbiol 1992301728ndash1733

52 K Murakami W Minamide K Wada E Nakamura H Teraoka S Wantanabe Iden-tification of methicillin-resistant strains of staphylococci by polymerase chain reac-tion J Clin Microbiol 1991292240ndash2244

53 G Arlet A Philippon Construction by polymerase chain reaction and use of intra-genic DNA probes for three main types of transferable beta-lactamases (TEMSHV CARB) FEMS Microbiol Lett 19916619ndash25

54 RC Levesque A Medeiros GA Jacoby Molecular cloning and DNA homology ofplasmid-mediated β-lactamase genes Mol Gen Genet 1987206252ndash258

55 GI Carter KJ Towner NJ Pearson RCB Slack Use of a non-radioactive hybridiza-tion assay for direct detection of gram-negative bacteria carrying TEM beta-lacta-mase genes in infected urine J Med Microbiol 198928113ndash117

56 PL Perine PA Totten KK Holmes EH Sng AV Ratman R Widy-Wersky HNsanze E Habte-Gabr WG Westbrook Evaluation of a DNA hybridization methodfor detection of African and Asian strains of Neisseria gonorrhoeae in men withurethritis J Infect Dis 198515259ndash63

57 FC Tenover MB Huang JK Rasheed DH Persing Development of PCR assaysto detect ampicillin resistance genes in cerebrospinal fluid samples containingHaemophilus influenzae J Clin Microbiol 1994322729ndash2737

58 C Mabilat P Courvalin Development of lsquolsquooligotypingrsquorsquo for characterization andmolecular epidemiology of TEM β-lactamases in members of the family Entero-bacteriaceae Antimicrob Agents Chemother 1990342210ndash2216

59 BW Catlin Iodometric detection of Haemophilus influenzae beta-lactamase Rapidpresumptive test for ampicillin resistance Antimicrob Agents Chemother 19757265ndash270

60 J Escamilla Susceptibility of Haemophilus influenzae to ampicillin as determinedby use of a modified one-minute beta-lactamase test Antimicrob Agents Chemother19769196ndash198

61 CH OrsquoCallaghan A Morris SM Kirby AH Shingler Novel method for detectionof β-lactamase by using a chromogenic cephalosporin substrate Antimicrob AgentsChemother 19721283ndash288

62 GV Doern HH Jorgensen C Thornsberry DA Preston T Tubert JS Redding LAMaher National collaborative study of the prevalence of antimicrobial resistance


among clinical isolates of Haemophilus influenzae Antimicrob Agents Chemother198832180ndash185

63 KGH Dyke Beta-lactamases of Staphylococcus aureus In JMT Hamilton-MillerJT Smith eds Beta-Lactamases London Academic Press 1979291ndash310

64 MC Roberts CD Swenson LM Owens AL Smith Characterization of chloram-phenicol resistant Haemophilus influenzae Antimicrob Agents Chemother 198018610ndash615

65 P Azemun T Stull M Roberts AL Smith Rapid detection of chloramphenicolresistance in H influenzae Antimicrob Agents Chemother 198120168ndash170

66 HW Matthews CN Baker C Thornsberry Relationship between in vitro suscepti-bility test results for chloramphenicol and production of chloramphenicol acetyl-transferase by Haemophilus influenzae Streptococcus pneumoniae and Aerococcusspecies J Clin Microbiol 1988262387ndash2390

67 L de la Maza SIL Miller MJ Ferraro Use of commercially available rapid chlor-amphenicol acetyl-transferase test to detect resistance in Salmonella spp J ClinMicrobiol 1990281867ndash1869

68 D Amsterdam Susceptibility testing of antimicrobials in liquid media In V Lorianed Antibiotics in Laboratory Medicine Baltimore Williams and Wilkins 199153ndash105

69 D Greenwood In vitro veritas Antimicrobial susceptibility tests and their clinicalrelevance J Infect Dis 1981144380ndash385

70 CC Sanders ARTs versus ASTs Where are we going J Antimicrob Chemother199128621ndash622

71 KS Thomson JS Bakken CC Sanders Antimicrobial susceptibility testing withinthe clinic In MRW Brown P Gilbert eds Microbiological Quality Assurance AGuide Towards Relevance and Reproducibility of Inocula New York CRC Press1995

72 JH Jorgensen Antimicrobial susceptibility testing of bacteria that grow aerobicallyIn JA Washington ed Infectious Disease Clinics of North America LaboratoryDiagnosis of Infectious Diseases Philadephia WB Saunders 1993393ndash409

73 CC Sanders KS Thomson PA Bradford Problems with detection of β-lactam resis-tance among nonfastidious gram-negative bacilli Infect Dis Clin N Am 19937411ndash425

74 C Thornsberry Antimicrobial susceptibility testing General considerations In ABalows WJ Hausler KL Herrmann HD Isenberg HJ Shadomy eds Manual ofClinical Microbiology Washington DC Am Soc Microbiol 19911059ndash1064

75 RB Thomson TM File RA Burgoon Repeat antimicrobial susceptibility testingof identical isolates J Clin Microbiol 1989271108ndash1111

76 WA Craig B Suh Protein binding and the antimicrobial effects Methods for deter-mination of protein binding In V Lorian ed Antibiotics in Laboratory MedicineBaltimore Williams and Wilkins 1991367ndash402

77 R Wise AP Gillet B Cadge Influence of protein binding on the tissue levels of6 β-lactams J Infect Dis 198014277

78 M Barza H Vine L Weinstein Reversibility of protein binding of penicillins Anin vitro study employing a rapid diafiltration process Antimicrob Agents Chemo-ther 19721427ndash432

364 Lister

79 J Blaser MN Dudley D Gilbert SH Zinner Influence of medium and method onthe in vitro susceptibility of Pseudomonas aeruginosa and other bacteria to ci-profloxacin and enoxacin Antimicrob Agents Chemother 198629927ndash929

80 NX Chin HC Neu Ciprofloxacin a quinolone caraboxylic acid compound activeagainst aerobic and anaerobic bacteria Antimicrob Agents Chemother 198425319ndash326

81 JF Chantot A Bryskier Antibacterial activity of ofloxacin and other 4-quinolonederivatives In vitro and in vivo comparison J Antimicrob Chemother 198516475ndash484

82 T Kumada HC Neu In vitro activity of ofloxacin a quinolone carboxylic acidcompared to other quinolones and other antimicrobial agents J Antimicrob Chemo-ther 198516563ndash574

83 J Child J Andrews F Boswell N Brenwald R Wise The in-vitro activity of CP99219 a new naphthyridone antimicrobial agent A comparison with other fluoro-quinolone agents J Antimicrob Chemother 199535869ndash876

84 MN Dudley CH Nightingale R Quintiliani RC Tilton In vitro activity of cefo-nicid ceforanide and cefazolin against Staphylococcus aureus and Staphylococcusepidermidis and the effect of human serum J Infect Dis 1983148178

85 HF Chambers J Mills TA Drake MA Sande Failure of a once-daily regimen ofcefonicid for treatment of endocarditis due to Staphylococcus aureus Rev InfectDis 19846 (suppl 4)S870ndashS874

86 D Andes R Walker S Ebert WA Craig Increasing protein binding of cefonicidenhances its in-vivo activity in an animal infection model Abstr A81 Programand Abstracts of the 34th Interscience Conference on Antimicrobial Agents andChemotherapy Washington DC Am Soc Microbiol 1994146

87 National Committee for Clinical Laboratory Standards Methods for dilution anti-microbial susceptibility tests for bacteria that grow aerobically Approved StandardM7-A4 Villanova PA National Committee for Clinical Laboratory Standards1997

88 PA Bradford CC Sanders Use of a predictor panel for development of a new diskfor diffusion tests with cefoperazone-sulbactam Antimicrob Agents Chemother199236394ndash400

89 FH Kayser G Morenzoni F Homberger Activity of cefoperazone against ampicil-lin-resistant bacteria in agar and broth dilution tests Antimicrob Agents Chemother19822215ndash22

90 M Rylander JE Brorson J Johnsson R Norrby Comparison between agar andbroth minimum inhibitory concentrations of cefamandole cefoxitin and cefuro-xime Antimicrob Agents Chemother 197915572ndash579

91 JM Casellas M Goldberg Incidence of strains producing extended spectrum β-lactamases in Argentina Infection 198917434ndash436

92 DF Sahm L Olsen In vitro detection of enterococcal vancomycin resistance Anti-microb Agents Chemother 1990341846ndash1848

93 BM Willey BN Kreiswirth AE Simior G Williams SR Scriver A Phillips DELow Detection of vancomycin resistance in Enterococcus species J Clin Microbiol1992301621ndash1624


94 National Committee for Clinical Laboratory Standards Performance Standards forAntimicrobial Susceptibility Testing Ninth Informational Supplement M100-S9Wayne PA National Committee for Clinical Laboratory Standards 1999

95 A Philippon SB Redjeb G Fournier AB Hassen Epidemiology of extended spec-trum β-lactamases Infection 199517347ndash354

96 PA Bradford CC Sanders Development of test panel of β-lactamases expressedin a common Escherichia coli host background for evaluation of new β-lactamantibiotics Antimicrob Agents Chemother 199539308ndash313

97 DM Schlaes A Bouvet C Devine JH Schlaes S Al-Obeid R Williamson Induc-ible transferable resistance to vancomycin in Enterococcus faecalis A256 Antimi-crob Agents Chemother 198933198ndash203

98 RN Jones SA Marshall Antimicrobial activity of cefepime tested against Bushgroup 1 β-lactamase-producing strains resistant to ceftazidime A multi-laboratorynational and international clinical isolate study Diagn Microbiol Infect Dis 19941933ndash38

99 AF Ehrhardt CC Sanders β-lactam resistance amongst Enterobacter species JAntimicrob Chemother 199332(suppl B)1ndash11

100 CC Sanders Cefepime The next generation Clin Infect Dis 199317369ndash379101 B Marchou M Michea-Hamzehpour C Lucain J-C Pechere Development of β-

lactam-resistant Enterobacter cloacae in mice J Infect Dis 1987156369ndash373102 KS Thomson CC Sanders Dissociated resistance among fluoroquinolones Antimi-

crob Agents Chemother 1994382095ndash2100103 GL Drusano DE Johnson M Rosen HC Standiford Pharmacodynamics of a flu-

oroquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sep-sis Antimicrob Agents Chemother 199337483ndash490

104 SL Preston GL Drusano AL Berman CL Fowler AT Chow B Dornsief V ReichlJ Natarajan M Corrado Pharmacodynamics of levofloxacin A new paradigm forearly clinical trials JAMA 1998279125ndash129

105 J Blaser BB Stone MC Groner SH Zinner Comparative study with enoxacin andnetilmicin in a pharmacodynamic model to determine importance of ratio of antibi-otic peak concentration to MIC for bactericidal activity and emergence of resis-tance Antimicrob Agents Chemother 1987311054ndash1060

106 CR Marchibanks JR McKiel DH Gilbert NJ Robillard B Painter SH ZinnerMN Dudley Dose ranging and fractionation of intravenous ciprofloxacin againstPseudomonas aeruginosa and Staphylococcus aureus in an in vitro pharmacoki-netic model Antimicrob Agents Chemother 1993371756ndash1763

107 MK Lacy W Lu X Xu PR Tessier DP Nicolau R Quintiliani CH NightingalePharmacodynamic comparisons of levofloxacin ciprofloxacin and ampicillinagainst Streptococcus pneumoniae in an in vitro pharmacokinetic model Antimi-crob Agents Chemother 199943672ndash677

108 CC Sanders WE Sanders Cycling of antibiotics An approach to circumvent resis-tance in specialized units of the hospital Clin Microbiol Infect 19981223ndash225

16

Basic Pharmacoeconomics

Mark A Richerson

Medical Service Corps United States Navy and Department of DefensePharmacoeconomic Center Fort Sam Houston Texas

Eugene Moore

Department of Defense Pharmacoeconomic Center Fort Sam Houston Texas

1 INTRODUCTION

Changes in the economic structure of the US health care delivery system havedriven the need for pharmacists physicians and others in health care to developexpertise in evaluating the economic implications of selecting among competinghealth care programs and drug therapies With the enactment of the Tax Equityand Fiscal Responsibility Act (TEFRA) of 1983 most hospital departments in-cluding pharmacy laboratory and radiology have transitioned from revenue-generating departments to cost centers within their institutions [1] Finite eco-nomic resources for health care combined with the increasing prevalence of man-aged care rapidly emerging new medical technologies and increasing demandon the part of health care consumers has resulted in a need for decision makersto carefully allocate these limited resources to those programs and products thatprovide the most value in terms of health benefits

All views expressed are those of the authors and do not necessarily represent those of the Depart-ments of the Army Navy Air Force or those of the Department of Defense

367


The medical community the pharmaceutical industry nursing and otherallied health fields have increasingly discovered the usefulness of various healtheconomic methodologies as they relate to making informed health care allocationdecisions As a result readers must understand how to interpret the ever-increas-ing amount of economic data appearing in the medical literature In a briefingto the Office of Health Economics (OHE) Prichart [2] described the Health Eco-nomics Evaluations Database (HEED) and the growth of health economics evalu-ations appearing in the literature In 1998 the HEED database contained over14000 references Between 1992 and 1996 there was a near doubling of thereferences added each year to the database [2]

So how is a chapter concerning itself with pharmacoeconomic analysisrelevant to a book about the pharmacodynamic properties of antimicrobials In-terestingly when the subject matter of published economic evaluations was ex-amined it was discovered that diseases of the circulatory system neoplasm andinfectiousparasitic diseases were the top three disease states studied between1992 and 1996 Furthermore in examining drug expenditures by therapeutic cate-gory it was found that annual total spending on antimicrobials rose 4 between1999 and 2000 and was ranked fourth within the 16 therapeutic categories evalu-ated (exceeded only by spending on cardiovascular alimentary and central ner-vous system agents) Patients with complaints associated with respiratory tractinfection constitute a large portion of those visiting primary care providers in theUnited States The 1995 National Ambulatory Medical Care Survey (NAMCS)identified acute respiratory tract infection as the most frequent principal diagnosis(31856976 in 1995) provided by office-based physicians [3] Taking into consid-eration the unique pharmacodynamic characteristics of various antimicrobials canlead to economic efficiencies in the treatment of a variety of infectious diseasestates

The purpose of this chapter is to provide the reader with an understandingof the basic principles of pharmacoeconomic analysis Specifically we will de-scribe the elements that define a pharmacoeconomic evaluation and the majortypes of analysis and attempt to demonstrate how these forms of analysis havebeen applied to the decision-making process

2 CHARACTERISTICS OF A PHARMACOECONOMIC

ANALYSIS

Pharmacoeconomics is a discipline concerned with comparing the costs and con-sequences of two or more competing alternatives The four major types of analy-sis include the evaluation of cost minimization cost effectiveness cost vs bene-fit and cost vs utility Most often pharmacoeconomic analysis is concerned withthe selection of particular drugs treatment protocols or therapeutic options Inaddition to the four basic types of analysis other frequently encountered types


of analysis include cost analysis and costndashconsequence analysis These types ofevaluations are frequently referred to as partial evaluations The basic differencebetween complete and partial evaluations is that partial analyses focus on eithercost or consequence and may or may not describe a comparison between compet-ing products or services The focus of this chapter will be on the complete formsof pharmacoeconomic analysis Although the major types of analysis differ inhow the therapeutic outcomes are quantified they all share one common elementthe inclusion of a cost measure

21 Cost and Perspective

Costs in health care economics studies are generally described as being directindirect or intangible Direct costs are further divided between direct medicalcosts and direct non-medical costs Direct medical costs are the costs of thoseitems that are used up in the course of providing medical services In the caseof pharmacoeconomic studies direct medical costs are typically associated withthe cost of drugs medical supplies laboratory tests hospitalizations and patientvisits Other direct medical costs that are particularly relevant when comparingcompeting antimicrobials include the additional costs associated with therapeuticdrug concentration monitoring treatment failure and adverse drug events

Direct non-medical costs include out-of-pocket expenses incurred by thepatient or by the patientrsquos family in the course of obtaining health care Forexample lost wages that a patient experiences while taking time away from workto attend a doctorrsquos appointment is considered a direct non-medical cost Indirectcosts from an economistrsquos viewpoint are those costs that result from lost produc-tivity The inclusion of indirect costs is controversial for a variety of reasonsparticularly the difficulty in placing a precise dollar value on productivity Whatis the dollar value of lost productivity associated with treatments directed towardchildren or stay-at-home spouses Including indirect costs assumes that a pa-tientrsquos medical condition by itself would not have resulted in lost productivityThere are a number of methods each associated with trade-offs to address theseissues No single method is universally accepted and a tremendous amount ofcontroversy remains about when and how to include indirect cost in health careeconomic studies

Intangible costs refer to those costs incurred outside the medical systemDrummond et al [4] describe a scenario where an occupational health programchanges the production cost of automobiles The increased costs associated withthe program are ultimately passed on to the purchasers of cars far removed fromthe companyrsquos new health program These costs are typically omitted from phar-macoeconomic evaluations because it is generally understood that they are usu-ally insignificant and that there is little to be gained by attempting to includethem in an analysis


The perspective of the ultimate user of the analysis should determine whichtypes of costs are included in the study From a managed care organizationrsquosperspective direct medical costs (cost of providing medical personnel equip-ment and supplies) may be of primary importance In addition to direct medicalcosts an analysis completed from a governmental or societal perspective mayneed to include patientsrsquo out-of-pocket expenses and lost productivity The majormessage for the reader is that the perspective of the analysis and the costs includedin the study must be fully understood before generalizing the results to any oneparticular setting

Evaluations of long-term drug therapies such as those for hypertension anddyslipidemia require that health care costs incurred over the multiple years ofthe study be discounted and reported in the current yearrsquos value Discounting inaddition to accounting for inflation considers the opportunity costs associatedwith investment in one option versus another As a rule of thumb discountingis required when completing an analysis that spans more than a one-year timeframe The evaluation of competing therapy options for acute infectious diseaseprocesses is somewhat straightforward in that the entire episode spans periodsranging from a few days to a few weeks and discounting is generally not requiredOn the other hand in an analysis of a more chronic infection human immunedeficiency infection for example the economic evaluation is likely to requirediscounting in order to express future expenditures in the most current yearrsquosdollar values [5]

22 Major Types of Pharmacoeconomic Evaluation

As stated previously the four major types of pharmacoeconomic evaluation in-clude cost-minimization cost effectiveness cost-benefit and cost-utility analysisEach type is unique in the way in which consequences or outcomes are measuredand compared A cost-minimization pharmacoeconomic evaluation refers to thecomparison of two or more drug alternatives where the choice of any one agentwill result in exactly the same outcome as selection of any of the others Clearlythe focus in this type of analysis becomes cost and the alternative associatedwith the lowest cost is generally the preferred agent This does not mean that theanalysis is concerned with drug acquisition cost only Other costs to considerdepending on the perspective taken in the analysis might include drug adminis-tration cost cost associated with therapeutic drug monitoring and the cost oftreating an adverse drug event Comparison of a brand name drug product andan FDA-approved AB rated generic product serves as a classic example of a cost-minimization study The comparison of two or more antimicrobials for whichrandomized clinical trials have demonstrated equal efficacy serves as anotherexample It cannot be overemphasized however that sufficient information mustbe available to demonstrate equal treatment outcomes with all the agents being


compared Too often equal efficacy is assumed without sufficient supportiveevidence

Cost-effectiveness studies employ an analytical technique that comparesboth costs and outcomes of competing agents or services This form of analysisdiffers from the others in that outcomes are measured in some form of naturalisticunit For example when comparing two drugs used to treat hypertension theoutcome of interest may be reduction in systolic and diastolic pressure In a cost-effectiveness analysis comparing two antimicrobials used to treat acute sinusitisthe outcome of interest is likely to be cure Quenzer et al [6] used complication-free cure as the outcome measure of interest when comparing the cost effective-ness of various antimicrobials used to treat lower respiratory tract infectionsThey contended that from the perspective of a managed care organization patientsatisfaction indirectly measured by a complication-free episode of care was animportant outcome of interest Life years saved is a frequently encountered out-come measure particularly when comparing drugs used for the treatment ofchronic diseases

The term lsquolsquocost-effectiversquorsquo has a very specific set of meanings As Lee andSanchez [7] point out the term has frequently been misused in the literature Itis commonly misinterpreted to mean that the least costly product (frequentlybased on drug acquisition cost only) or decision is the product or decision ofchoice From a pharmacoeconomic decision-making perspective however thereare three different situations that arise in which one decision is more cost-effec-tive than another The most intuitive situation occurs when product A is preferredover product B because it results in lower costs while also being clinically moreeffective than product B Product A would be the decision of choice and wouldbe considered dominant to product B Another fairly straightforward example isa situation where product B is discovered to be equally as effective as productA yet product A results in lower costs Again product A would be preferredThe final description of cost effectiveness is frequently overlooked In a situationwhere product A is both more costly and more effective than product B productA may still be the most cost-effective choice because the extra clinical benefitassociated with product A may be judged to be worth the additional incrementalcosts This illustrates the point that although pharmacoeconomic analysis cancontribute to more informed decisions value judgments will continue to be neces-sary in many situations

Cost-benefit analysis expresses the benefits (or consequences) of a decisionchoice in terms of dollar value By measuring the cost and benefits in the sameunits (currency) it forces decision makers to make explicit decisions aboutwhether the benefits of a drug or program choice are worth the investment (cost)This form of analysis possesses the advantage of allowing comparison of anycombination of drugs and program options available in a particular institutionCost-benefit analysis is particularly useful in situations where the health care


system is funding multiple competing programs or where treatment options arecompeting for the same finite amount of money As long as the benefits of eachchoice can be expressed in currency any combination of programs services ortherapies can be compared One of the most difficult barriers to using this formof analysis is the need to place an acceptable dollar value on a particular levelof health

Cost-utility is a form of analysis where the outcome of interest is the pa-tientrsquos preference or that of a population of patients for a particular state ofhealth status or improvement in health status Utility scores are further multipliedby expected life years to yield quality-adjusted life years (QALY) gained Interms of methodologies a cost-utility analysis is conducted in much the samefashion as a cost-effectiveness analysis Unlike cost-effectiveness analysis wheretwo or more alternatives are compared for the same condition cost-utility analysisallows the comparison of different programs or treatment options directed towarddifferent medical conditions as long as those programs or treatment options resultin changes in patient quality of life The role of cost-utility analysis is controver-sial and the measurement methods continue to evolve Although there are a vari-ety of methods and measure tools designed to collect utility scores none havebeen universally accepted

3 OUTCOMES IN PHARMACOECONOMIC ANALYSIS

Pharmacoeconomics is a discipline concerned with the costs and consequencesof selections of particular drugs treatment protocols or therapeutic options [8]Frequently the consequences of such selections in health care are termed out-comes An outcome in the strictest definition is the final consequence of anaction [9] In health care economic research and decision making there are usu-ally different types of outcome measures describing different markers of interestthat can be studied or analyzed for a given disease state [1011] Outcome mea-sures can describe the efficacy of a drug or treatment option the effectivenessor the economic benefit Outcome measures can be surrogate or intermediate(eg a laboratory value) or absolute (eg absence of disease) The best or mostvalid outcome to study varies with the disease state being analyzed and with theinterests (perspective) of the persons conducting the study Outcomes may beclinical economic or humanistic [134]

31 Outcome Measures in the Treatment

of Infectious Disease

Outcome measures of drug therapy in any disease state are generally driven bypharmacodynamic considerations In the case of infectious disease pharmacody-namics refers to the relationship between the concentration of the drug at its


target tissues and its microbiological effect [12] The microbiological effect ofinterest is of course the eradication of the pathogenic invasion and lsquolsquocurersquorsquo ofthe infection For any given antimicrobial drug administration will result in itsselective distribution to tissues in concentrations that vary according to pharma-cokinetic properties of the drug and the drugndashhostndashdisease interaction [5] Thereis also a specific minimum concentration at which the drug will produce its anti-microbial action with respect to a given pathogen In vitro this is called the mini-mum inhibitory concentration (MIC) and is a measure of the drugrsquos efficacy [5]A drugrsquos MIC is inversely related to its antimicrobial activity The lower a drugrsquosMIC the greater the drugrsquos activity against the given pathogen An antimicrobialdrugrsquos effectiveness is determined by the interplay between its pharmacokineticsand its pharmacodynamics The drug must distribute to the target tissues (site ofinfection) in concentrations that meet or exceed the MIC be capable of killingthe pathogen and do so without causing serious adverse events in the patientThe result is a measurable outcome for the treatment of the infectious diseaseExamples of outcome measures in infectious disease are provided in Table 1

In general outcomes measurement in pharmacoeconomic analysis of infec-tious disease therapy is fairly straightforward The acute nature of most infectiousdiseases (HIV being the exception) makes it easy to define a desired outcomeAn acute infection is a short-duration event with obvious measurable signs of

TABLE 1 Types of Outcomes in Pharmacoeconomic Analysis

Type of outcome Examples applicable to infectious disease

Clinical Cure of infection (confirmed by culture)Absence of opportunistic infection (in immunocom-

promise)Resolution of febrile illnessResolution of pneumonia symptoms (decreased sputum

production lsquolsquoclearingrsquorsquo of sputum color)Economic Decreased length of stay (LOS)

Decreased total health care costDecreased drug acquisition costDecreased intensity of care (nursing and ancillary ser-

vices supplies)Decreased resource utilization

Humanistic Years of life addedQuality-adjusted life years savedDecreased incidence of adverse drug reactionDecreased pain and sufferingQuality of life


morbidity and in many cases the potential for death or serious complications ifinadequately treated The treatment goal is resolution of the infection and in thecase of hospitalized patients discharge or stepdown to a less intensive therapyThe outcome measure that provides the most valuable information in a pharma-coeconomic analysis is cure of the infection Indeed most research studies indrug therapy of infectious diseases measure efficacy andor effectiveness by theprobability that the therapy will result in elimination of the infection In a sensecure of the infection can be seen as the effectiveness measure that is of greatestinterest to the patient Itrsquos frequently the reason for the encounter with the healthcare system in the first place

The health care system or the payer while concerned with clinical out-comes may also be interested in economic outcomes [1] An economic analysisdone from the payerrsquos perspective may focus on decreasing hospital lengths ofstay or intensity of care These variables are directly related to the drugrsquos clinicaloutcomes (ie drugs with greater efficacy might produce more desirable eco-nomic outcomes) but the decision makerrsquos focus might be on the economic out-come for obvious reasons In general payers look to increase economic efficien-cies so that they can provide more care for the same costs or provide the samecare at a greater lsquolsquoprofitrsquorsquo (ie less cost) [11314]

Both the patient and the health care system (or payer) are concerned withhumanistic outcomes Humanistic outcomes are those that describe the impactof a therapy on some aspect of human quality of life longevity pain and suffer-ing or emotional satisfaction [1] Quality of life can be defined as an individualrsquosoverall satisfaction with life and general personal well-being [15] Health-relatedquality of life (HRQOL) includes those aspects of quality of life that are affectedby onersquos health From the patientrsquos perspective drug therapy of infection shouldpreserve or improve HRQOL by maximizing positive humanistic outcomes andminimizing negative humanistic outcomes In acute infection the obvious payoffoccurs when the infection is resolved and the patient is discharged from the hospi-tal with no permanent sequelae In chronic infection such as that which occurswhen an individual has contracted the human immunodeficiency virus (HIV)discussion of humanistic outcomes take on more than one dimension Currenttreatment regimens such as HAART (highly active antiretroviral therapy) do noteradicate the infection but instead suppress viral activity to the point where itsimpact on the bodyrsquos T-helper cells is minimal This comes at a price that pricebeing that the patient must be willing to tolerate side effects that range fromannoying to uncomfortable to life-threatening The patient and physician mustattempt to find the trade-off between the HAART combination that offers thebest promise for longevity with the least impact on quality of life

Humanistic outcomes can be difficult to use in a pharmacoeconomic analy-sis Although most health care providers administrators and researchers agreethat it is important to assess humanistic aspects of care there is no consensus


on standard measurement techniques [8] Because of the subjective nature ofquality of life it is difficult to correlate improvements in HRQOL to clinicalimprovements [8] In the HAART example above for instance the patientrsquos viralload may be brought to undetectable levels (clinical improvement) while the pa-tient reports a decrease in HRQOL (from side effects) Another difficulty withapplying humanistic outcomes to pharmacoeconomic analysis is that it is fre-quently difficult to place an economic value on them Although everyone canagree that prolonging life is mostly desirable it is nonetheless very difficult toanswer the question What is the dollar value of an extra year of life The answeragain depends on the perspective of the answerer A third-party payer might valuethe extra year of life differently than a patient or a health care provider Entiresubdisciplines in health economics are devoted to the issue of assessing and plac-ing value on humanistic and quality of life outcomes in health care Althoughthe subjects are beyond the scope of this chapter two such areas of study arecontingent valuation methodology (CVM) [16] and time trade-off (standard gam-ble) [17] analysis Readers desiring to learn more on these subjects are directedto the vast body of information in the medical literature

4 METHODOLOGICAL CONSIDERATIONS IN

CONDUCTING PHARMACOECONOMIC ANALYSES

The validity of a pharmacoeconomic analysis is influenced a great deal by themethodology one chooses to employ in doing the analysis To be valid and usefulan analysis must use methods that are most appropriate to the disease state inquestion in the health setting of interest Selection of the type of analysis andsecuring the data inputs on which the analysis is based are critical steps in con-ducting a good sound pharmacoeconomic analysis

As stated elsewhere in this chapter there are at least four major types ofpharmacoeconomic analyses cost minimization cost-benefit cost-utility andcost effectiveness are all analysis types [18] In analyzing the pharmacoeconom-ics of an infectious disease agent or protocol one of the four types is usuallymore suitable than the others However in some cases two or more methods areequally valid Some methods may in theory produce results with greater validitybut may be too difficult to put into practice or may be judged unethical

Choice of the analysis type should be based on the goals of the analyst ororganization doing the analysis A government entity concerned with formulatingpublic policy or allocating scarce resources will have different goals and require-ments than a managed care organization desiring to enhance economic efficiencyin its hospitals Researchers seeking to advance the body of knowledge in thediscipline will have still different goals and requirements Organizations seekingto formulate public policy or allocate resources are likely to find cost-benefitanalysis to be more useful [18] If two treatment choices are deemed to be equal


in outcomes an organization is likely to find cost minimization analysis to beuseful [18] In most cases the organization is seeking to provide the most effec-tive treatment for the least expenditure In this case it is likely that cost-effective-ness analysis is most useful

Regardless of which overall analysis type is chosen there remains the ques-tion of which methodology to use in conducting the analysis The analyst has anumber of methodological techniques to choose from each with its own optimalapplications strengths and weaknesses These techniques include cost compari-sons mathematical models simulations and decision analysis

Cost comparison is the simplest methodological technique and it is mostapplicable to cost-minimization analysis This technique is also sometimes calledcost identification When all the costs of therapy are known or identified theyare tabulated and compared Because all therapies are deemed to have equal out-comes the therapy with the lowest identified cost is considered to have an eco-nomic advantage

Mathematical models and simulations are very frequently used in costeffectiveness and cost-benefit analyses [18] A mathematical model is a modelthat seeks to precisely describe in mathematical terms all of the costs and conse-quences of a particular therapeutic option Simulations are quite similar to mathe-matical models except in one critical aspect Generally speaking mathematicalmodels represent static points in time whereas simulations attempt to showchange over a time course [11] Hence a lsquolsquosimulation clockrsquorsquo (ie a mechanismto account for time change in regular increments) is a critical part of a simulationSimulations when done on a computer allow changing events to be representedgraphically perhaps making it easier for the intended audience to lsquolsquoseersquorsquo thedifferences among competing alternatives Mathematical models and simulationsare more alike than different in that they both require precise identification ofcosts and consequences in mathematical terms Generally speaking mathematicalmodels are used more often in pharmacoeconomics than simulations though afew exceptionally well done simulations can be found in the literature [19] Thereader seeking to learn more about simulations and their application to medicaldecision making should consult the references at the end of this chapter [18ndash22]

As stated above the mathematical model expresses the costs and conse-quences of treatment choices in mathematical terms When applied to cost-effec-tiveness analysis this means that the analyst must develop an equation that ex-presses as precisely as possible the relationship between the variables (inputs)that influence the costs and outcomes of each treatment course Variables willof course differ with each disease state or drug type being analyzed Some poten-tially important variables to consider when developing a mathematical modelinclude the acquisition cost of the drug the cost of administering the drug nursingcosts the probability of incurring additional visits additional visit costs the prob-ability of hospitalization hospitalization costs the probability of adverse drug


TABLE 2 Advantages and Disadvantages of Modeling

Advantages Disadvantages

Allows evaluation of multiple op- It may be difficult to validate find-tions and prediction of outcomes ings

Can be used to predict outcomes as- Decision makers unfamiliar with thesociated with rare events technique may be skeptical

Does not endanger or inconve- Data may be difficult to get andornience patients or providers apply

Costs are much lower than an obser- It usually requires assumptionsvational study

Source Adapted from Ref 18

events (ADE) the cost of ADE the probability of treatment failure the cost oftreatment failure the probability of additional laboratory monitoring the costsof additional laboratory monitoring the probability of treatment success andbiological measures of outcome

Mathematical models can be further categorized as stochastic or determin-istic [11] If none of a modelrsquos inputs involve probabilities the model is saidto be deterministic If one or more of the modelrsquos inputs involve probabilitydistributions the model is said to be stochastic For example in a pharmacoki-netic model the mathematical relationships describing the drugrsquos absorption dis-tribution and elimination are known and can be represented by specific equationsand the model is considered deterministic In a model such as one for the treat-ment of community-acquired pneumonia several variables may involve probabil-ity distributions The likelihood of successful resolution of infection of seekingretreatment after treatment failure of experiencing an ADE severe enough torequire treatment or of presenting with an infection caused by a resistant organ-ism are all governed by probabilities of occurrence and the model is consideredstochastic A stochastic model in which the inputs from one stage are dependenton the outputs from another stage is termed a Markov chain or Markov model[11]

Still another type of mathematical model which can be either stochasticor deterministic is the analytical hierarchy model This type of model is alsocalled a multiattribute utility theory (MAUT) model [2021] The key feature ofthis model is the fact that all categories of model inputs are weighted by relativeimportance to the decision maker prior to running the model For example ahealth care system choosing among new antibiotics for formulary inclusion mayplace a higher weight on the attribute lsquolsquocoverage of resistant organismsrsquorsquo thanon the attributes of price probability of ADE or cost of administration In thiscase the lsquolsquoutilityrsquorsquo or health systemrsquos valuation will have additional influence


TABLE 3 Types of Mathematical Models

Type Description

Stochastic Involves probability distributions uncertainty in val-ues of variables

Deterministic No probabilities involved all variables specificallystated and all mathematical relationships known

Analytical hierarchy Involves valuation weighting of specific model attri-butes by end users

on the modelrsquos behavior and results The types of mathematical models and theircharacteristics are described in Table 3

41 Decision Analytic Modeling

Another type of modeling that is very useful in pharmacoeconomics is decisionanalytic (DA) modeling Decision analytic models seek to quantify the expectedvalue of a particular course of action by examining the probabilities of eventsoccurring after the decision and the outcomes (payoff) that result from the event[1122] These models involve the construction of a decision tree with eachbranch representing an outcome probability The analyst can compare competingchoices and select the decision that offers the best expected value for the healthsystem In cost-effectiveness analysis the lsquolsquowinnerrsquorsquo is usually the treatment paththat yields the lowest expected value in dollars In cost-benefit analysis the lsquolsquowin-nerrsquorsquo is usually the treatment path that yields the highest expected value in dollarsA simplified example of a decision tree is shown in Fig 1

The case in Fig 1 illustrates the basic principles of decision trees In stan-dard notation a square box represents a decision node (eg treat with A or B)circles represent chance (probability) nodes and triangles represent terminal

FIGURE 1 Antibiotic A vs antibiotic B in acute infection


(payoff) nodes Multiplying the probabilities at each chance node by the payoffsand summing the results gives the expected value of each choice In the exampleantibiotic B has a 5 greater probability of cure but costs a little more for eachcourse of therapy Although it is not necessarily obvious the most cost-effectivechoice in the above example is antibiotic B Although it would appear that B ismore expensive by $25 per course of therapy when one factors in the greaterprobability of cure (assuming an equal cost of failure of $1100) the expectedvalue of B becomes $380 [(90 $300) (10 $1100)] versus an expectedvalue of $399 [(85 $275) (15 $1100)] for A Thus one can say that onaverage over a large number of cases B will actually work out to be less expensivethan A by approximately $19 per case In practice decision trees are usuallymore complex than the example shown above There may be many branchesrepresenting probabilities and associated payoffs for ADEs complications re-treatment etc Decision trees are nonetheless very useful when one can obtainthe proper data (probabilities and the associated costs) with which to construct themodel A more detailed example of a decision analysis applied to the economicevaluation of competing antibiotic regimens can be seen in a recent study byRicherson et al [23]

42 Data Sources for Pharmacoeconomic Studies

The decision on which type of model to use in a pharmacoeconomic analysis isdependent upon the data available for inputs In turn the data available for inputsdepends in many cases on the pharmacoeconomic question one is trying to an-swer Certain data lend themselves to answering some questions more accuratelythan others Certain data are inappropriate for answering some types of questionsThus it is wise for the analyst not to approach the matter of data considerationstoo cavalierly when planning a pharmacoeconomic study

In order to increase the scientific quality and integrity of pharmacoeco-nomic studies consensus guidelines have been developed by some countries andby professional and academic organizations Australia and Canada adopted na-tional guidelines on health economic analysis [2425] and the International So-ciety for Pharmaceutical Outcomes Research has developed an internationalconsensus statement on pharmacoeconomic methodology [26] Each of these doc-uments addresses in detail the issue of data validity and applicability of data tovarious types of analyses

There are a number of data sources available to use in constructing a phar-macoeconomic study or model Data can generally tell the researcher three thingsthat may be used in a pharmacoeconomic study the likelihood of outcomes asso-ciated with the treatment or nontreatment of disease the treatment patterns associ-ated with the therapy and the costs associated with the therapy andor diseasestate [27] Some researchers have coined the term lsquolsquotransition probabilitiesrsquorsquo to


refer to the first of these The data must be incorporated into the analysis in amanner that is rational and appropriate to the assumptions of the analysis

Table 4 shows the data types that may be used in conducting a pharmaco-economic analysis or constructing a pharmacoeconomic model The data typeswill vary in their applicability to different types of pharmacoeconomic analysesObviously one of the best sources for data in a health care system is local research[8] An advantage of using local research data is that a well-designed local re-search project can tell the analyst more about the probable costs and consequencesof treatment decisions at his or her hospital than data from other facilities that mayor may not have had similar patients and prescribing practices A disadvantage tousing local research data is that it takes considerable time and resources to con-duct a good study [828] By the time the study is designed and implementedand the findings analyzed market conditions may have changed and renderedthe results nonapplicable Also many hospitals are under budgetary pressuresthat do not allow the staffing or other resources needed to conduct a well-designedstudy

43 Evaluating Pharmacoeconomic Studies

Often those in health care will need to evaluate other pharmacoeconomic studieseither for the purpose of making a formulary or treatment decision or for thepurpose of gathering data for their own models In those cases it is importantto make sure that the pharmacoeconomic study is valid and applicable to thehealth care system in question There are several key questions that one mustask in evaluating a pharmacoeconomic analysis these are listed in Table 5

The first question listed in Table 5 is especially important when reviewinga study or analysis that makes conclusions about the pharmacoeconomics of agiven treatment option A clearly defined clinical question that is scientificallyrelevant and capable of being answered by the study design is a must It simplydoes not make sense to attempt to ascertain the economic advantage of a therapyif the study was not properly designed to determine a relevant clinical outcomeFor example it is common to find articles in which one antibiotic is claimed tobe economically superior to another but the data supporting the conclusion comefrom analyzing a subgroup that just happened to respond better to treatment be-cause they had a particular variant of the infecting organism that was unantici-pated at the start of the trial Similarly it is also important to know whether allof the interventions compared have been shown to be clinically effective Forexample it may be invalid to determine that a very low cost antibiotic is lsquolsquomorecost-effectiversquorsquo than another when in fact the low cost antibiotic is no longerconsidered clinically effective because of increasing microbial resistance

Other important questions surround economic models First and foremostthe modelrsquos assumptions must be rational and a realistic representation of real-

Basic Pharmacoeconomics 381T

AB

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TABLE 5 Key Questions for Evaluating Pharmacoeconomic Studies

Is the analysis based on a study that answers a clearly defined clinicalquestion about an economically important issue

Does the type of analysis match the objectives of the organization orperson conducting the study Is it appropriate

Does the analysis seek to answer questions for which it is not structuredIs the quality of the data appropriate to the conclusions of the analysisIs there real or perceived bias in the way in which the analysis was

structuredIf an economic model was the model tested for sensitivity of its inputsWere the modelrsquos assumptions realistic in comparison to lsquolsquoreal lifersquorsquoWhose viewpoint are costs and benefits being considered fromHave all the interventions compared been shown to be clinically effectiveHow were costs and benefits measured

Source Adapted from Refs 14 and 22

life occurrences [715] Failure and retreatment rates probability of laboratorymonitoring and likelihood of additional visits are some areas where modelerssometimes make unrealistic assumptions that may bias a modelrsquos results Anotherimportant consideration for economic models is sensitivity testing [151829]The modelrsquos inputs should be tested around a range of values to determine howchanging these values affect the modelrsquos results If the modelrsquos results appear tobe similar across the likely range of possible input values then the model is saidto be robust On the other hand if the modelrsquos results vary greatly when smallbut realistic changes are made in its inputs then it is less valid A final consider-ation that should be given to economic models is whether or not the model istransparent and reproducible All of the major governmental and academic bodiesthat have developed guidelines have included recommendations that the modelrsquosinputs and mathematical relationships be transparent (ie equations and variablesclearly shown) They also recommend that the model be constructed in a mannerthat would make it possible for other researchers or analysts to duplicate themodel and its findings [16ndash18] Models that have hidden (lsquolsquoblack boxrsquorsquo) relation-ships may be considered of questionable validity

The rapid development of new drug technologies combined with direct-to-consumer advertising increased patient access to care and finite resources willrequire that decision makers have the analytical tools necessary to make impor-tant health-related resource allocation decisions This chapter has attempted tointroduce the reader to some of the available pharmacoeconomic methodologies


used to assess the cost and outcomes of competing drug therapy options includ-ing effective antimicrobial therapy

REFERENCES

1 Juergens JP Szeinbach SL Smith MC Will future pharmacists understand pharma-coeconomic research Am J Pharm Educ 199256135ndash140

2 Pritchard C Trends in economic evaluation Office of Health Economics BriefingNo 36 April 1998

3 Vital and Health Statistics Series 13 No 1294 Drummond MF Stoddart GL Torrance GW Methods for the Economic Evaluation

of Health Care Programmes New York Oxford Univ Press 19875 Eisenberg JM Clinical economics A guide to the economic analysis of clinical

practice JAMA 19892622879ndash28866 Quenzer RW Pettit KG Arnold RJ Kaniecki DJ Pharmacoeconomic analysis of

selected antibiotics in lower respiratory tract infection Am J Managed Care 199731027ndash1036

7 Lee JT Sanchez LA Interpretation of lsquolsquocost-effectiversquorsquo and soundness of economicevaluations in the pharmacy literature Am J Hosp Pharm 199148622ndash627

8 Bootman L Pharmacoeconomics (lsquolsquoThe Green Bookrsquorsquo)9 Webstersrsquo New Riverside University Dictionary Boston Riverside 1988

10 Rupp MT Kreling DH The impact of pharmaceutical care on patient outcomesWhat do we know Drug Benefit Trends 19979(2)35ndash47

11 Tse CS Outcomes measurement across the continuum of care Med Interface 199710(11)103ndash107

12 Kenreigh CA Wagner LT Medscape Pharmacists Treatment Update HillsboroMedical Education Collaborative Inc June 1999

13 McDonald RC Coons SJ An Introduction to Health Economics Indianapolis EliLilly 1993

14 Greenhalgh T Papers that tell you what things cost (economic analysis) Br Med J1997315596ndash599

15 Jones AJ Sanchez LA Pharmacoeconomic evaluation Applications in managedhealth care formulary decision making Drug Benefit Trends 19957(9)121519ndash2232ndash34

16 Morrison GC Gyldmark M Appraising the use of contingent valuation Health Econ19921(4)233ndash243

17 Dolan P Gudex C Kind P Williams A The time trade-off method Results froma general population study Health Econ 19965(2)141ndash154

18 Dean BS Gallivan S Barber ND Van Ackere A Mathematical modeling of phar-macy systems Am J Health Syst Pharm 1997542491ndash2499

19 Lightwood JM Glantz SA Short-term economic and health benefits of smokingcessation Myocardial infarction and stroke Circulation 1997961089ndash1096

20 Schumacher GE Multiattribute evaluation in formulary decision making as appliedto calcium channel blockers Am J Hosp Pharmacy 199148(2)301ndash308


21 McCoy S Chandramouli J Mutnick A Using multiple pharmacoeconomic methodsto conduct a cost-effectiveness analysis of histamine H2 receptor antagonists AmJ Health Syst Pharm 199855(suppl 4)S8ndashS12

22 Summers KH Hylan TR Edgell ET The use of economic models in managed carepharmacy decisions J Managed Care Pharm 1998442ndash50

23 Richerson MA Ambrose PG et al Pharmacoeconomic evaluation of alternativeantibiotic regimens in hospitalized patients with community acquired pneumoniaInfect Dis Clin Pract 19987227ndash233

24 Commonwealth of Australia Dept of Human Services amp Health Guidelines for thepharmaceutical industry on preparation of submissions to the Pharmaceutical Bene-fits Advisory Committee including major submissions involving economic analysisCanberra Australian Govt Pub Service 1995

25 Canadian Coordinating Office for Health Technology Guideline for Economic Eval-uation of Pharmaceuticals Canada Ottawa CCOHTA 1994

26 International Society for Pharmaceutical Outcomes Research Consensus Statementon Pharmacoeconomic Studies Philadelphia ISPOR 2000

27 Nuijten MJC The selection of data sources for use in modeling studies Pharmaco-economics 199813(3)305ndash315

28 Else BA Armstrong EP Cox ER Data sources for pharmacoeconomic and healthservices research Am J Health-Syst Pharm 1997542601ndash2608

29 Brennan A Akehurst R Modelling in health economic evaluation What is its placeWhat is its value Pharmacoeconomics 200017(5)445ndash459

17

Utilizing Pharmacodynamics andPharmacoeconomics in Clinicaland Formulary Decision Making

Paul G Ambrose


Annette Zoe-Powers

Bristol-Myers Squibb Company Plainsboro New Jersey

Rene Russo

Rutgers University Piscataway New Jersey

David T Jones

University of California at Davis Sacramento California

Robert C Owens Jr

Maine Medical Center Portland Maineand University of Vermont College of Medicine Burlington Vermont

1 INTRODUCTION

Managed care organizations (MCOs) community hospitals and university teach-ing hospitals are challenged to control total organizational cost while maintaininghigh quality patient care and services Most health care institutions have imple-

385

386 Ambrose et al

mented processes including pharmacy and therapeutics committees (PampTs) andformularies in an effort to control drug costs In essence a formulary is a listof products that have been reviewed and approved by the Pharmacy and Thera-peutics Committee Moreover the goal of the PampT and formulary process is tooptimize clinical outcomes while controlling or minimizing cost Despite theseefforts drug and organizational expenditures continue to increase Because manyantibiotic regimens could be considered appropriate for the treatment of a giveninfectious disease the challenge is to find the most cost-effective regimens Itshould be realized however that a low drug cost does not necessarily correlateto a low organizational cost For instance although a drug with a high acquisitionprice compared with standard therapy may increase total drug costs it may sig-nificantly decrease the institutional expenditures in other areas Unfortunatelyfew prospective randomized clinical data are available comparing new therapeu-tic modalities and standard regimens under usual care conditions

Consequently it is crucial to develop methods to treat infections that areboth clinically sound and economically effective The application of pharmacody-namic and pharmacoeconomic concepts to issues involving the use of antimicro-bial agents provides a data-driven paradigm to achieve these goals This approachoften identifies the best agent to maximize bacterial killing the appropriate doseand the most cost-effective agent to select for formulary inclusion and clinicaluse In order to understand the discussion contained in this chapter a basic under-standing of pharmacodynamic theory and pharmacoeconomic concepts is essen-tial For a complete review of these concepts the reader is encouraged to reviewChapters 5 7 9 and 16 as they form the basis for the discussion that follows

In this chapter pharmacodynamic and pharmacoeconomic principles areused to guide clinical and formulary decisions Through the use of contemporaryantimicrobial agents as real-life illustrations the reader will become cognizanton how these concepts can be applied to formulary decision making and in theclinical practice of medicine and pharmacy Moreover agents from a variety ofantimicrobial classes (eg cephalosporins fluoroquinolones and macrolides)and modes of bacterial killing (time-dependent versus concentration-dependent)will be used as examples Obviously the following evaluations are not meantto be complete unto themselves but rather are intended to outline the use ofpharmacodynamic and pharmacoeconomic data in the decision-making process

2 CEFEPIME VERSUS CEFTAZIDIME

21 Background

Cefepime and ceftazidime are intravenously administered β-lactam antibioticsthat exhibit a broad spectrum of antibacterial activity that includes aerobic gram-positive and gram-negative bacteria Due to their spectrum of activity these


agents have emerged as standard therapy for a number of serious nosocomialinfections such as hospital-acquired pneumonia and the empirical therapy of fe-brile neutropenia

22 Pharmacokinetics

The pharmacokinetic parameters of cefepime and ceftazidime in adults are pre-sented in Table 1 [12] From these data it is apparent that the two agents aresimilar pharmacokinetically in terms of volume of distribution clearance percenturinary excretion and protein binding However cefepime has an approximately25 longer serum half-life and a slightly larger area under the serum concentra-tionndashtime curve (AUC) For this reason cefepime is dosed on a twice-dailyschedule whereas ceftazidime must be dosed three times daily in those patientswith normal or moderately reduced renal function (Clcr 50ndash60 mLmin) Thepharmacokinetics of both agents vary with degree of renal function Followinga 1000 mg dose of cefepime in patients with glomerular filtration rates (GFRs)of less than 60 mLmin serum half-life and AUC increase from 23 to 49 h andfrom 131 to 292 microg sdot hmL respectively [3] Similarly following a 1000 mgdose of ceftazidime in patients with GFRs less than 50 mLmin serum half-lifeand AUC increase from approximately 19 to 40 h and 130 to 270 microg sdot hmLrespectively [14]

23 Microbiology

Comparative antimicrobial activities of cefepime and ceftazidime against variouspathogens are presented in Table 2 [5] From these data it appears that cefepimehas lower minimal inhibitory concentration (MIC) values than ceftazidimeagainst most gram-positive aerobes especially Streptococcus pneumoniae andStaphylococcus aureus This is significant because gram-positive aerobes pri-marily staphylococci are a leading cause of serious nosocomial infection In factS aureus is the second most common cause of hospital-acquired pneumonia

Against aerobic gram-negative microorganisms including Pseudomonasaeruginosa the microbiological activity of cefepime is similar to that of ceftazi-dime however notable exceptions exist Unlike ceftazidime cefepime has beenshown to have enhanced stability in the presence of Bush-Jacoby-Mederios group1 β-lactamases which when induced can render all other currently availablecephalosporins inactive [67] Not unexpectedly therefore cefepime demon-strates significantly lower MICs against isolates such as Enterobacter species andCitrobacter freundii that elaborate this enzyme In addition cefepime exhibitsappreciably more activity against Klebsiella pneumoniae These observationscarry considerable clinical relevance because P aeruginosa K pneumoniaeEnterobacter species and Citrobacter species are the first third fourth and fifthmost likely pathogens in hospital-acquired pneumonia respectively Although it

388 Ambrose et al

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epim

e

012

0

1210

00

Cef

tazi

dim

e

012

0

1210

00

Ser

rati

am

arce

scen

s(6

8)C

efep

ime

025

05

985

Cef

tazi

dim

e

012

05

971

Pse

ud

om

on

asae

rug

ino

sa(5

69)

Cef

epim

e2

890

0C

efta

zid

ime

216

877

Ste

no

tro

ph

om

on

asm

alto

ph

ilia

(51)

Cef

epim

e

16

1619

6C

efta

zid

ime

8

1654

9

So

urc

eR

ef

5

390 Ambrose et al

is tempting to conclude from in vitro microbiological observations that cefepimeis a preferential choice over ceftazidime the significance if any of these observa-tions will become apparent in the pharmacodynamic discussion below

24 Pharmacodynamics

An accurate prediction of clinical outcome requires the use of pharmacodynamicconcepts that integrate microbiological and pharmacokinetic data Cefepime andceftazidime like all other β-lactam antimicrobial agents kill bacteria in a time-dependent fashion Once the drug concentration exceeds the MIC of the pathogenkilling occurs at a zero-order rate and increasing drug concentration does notresult in a proportionally increased rate of kill Thus the goal with β-lactamantimicrobial agents is to maintain their duration or time above the MIC for aslong as possible over any one dosing interval As mentioned in Chapter 5 areasonable time period for β-lactam antimicrobial agents to exceed the MIC is40ndash60 of any single dosing interval [8] Earlier comparisons of in vitro activityagainst several clinically relevant pathogens between cefepime and ceftazidimewere made In the following analysis we will explore the potential clinical sig-nificance of these observations

Time versus plasma concentration curves of cefepime and ceftazidime arepresented in Figs 1 and 2 respectively Following a 1000 mg intravenous doseof cefepime every 12 h serum levels remain above the MIC of most organismsover the entire dosing interval Against P aeruginosa however cefepime serumconcentration time above the MIC (T MIC) is approximately 50 of the dosinginterval Following a 1000 mg intravenous dose of ceftazidime every 8 h a some-what different picture emerges The serum concentration of ceftazidime remainsabove the MIC over the entire dosing interval for only E coli and K pneumoniaeAgainst P aeruginosa and S aureus serum ceftazidime T MIC is approxi-mately 50 Moreover against C freundii and E cloacae for only 25 of thedosing interval does the ceftazidime serum concentration remain above the MIC

Based on these observations one would expect similar clinical outcomeswhen either agent is used to treat infections involving E coli or K pneumoniaebecause both agents maintain drug concentrations over the entire dosing intervalHowever against C freundii and E cloacae it would be predicted that cefepimeis the superior agent because cefepime maintains levels over 100 of the dosinginterval compared with 25 for ceftazidime Indeed the literature is replete withcase reports describing failure of ceftazidime when used to treat serious infectionsinvolving these pathogens [9] In fact because these organisms can elaborateBush-Jacoby-Mederios group 1 β-lactamases third generation cephalosporins arenot recommended for infections when these organisms are likely to be involvedOn the other hand cefepimersquos advantage against S aureus would not be expectedto be of clinical relevance because ceftazidime maintains serum levels above the


FIGURE 1 Concentrationndashtime profile of cefepime following a 1 g dose every12 h with MIC values of selected bacteria superimposed

MIC for approximately 50 of the dosing interval Moreover one would predictsimilar outcomes against P aeruginosa for the same reason [10]


As mentioned earlier few prospective randomized clinical data are availablecomparing new therapeutic modalities and standard regimens under usual careconditions Naturalistic trials are a study design approach that compares one drugregimen such as a new drug with one or more commonly used therapies underusual care conditions In other words naturalistic studies compare clinical andother outcome measures of competing therapeutic alternatives based upon howthe drugs are actually used in clinical practice rather than in the strict confinesof traditional clinical trials This information is crucial especially as new prod-ucts are introduced to the market because health care providers must make clini-cal and economic decisions concerning these agents Two such studies have di-rectly compared cefepime and ceftazidime and are reviewed briefly below

Ambrose et al [1] compared the cost effectiveness of cefepime and ceftazi-dime in intensive care unit patients with hospital-acquired pneumonia The effi-cacy safety and cost effectiveness of each agent were evaluated in a prospective

392 Ambrose et al

FIGURE 2 Concentrationndashtime profile of ceftazidime following a 1 g dose ev-ery 8 h with MIC values of selected bacteria superimposed

noninterventional investigator-blinded study involving 100 patients Clinicalsuccess rates were 60 and 78 for patients treated with ceftazidime and cefe-pime respectively (P 005) Microbiological eradication rates were 55 forceftazidime and 77 for cefepime (P 004) In those patients in whom Paeruginosa were isolated the organism was eradicated in 70 (1420) of cefe-pime patients and 50 (714) of ceftazidime patients Interestingly the authorsnoted that the MICs of P aeruginosa and S aureus were higher for ceftazidimethan for cefepime at the study institution and that the resultant pharmacodynamicdifferences may be the reason for their observations The frequency of concomi-tant antibiotic use was less in the cefepime group [ceftazidime 74 (3750)cefepime 44 (2250) P 0004] particularly with the use of vancomycinCefepime was more cost effective than ceftazidime (ceftazidime $2452810 cef-epime $1999621) Sensitivity analysis of efficacy rates demonstrated that ceftaz-idime would have to be 50 more effective than cefepime to change the eco-nomic outcome The authors concluded that cefepime was clinically moreefficacious and more cost effective than ceftazidime in similar patients withhospital-acquired pneumonia [11]


Owens et al [12] compared the cost effectiveness of cefepime dosed 1000ndash2000 mg every 12 h and ceftazidime dosed 1000ndash2000 mg every 8 h as empiricalmonotherapy for febrile neutropenia The efficacy safety and cost effectivenessof each agent were evaluated in a prospective noninterventional evaluator-blinded study involving 99 patients

Patients were stratified by risk for infection in accordance with the IDSAFDA and HIS guidelines (ie cancer type age and fever) Clinical assessmentswere made at 72 h of therapy and at 7 days post-therapy or at discharge fromthe hospital Clinical success was defined as apyrexia maintained clinical signsand symptoms improved and organism eradicated (when applicable) no relapseor new infection and survival Failure was defined as inability to eradicate thepathogen death due to infection and discontinuation of study agent because ofnonresponse

Clinical success rates were 80 and 96 for patients treated with ceftazi-dime and cefepime respectively (P 0028) Moreover the frequency of CDCIDSA-unsupported vancomycin addition to therapy occurred significantly (P 0001) less often in the cefepime group (14) than in the ceftazidime group(46)

The authors found that cefepime was more cost effective than ceftazidime(ceftazidime $50128 cefepime $31407) Sensitivity analysis of efficacy ratesdemonstrated that cefepime efficacy would have to decrease by 50 to changethe economic outcome The authors also noted that the elimination of ceftazidimefrom the formulary was associated with a favorable impact on Enterobacter cloa-cae susceptibility in the study units

26 Conclusion

Based upon this pharmacodynamic analysis it would be predicted that clinicaloutcomes would be improved if cefepime were chosen over ceftazidime for thetreatment of nosocomial infection This prediction is supported by comparativepharmacoeconomic data obtained from studies naturalistic in design Thereforeit may be reasonable to select cefepime over ceftazidime for formulary inclusion

3 CLARITHROMYCIN VERSUS AZITHROMYCIN

31 Background

Azithromycin and clarithromycin are two of the most commonly prescribed anti-biotics in the United States Because of their convenient dosing schedule andactivity against respiratory tract pathogens azithromycin and clarithromycin areprimarily used to treat infections such as community-acquired pneumonia sinus-itis and bronchitis

394 Ambrose et al

32 Pharmacokinetics

The pharmacokinetics parameters in adults of both macrolides are shown in Table3 The bioavailability and volume of distribution are similar between the twodrugs However azithromycin has a markedly longer half-life (t12) than clarithro-mycin 68 h and 3ndash4 h respectively [1314] As a result azithromycin can bedosed just once daily for 5 days Clarithromycin undergoes metabolism throughoxidative and hydrolytic pathways that produce several metabolites Converselyazithromycin is not highly metabolized with 75 of the parent compound beingexcreted unchanged [15] Because the macrolides rapidly distribute into tissuesresulting in high concentrations within the cells the serum concentration of mac-rolides is markedly lower than that of β-lactam antibiotics

33 Microbiology

The microbiological activity of macrolides against various bacteria is shown inTable 4 In vitro studies have shown that both agents are active against S pneu-moniae and M catarrhalis Based solely on MIC data azithromycin appears tobe more active against H influenzae than clarithromycin However the combina-tion of clarithromycin and its active metabolite 14-hydroxyclarithromycin hasbeen demonstrated to have additive or even synergistic activity against H in-fluenzae [16]

34 Pharmacodynamics

As mentioned earlier macrolides such as erythromycin and clarithromycin andazilides like azithromycin are generally considered concentration-independentkilling agents As with β-lactam agents the duration of time that the drug concen-tration exceeds the MIC of the infecting pathogen at the site of infection is theprimary determinant of efficacy for macrolides and azilides [4] How long thelevels of these agents should remain above the MIC remains controversial asthese agents have a prolonged post-antibiotic effect (PAE) with gram-positivecocci and Haemophilus influenzae [1718] Craig et al [17] postulated that thePAE allows these drugs to yield maximal efficacy in murine thigh infectionswhen concentrations exceed the MIC for significantly less than 50 of the dosinginterval (ie 30ndash35) Due to an extended PAE and long serum half-life theproper pharmacodynamic correlate for azithromycin may be the AUCMIC ratiorather than T MIC

Other investigators suggest that the concentration of the drug at the site ofinfection specifically where bacteria attach to the mammalian cell is significantlyhigher than the concentration in the serum This is due to macrolides and azilidesespecially azithromycin being highly concentrated inside mammalian cells Asthe drug egresses out of the cell a drug concentration gradient exists that is


TA

BLE

3S

tead

y-S

tate

Ph

arm

aco

kin

etic

Par

amet

ers

of

Azi

thro

myc

inan

dC

lari

thro

myc

inFo

llow

ing

500

mg

(Day

1)an

d25

0m

g(D

ays

2ndash5

)D

ose

san

d50

0m

gD

ose

sR

esp

ecti

vely

a

Cm

axH

alf-

life

AU

CV

ssC

lear

ance

U

rin

ary

P

rote

inA

gen

t(micro

gm

L)(h

)(h

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mL)

(L)

(mL

min

)ex

cret

ion

bin

din

g

Azi

thro

myc

in0

2468

21

2160

630

65

7ndash5

1C

lari

thro

myc

in2

854

820

84

191

437

3042

ndash70

aC

max

p

eak

seru

mco

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ntr

atio

n

AU

C

area

un

der

the

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mco

nce

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atio

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ime

curv

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yst

ate

bP

rote

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ind

ing

vari

esat

dif

fere

nt

con

cen

trat

ion

s

396 Ambrose et al

TABLE 4 Comparative In Vitro Microbiological Activity of Azithromycinand Clarithromycin


Gram-positiveStreptococcus pyogenes (105) Azithromycin 003 003

Clarithromycin 0016 0016Staphylococcus aureus BLA neg Azithromycin 013 013

(54) Clarithromycin 006 006

Streptococcus pneumoniae penicil- Azithromycin 003 4lin-susceptible (26) Clarithromycin 0016 2

Penicillin resistant (15) Azithromycin 05 16Clarithromycin 025 16

Gram-negativeHaemophilus influenzae (1137) Azithromycin 2 2

Clarithromycin 8 16Moraxella catarrhalis (723) Azithromycin 006 006

Clarithromycin 006 012

Source Refs 40ndash42

highest at the mammalian cell surface and lowest in the serum In this paradigmthe drug concentration in the serum is a poor correlate for that at the site ofinfection [19]

Azithromycinrsquos long serum half-life (approximately 68 h) which resultsin low serum concentration over an extended period of time has raised concernregarding its propensity to select for macrolide-resistant isolates [20] Leach etal [21] prospectively studied the impact of community-based azithromycin treat-ment on carriage and resistance of Streptococcus pneumoniae Single-doseazithromycin (20 mgkg) was given to children in a remote Aboriginal commu-nity in Australia with trachoma and their household contacts who were childrenCarriage rates of pneumococci resistant to azithromycin immediately before treat-ment with azithromycin and 2ndash3 weeks 2 months and 6 months after treatmentwere 19 545 345 and 59 respectively In this study the authors con-cluded that the selective pressure of azithromycin allowed the growth and trans-mission of pre-existing azithromycin-resistant strains

Similarly azithromycin was evaluated in an open clinical microbiologicaland pharmacokinetic study in hospitalized patients with acute exacerbation ofchronic bronchitis [22] Patients received 500 mg on the first day followed by250 mg once daily for 4 days Cultures of Haemophilus influenzae during andafter treatment showed many infections persisting Geometric mean MICs of


FIGURE 3 Pharmacodynamic concept Darwinrsquos window of natural selectionSchematic illustration of the serum pharmacokinetic profile of two oral drugregimens over one dosing interval If the serum concentrationndashtime curvesof two drugs one each with a short or long serum half-life are compared withan MIC value superimposed a period or lsquolsquowindowrsquorsquo of potential Darwinianselection appears For the agent with a short half-life the duration of timebetween when the drug concentration falls below the MIC and its total elimi-nation from the body is short relative to that of the agent with a much longerhalf-life

azithromycin for these organisms rose from 123 mgL pretreatment to 487 mgL a week after the end of treatment

One explanation for these observations relates to azithromycinrsquos long se-rum half-life and the duration of time subinhibitory concentrations of drug exist[23] If the serum concentrationndashtime curves of two drugs one each with shortand long serum half-lives are compared with an MIC value superimposed aperiod or lsquolsquowindowrsquorsquo of potential Darwinian selection appears (Fig 3) For theagent with a short half-life the duration of time between when the drug concentra-tion falls below the MIC and its total elimination from the body is short relativeto that of the agent with a much longer half-life For an agent with a 68 h half-life such as azithromycin the drug is not totally eliminated from the body for5ndash7 half-lives or 14ndash20 days after the end of therapy Therefore this period ofnatural selection or lsquolsquoDarwinrsquos windowrsquorsquo may be the pharmacodynamic explana-tion for the aforementioned observations

398 Ambrose et al


Health economic models have been used to investigate the cost consequencesof different treatment options in community-acquired respiratory tract infections[112425] Two recent studies evaluated the relationship between cost of careand either first-line antibiotic clinical efficacy or microbiological eradication ratefor the treatment of community-acquired pneumonia and acute exacerbation ofchronic bronchitis [2526] These studies used a Markov model that was popu-lated with health care resource estimates obtained from a panel of physicians(including general practitioners and pulmonology specialists) Cost was deter-mined by multiplying utilized resource items by the price of each item and evalu-ated from the perspective of the third-party payer

Use of these models has led to several significant observations First theleast expensive antibiotic is not necessarily the most cost-effective treatment mo-dality second not unexpectedly treatment failure resulted in the increased utili-zation of health care resources These resources included physician office visitslaboratory tests antibiotics and hospital admissions Consequently clinical effi-cacy rate was found to be a key cost driver due to the high cost of treatmentfailure This was especially true of treatment failures that resulted in hospitaladmissions

These observations are supported by a retrospective review of the medicalliterature since 1990 which investigated the relationship between clinical efficacyand microbiological eradication rates The review found 12 comparative studiesof acute exacerbation of chronic bronchitis in which both clinical and microbio-logical success rates were reported for 27 treatments involving 15 different antibi-otics A significant correlation (r 090) was found between clinical failurerates and microbiological eradication failure rates Hence a key cost driver inthe treatment of acute exacerbation of chronic bronchitis is the eradication rateof the causative bacterial pathogens by the first-line antibiotic Failure to eradicatethe causative pathogens increases the risk of treatment failure with its associatedadditional treatment costs

In a prospective randomized comparative study Weiss [26] evaluated theefficacy and safety of azithromycin clarithromycin and erythromycin in the ther-apy of patients with the clinical diagnosis of acute exacerbation of chronic bron-chitis Although the clinical efficacy rates were higher for azithromycin (80)and clarithromycin (85) compared with erythromycin (70) 40 of theazithromycin group requested and received prescription refills The financial im-pact of this observation is not limited to the mere cost of an additional prescriptionfor azithromycin which is minimal compared to the burden placed on the healthcare system in the form of additional physician office visits and potential hospital-izations


36 Conclusion

Based upon the foregoing pharmacodynamic analysis it would be predicted thatthe use of azithromycin as first-line antibiotic treatment might result in the morerapid selection of resistant strains of respiratory tract pathogens and treatmentfailure Moreover clinical and microbiological failure of first-line therapy re-mains the key cost driver in patients with community-acquired respiratory tractinfections For these reasons it may be reasonable to select clarithromycin overazithromycin for formulary inclusion and clinical use

4 GATIFLOXACIN VERSUS LEVOFLOXACIN

41 Background

Fluoroquinolones such as gatifloxacin and levofloxacin have become popularchoices for the empirical treatment of community-acquired pneumonia acute ex-acerbation of chronic bronchitis and sinusitis These agents are different fromearlier quinolones due to their increased in vitro activity against Streptococcuspneumoniae which is the most common and difficult pathogen associated withcommunity-acquired respiratory tract infection The increased popularity of gati-floxacin and levofloxacin has paralleled the rising resistance rates among pneu-mococci and H influenzae to many traditional antimicrobial agents such as thecephalosporins and macrolides

42 Pharmacokinetics

The pharmacokinetic profiles of gatifloxacin and levofloxacin are presented inTable 5 These two agents are similar with regard to volume of distributionpercent urinary excretion and protein binding Gatifloxacin demonstrates aslightly longer half-life whereas levofloxacin has a slightly larger area under theserum concentrationndashtime curve (AUC) Both agents are administered once dailyBoth agents are primarily excreted as unchanged drug from the kidney with negli-gible hepatic metabolism Both agentsrsquo pharmacokinetics vary with renal func-tion and doses should be decreased accordingly in those patients with renal insuf-ficiency As with many of the newer generation fluoroquinolones the oralbioavailabilities of gatifloxacin and levofloxacin are near 100 making theseideal agents for oral antimicrobial therapy

43 Microbiology

Comparative antimicrobial in vitro activities of gatifloxacin and levofloxacinagainst various pathogens are presented in Table 6 One of the notable differences

400 Ambrose et al

TA

BLE

5P

har

mac

oki

net

icP

aram

eter

so

fG

atifl

oxa

cin

and

Levo

flo

xaci

nFo

llow

ing

400

mg

and

500

mg

Do

ses

Res

pec

tive

lya

Cm

axH

alf-

life

AU

CV

ssC

lear

ance

U

rin

ary

P

rote

inA

gen

t(micro

gm

L)(h

)(h

sdotmicrog

mL)

(L)

(mL

min

)ex

cret

ion

bin

din

g

Gat

iflo

xaci

n4

28

ndash10

513

140

161

835

20Le

vofl

oxa

cin

87

772

511

199

726

30

Cm

ax

pea

kse

rum

con

cen

trat

ion

A

UC

ar

eau

nd

erth

ese

rum

con

cen

trat

ion

ndashtim

ecu

rve

Vss

volu

me

of

dis

trib

uti

on

atst

ead

yst

ate

TABLE 6 Comparative In Vitro Microbiological Activity of Gatifloxacinand Levofloxacin


Gram-positiveEnterococcus faecalis (100) Gatifloxacin 025 2

Levofloxacin 1 2Enterococcus faecium (40) Gatifloxacin 2 4

Levofloxacin 2 8

Staphylococcus aureus Gatifloxacin 006 013Meth-S (90) Levofloxacin 013 025


Staphylococcus epidermidis Gatifloxacin 013 025Meth-S (39) Levofloxacin 05 05


Streptococcus pneumoniae Gatifloxacin 025 1Pen-S (30) Levofloxacin 1 2

Pen-R (15) Gatifloxacin 025 05Levofloxacin 1 2

Streptococcus pyogenes (47) Gatifloxacin 025 05Levofloxacin 1 1

Streptococcus agalactiae (38) Gatifloxacin 025 05Levofloxacin 05 1

Gram-negativeCitrobacter freundii (52) Gatifloxacin 006 1

Levofloxacin 003 05Enterobacter cloacae (63) Gatifloxacin 0016 006

Levofloxacin 003 006Escherichia coli (30) Gatifloxacin 0008 0016

Levofloxacin 0016 003Haemophilus influenzae (46) Gatifloxacin 0008 0016

Levofloxacin 003 006Klebsiella pneumoniae (61) Gatifloxacin 003 013

Levofloxacin 003 013Moraxella catarrhalis (11) Gatifloxacin 0008 016

Levofloxacin 0008 006Morganella morganii (41) Gatifloxacin 006 025

Levofloxacin 003 006Proteus mirabilis (37) Gatifloxacin 013 025

Levofloxacin 003 025Pseudomonas aeruginosa (50) Gatifloxacin 4 32

Levofloxacin 2 32Serratia marcescens (55) Gatifloxacin 025 4

Levofloxacin 025 8Stenotrophomonas maltophilia (50) Gatifloxacin 025 4

Levofloxacin 025 8

Source Refs 43 44

402 Ambrose et al

in these in vitro data involves the pneumococci Gatifloxacin exhibits up to fourtimes greater potency than levofloxacin against isolates of S pneumoniae whichas mentioned earlier is the most common and most virulent pathogen associatedwith community-acquired respiratory tract infection Although it is tempting toconclude from this in vitro microbiological observation that gatifloxacin is a pref-erential choice over levofloxacin the clinical relevance if any of this observationwill become apparent in the following pharmacodynamic discussion

44 Pharmacodynamics

The pharmacodynamic properties of fluoroquinolone antibiotics have been wellelucidated Gatifloxacin and levofloxacin like all fluoroquinolones eliminatebacteria most rapidly when their concentrations greatly exceed the MIC of thetargeted organism This type of activity is referred to as concentration-dependentor time-independent killing [27ndash30] Although peakMIC ratios of greater than10ndash121 correlate with optimal bactericidal activity the AUCMIC ratio is theimportant parameter determining the efficacy of fluoroquinolones

Data obtained from animal models in vitro pharmacodynamic studies andclinical outcome studies indicate that the magnitude of the AUCMIC ratio canbe used to predict response Forrest et al [30] demonstrated that an AUCMICratio of 125 was associated with the best bacterial eradication rates in the treat-ment of infections caused by gram-negative pathogens For gram-positive bacte-ria it appears that the AUCMIC ratio may be lower An in vitro model of infec-tion demonstrated that for levofloxacin and ciprofloxacin an AUCMIC ratio ofapproximately 30 was associated with a 4 log kill while ratios less than 30 wereassociated with a significantly reduced extent of bacterial killing and in someinstances bacterial regrowth [31] In like fashion Lister and Sanders [32] reportedthat for levofloxacin and ciprofloxacin an AUCMIC ratio of 32ndash64 was associ-ated with maximal eradication of S pneumoniae in an in vitro model of infectionThese data are supported by data from animal models of infection where survivalwas associated with an AUCMIC ratio of 25ndash30 against pneumococcus [33]Finally for gatifloxacin and levofloxacin bacteriological eradication was associ-ated with an AUCMIC ratio greater than 33 in patients with community-acquiredpneumonia and acute exacerbation of chronic bronchitis [34]

Clinicians often make such interquinolone comparisons based upon AUCMIC ratios Typically this is accomplished by using mean AUC values usuallyobtained from normal healthy volunteers and MIC90 values from the literatureWhen making comparisons in this manner variability in pharmacokinetic (AUC)and microbiological (MIC) data is not accounted for [35] Thus the metrics devel-oped applies only to a minimum number of clinical instances

Pharmacodynamic modeling using Monte Carlo simulation is one approachthat takes into account variability in microbiological and pharmacokinetic data


Monte Carlo simulation estimates the probability of achieving a pharmacody-namic outcome This is accomplished by selecting an AUC value from a popula-tion of actual patients (in proportion to their probability of occurrence) and pair-ing it with an MIC value from a clinical isolate (also in proportion to theprobability of occurrence) This is done for thousands of iterations resulting ina probability distribution that reflects the chance that a pharmacodynamic targetwill be achieved in a patient treated with a given drug

Ambrose et al performed such an analysis using gatifloxacin and levoflox-acin pharmacokinetic data obtained from adult patients (18 years or older) en-rolled in clinical trials conducted at multiple centers [36] and susceptibility datafor S pneumoniae from almost 2000 isolates [37] The gatifloxacin data set in-cluded 64 acutely ill patients all of whom had community-acquired infectionsthe pharmacokinetic data set for levofloxacin included 172 acutely ill patientsall of whom had community-acquired infections [38] AUC and MIC probabilitydistributions based on these data were then plotted for both agents and a 5000-patient Monte Carlo simulation was performed

Following standard 500 mg doses of levofloxacin the probability of achiev-ing an AUCMIC ratio of greater than 30 against S pneumoniae was 80(Fig 4) With standard 400 mg doses of gatifloxacin the probability of achieving

FIGURE 4 Pharmacodynamics of levofloxacin against Streptococcus pneu-moniae Results of a 5000 patient Monte Carlo simulation based upon thevariability in the pharmacokinetics in the population of patients modeled andthe MIC variability observed in a large susceptibility surveillance programThe dark-colored bars represent the number of simulated patients with AUCMIC ratios less than 30 whereas the light-colored bars represent the numberof simulated patients with AUCMIC ratios of 30 or greater The probabilityof attaining an AUCMIC ratio of at least 30 is approximately 80

404 Ambrose et al

FIGURE 5 Pharmacodynamics of gatifloxacin against Streptococcus pneu-moniae Results of a 5000 patient Monte Carlo simulation based upon thevariability in the pharmacokinetics in the population of patients modeled andthe MIC variability observed in a large susceptibility surveillance programThe dark-colored bars represent the number of simulated patients with AUCMIC ratios less than 30 whereas the light-colored bars represent the numberof simulated patients with AUCMIC ratios of 30 or greater The probabilityof attaining an AUCMIC ratio of at least 30 is approximately 94

an AUCMIC ratio of greater than 30 for S pneumoniae was 94 (Fig 5) Thesedata suggest that in treating patients with community-acquired pneumonia dueto S pneumoniae the pharmacodynamic target hit rate is greater for gatifloxacinthan for levofloxacin Interestingly these pharmacodynamic target hit rates mir-rored the bacteriological eradication rates observed in randomized double-blindmulticenter phase III clinical trials involving these two agents [39] Unfortu-nately currently there are no comparative pharmacoeconomic data available forthese two agents

45 Conclusion

Based upon the foregoing pharmacodynamic analysis it would be predicted thatthe use of gatifloxacin would achieve a greater pharmacodynamic target hit rateagainst pneumococci than levofloxacin In the era of multidrug-resistant pneumo-cocci this may result in the less rapid selection of resistant strains if gatifloxacinis used preferentially over levofloxacin However more clinical data will beneeded to confirm or refute this hypothesis Nonetheless due to the aforemen-tioned pharmacodynamic differences between these two agents it may be reason-able to select gatifloxacin over levofloxacin for the treatment of community-acquired respiratory tract infections


5 SUMMARY

In this chapter pharmacodynamic theory and pharmacoeconomic principles wereused as a guide for rational decision making The use of these principles can beapplied to any drug or drug class and should be considered an aid in a data-driven decision-making paradigm Our purpose was not to convince the readerto select one antimicrobial agent over the other in the examples given but toillustrate how pharmacodynamic and other data can and have been used for deci-sion making

REFERENCES

1 Product information Fortaz (Ceftazidime sodium) Glaxo Pharmaceuticals June1994

2 Product information Maxipime (Cefepime hydrochloride) Bristol-Myers SquibbCompany January 1996

3 Barbhaiya RH Knopp CA Forgue ST Matzke GR Guay DRP Pittman KA Phar-macokinetics of cefepime in subjects with renal insufficiency Clin Pharmacol Ther199048268ndash276

4 Ambrose PG Ceftazidime Antibio Clin 1997144ndash495 Marshall SA Aldridge KE Allen SD Fuchs PC Gerlach EH Jones RN Compara-

tive antimicrobial activity of piperacillin-tazobactam tested against more than 5000recent clinical isolates from five medical centers Diagn Microbiol Infect Dis 199521153ndash168

6 Hiraoka M Inoue M Mitsuhashi S Hydrolytic rate at low drug concentration as alimiting factor in resistance to newer cephalosporins Rev Infect Dis 198810746ndash751

7 Chong Y Lee K Kwon OH In-vitro activities of cefepime against Enterobactercloacae Serratia marcescens Pseudomonas aeruginosa and other gram-negativebacilli J Antimicrob Chemother 199332(suppl 3)21ndash29

8 Quintiliani R Nicolau DP Nightingale CH Clinical relevance of penicillin-resistantStreptococcus pneumoniae Infect Dis Clin Prac 19965S37ndashS41

9 Chow JW Fine MJ Shales DM Quinn JP Hooper DC Johnson MP et al Entero-bacter bacteremia Clinical features and emergence of antibiotic resistance duringtherapy Ann Intern Med 1991115585ndash590

10 Owens RC Ambrose PG Quintiliani R Ceftazidime to cefepime formulary switchPharmacodynamic and pharmacoeconomic rationale Conn Med 1997225ndash227

11 Ambrose PG Richerson MA Stanton ME Bui K Nicolau DP Nightingale CH etal Cost-effectiveness analysis of cefepime compared with ceftazidime in intensivecare unit patients with hospital-acquired pneumonia Infect Dis Clin Prac 19998245ndash251

12 Owens RC Owens CA Holloway WJ Comparative evaluation of the cefepime Q12hour dosing interval as empirical therapy for febrile neutropenic patients Pharmaco-therapy 199919496ndash497

406 Ambrose et al

13 Product information Zithromax (azithromycin) Pfizer Inc June 199614 Product information Biaxin (clarithromycin) Abbott Laboratories August 199415 Shepard RM Fouda HG Johnson RC et al Disposition and metabolism of azithro-

mycin in rats dogs and humans Int Congr Infect Dis Montreal Quebec July 15ndash19 1990 abstr 184

16 Jorgenson JH Maher LA Howell AW Activity of clarithromycin and its principalhuman metabolite against Haemophilus influenzae Antimicrob Agents Chemother1994351524ndash1526

17 Craig WA Rikardsdottir S Watanable Y In vivo and in vitro post-antibiotic effectsof azithromycin Abstracts of the 32nd Interscience Conference on AntimicrobialAgents and Chemotherapy Anaheim September 1992

18 Odenholt-Tornqvist I Lowdin E Cars O Post-antibiotic effects and post-antibioticsub-MIC effects of roxithromycin clarithromycin and azithromycin on respiratorytract pathogens Antimicrob Agents Chemother 199539221ndash226

19 Nightingale CH This volume Chapter 920 Jackson MA Burry VF Olson LC Duthie SE Breakthrough sepsis in macrolide-

resistant pneumococcal infection Pediatr Infect Dis 1996151049ndash105021 Leach AJ Shelby-James TM Mayo M Gratten M Laming AC Currie BJ Mathews

JD A prospective study of the impact of community-based azithromycin treatmentof trachoma on carriage and resistance of Streptococcus pneumonia Clin Infect Dis199724356ndash362

22 Davies BI Maesen FPV Gubbelmans R Azithromycin (CP-62993) in acute exacer-bations of chronic bronchitis An open clinical microbiological and pharmacokineticstudy J Antimicrob Chermother 198823743ndash751

23 Guggenbichler JP Kastner U Influence of antibiotics on the normal flora InfectMed 199815(suppl A)15ndash22

24 van Barlingen H Volmer T Lacey LF The clinical failure rate of first-line antibiotictreatment is the key cost driver in the treatment of patients with severe community-acquired lower respiratory tract infections Abstracts 6th Intl Symp on QuinolonesDenver CO November 1998

25 Lacey LF Volmer T Harris AM The microbiological eradication rate of first-lineantibiotic treatment is a key cost driver in the treatment of acute exacerbation ofchronic bronchitis Abstracts 6th Intl Symp on Quinolones Denver CO November1998

26 Weiss LR A clinical study of macrolide therapy in a select patient population Ab-stracts 4th Intl Conf on the Macrolides Azilides Streptogramins and KetolodesBarcelona Spain January 1998


28 Drusano GL Johnson DE Rogan M Standford HC Pharmacodynamics of a fluor-oquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sepsisAntimicrob Agents Chemother 199337483ndash490

29 Dudley MN Pharmacodynamics and pharmacokinetics of antibiotics with specialreference to the fluoroquinolones Am J Med 1991 91(suppl 6A)45Sndash50S


30 Forrest A Nix DE Ballow CH Schentag JJ Pharmacodynamics of intravenous ci-profloxacin in seriously ill patients Antimicrob Agents Chemother 1993371073ndash1081

31 Lacy ML Lu W Xu X Nicolau DP Quintiliani R Nightingale CH Pharmacody-namic comparisons of levofloxacin and ciprofloxacin against Streptococcus pneu-moniae in an in vitro model of infection Antimicrob Agents Chemother 199943672ndash677


33 Vesga O Craig WA Activity of levofloxacin against penicillin-resistant Strepto-coccus pneumoniae in normal and neutropenic mice Abstracts 36th InterscienceConf on Antimicrobial Agents and Chemotherapy New Orleans Sept 15ndash181996

34 Ambrose PG Grasela DM Pharmacodynamics of fluoroquinolones against Strep-tococcus pneumoniae Analysis of phase-III clinical trials Abstracts 40th Inter-science Conf on Antimicrobial Agents and Chemotherapy Toronto Sept 17ndash202000

35 Ambrose PG Quintiliani R Limitations of single-point pharmacodynamic analysisPediatr Infect Dis J 200019769

36 Ambrose PG Grasela DM Effect of pharmacokinetic and microbiological variabil-ity on the pharmacodynamics of gatifloxacin and levofloxacin against Streptococcuspneumoniae Abstracts 39th Interscience Conf on Antimicrobial Agents and Che-motherapy San Francisco Sept 26ndash29 1999

37 Jones RN Pfaller MA Doern GV Beach M Antimicrobial activity of gatifloxacina newer 8-methoxy fluoroquinolone tested against over 23000 recent clinical iso-lates from the SENTRY antimicrobial surveillance program Abstracts 37th Intersci-ence Conf on Antimicrobial Agents and Chemotherapy San Diego Sept 24ndash271997


39 Mayer H Pierce P Ambrose PG Bacteriologic eradication of gram-positive organ-isms Gatifloxacin and levofloxacin Abstracts Am Thoracic Soc Chicago May 5ndash10 2000

40 Fung-Tomc J Huczko E Stickle T Minassian B Kolek K Denbleyker KBonner D Kessler R Antibacterial effects of cefprozil compared with those of13 oral caphems and 3 macrolides Antimicrob Agents Chemother 199539(2)533ndash538

41 Doern GV Brueggemann AB Pierce G Hogan T Holley HP Rauch A Prevalenceof antimicrobial resistance among 723 outpatient clinical isolates of Moraxella cat-tarhalis in the United States in 1994 and 1995 Antimicrob Agents Chemother 199640(12)2884ndash2886

42 Doern GV Brueggeman AB Pierce G Holley HP Rauch A Antibiotic resistanceamong clinical isolates of Haemophilus influenzae in the United Sataes in 1994 and1995 Antimicrob Agents Chemother 199741(2)292ndash297

408 Ambrose et al

43 Bauerfeind A Comparison of the antibacterial activities of the quinolones BAY12-8039 gatifloxacin trovefloxacin clinafloxacin levofloxacin and ciprofloxacin JAntimicrob Therapy 199740639ndash651

44 Goldstein EJC Citron DM Merriam CV Tyrell K Warren Y Activity of gatifloxa-cin compared to those of five other quinolones versus aerobic and anaerobic isolatesfrom skin and soft tissue samples of human and animal bite wound infections Anti-microb Agents Chemother 199943(6)1475ndash1479

409

Index

Aminoglycosides 125clinical use and application 139mechanism of action 126microbiologic spectrum 127PAE 133pharmacodynamics 129pharmacokinetics 128resistance 137synergy 137toxicodynamics 138

Animal models (studies) 10 67beta-lactams 105changing drug elimination 76changing drug input 76design issues 73 81

comparison to humanpharmacokinetics 75

drug assay 74modeling 74sampling 73

endocarditis 86endpoints 68 82mathematical indices 79meningitis model 85models 83

thigh infection 83

[Animal models (studies)]osteomyelitis 86quantifying 72quinolones 163peritonitissepticemia model 84pharmacokinetics 72pneumonia animal model 83pyelonephritiskidney model 84relevance to humans 68resistance 89selection of appropriate PK-PD

models 82streptogramins 236vancomycin 188

Antibacterial resistance 327Antifungals 285

Amphotercin B 287in vitro data 288in vivo data 290

animal models 286antifungal combinations 296azoles 292

in vitro data 292clinical studies 294

clinical data 287flucytosine 294

410 Index

[Antifungals]in vitro data 294in vivo data 295

in vitro data 286Antivirals 259

clinical studies of pharmacodynamics272

in vitro systems 260protein binding 262

Area under the concentration time curve(AUC) 159

AUCquinolones 160

Azalides 205pharmacodynamic model 213pharmacodynamics 207tissue distribution 208

Biofilm-catheter model 54Beta-lactamase inhibitor combinations

111Beta-lactams 99Breakpoints 89 155

Clindamycin 227antimicrobial activity 227chemistry 227in vitro studies 228mechanism of action 227pharmacodynamics 228pharmacokinetics 228PAE 230serum bactericidal titer 229serum inhibitory titer 229

Clinical decision making 385azithromycin 393

microbiology 394pharmacodynamics 394pharmacokinetics 394


cefepime 386microbiology 387pharmacodynamics 390

[Clinical decision making]pharmacoeconomics 391pharmacokinetics 387




Clinical efficacy 33Clinical trial data

drug exposure 308endpoints 304human pharmacodynamics 303pathogen identification 304susceptibility determination 304

Clinical pharmacodynamic targets 28160

Clinical practice 35application of pharmacodynamic pa-

rameters 35Clinical studies 106Combination therapy in vitro model 51Concentration-dependent killing 158

aminoglycosides 125metronidazole 225quinolones 155

Continuous infusionbeta-lactams 106

Doxycycline 249Drug combinations 12

Effect of antimicrobial agents on micro-organisms 24

Endocarditis model 86Experimental infection models 235

streptogramins 235

Index 411

Fibrin clot in vitro model 55Formulary decision making 385

azithromycin 393microbiology 394pharmacodynamics 394pharmacokinetics 394


cefepime 386microbiology 387pharmacodynamics 390pharmacoeconomics 391pharmacokinetics 387




Free drug 163

Glycylcyclines 250

Human infections 12Human studies

quinolones 165vancomycin 188

Intracellular pathogens in vitro model57

In vitro models 41bacterial adherence 62beta-lactams 104clindamycin 228dilutionary artifacts 62limitations 59

altered bacterial growthcharacteristics 60

[In vitro models]morphology 60physiology 60sensitivity to serum 61virulence 60

effects of immune system 59microorganism viability 61protein binding 61quinolones 163resistance 113streptogramins 236vancomycin 185

In vitro studies 104

Ketolides 205Killing

bacterial activity 2Kinetic bacterial killing properties 26

LY333328 197

Macrolides 205azithromycin 393 394 398clarithromycin 393 394 398pharmacodynamic model 213pharmacodynamics 207tissue distribution 208

MBC 25Meningitis animal model 85Metronidazole 221

absorption 223antimicrobial activity 222

bacteria 223protozoa 223

bactericidal effect 225chemistry and mechanism of action

222concentration dependent killing 225distribution 224excretion 225metabolism 224PAE 226pharmacodynamics 225pharmacokinetics 223serum bactericidal titers 226minocycline 249

412 Index

MIC 25Microbiology

cefepime 387ceftazidime 387gatifloxacin 399levofloxacin 399

Minimum bactericidal concentration 2Minimum inhibitory concentration 2

New antimicrobials and formulations 15

One-compartment in vitro model 43Osteomyelitis animal model 86Outcome data

quinolones 163

Parameters for microbiological activityof antimicrobial agents 25

Patterns of antimicrobial activity 5Peak concentration ratio (CpMIC) 160PeakMIC

resistance 352quinolones 160

PeritonitisSepticemia animal model 84Pharmacodynamic analysis 169

single point 170Monte Carlo 171

Pharmacodynamic concepts 1 158Pharmacodynamics

aminoglycosides 125 129animal models 82azithromycin 394beta-lactams 99

correlation to in vitro resistance 113cefepime 391ceftazidime 391clarithromycin 394clindamycin 228clinical efficacy 33 35clinical use and application 139gatifloxacin 402human clinical trials 303levofloxacin 402magnitude required for clinical

efficacy 9

[Pharmacodynamics]metronidazole 225new dosage regimens 14parameters 5 29pharmacokinetics versus pharmacody-

namics 100quinolones 167resistance 349 352

peakMIC 352streptogramins 234tetracyclines 251

Pharmacoeconomics 367 368azithromycin 398cefepime 391ceftazidime 391clarithromycin 398cost 369data sources 379decision analysis 378evaluating studies 380methodological considerations

375outcomes 372

measures 372perspective 369types 370

cost benefit 371cost effectiveness 371Pharmacokinetics 28

aminoglycosides 125 128animal models 72 82

comparison to humans 75basic principles 184cefepime 387ceftazidime 387clindamycin 228gatifloxacin 399glycylcyclines 250doxycycline 249levofloxacin 399minocycline 249quinolones 161magnitude required for clinical

efficacy 9metronidazole 223parameters 5

Index 413

[Pharmacokinetics]pharmacodynamics versus pharmaco-

kinetics 100streptogramins 233tissue fluid 28tetracyclines 248

Pneumonia animal model 83Post-Antibiotic Effect (PAE) 3 87

101 133 159clindamycin 230metronidazole 226

Post antibiotic leukocyte enhancement4

Protein binding 89 103Prevention of resistance 14Pyelonephritiskidney animal model

84

Quinolones 155animal models (studies) 163AUC 160human studies 165in vitro models (studies) 163outcome data 163peakMIC 160

Quinolone generation 156

Resistance 89 137 327acquired 350antibacterial 327automated susceptibility systems 344detection in clinical laboratory 333

clinical relevance susceptibilitytests 333

emerging problems 328gram-negative bacteria 329

E coli 330H influenzae 329K pneumoniae 330other enterobacteriaceae 331P aeruginosa 333

gram-positive bacteria 328enterococcus 328S aureus 328S pneumoniae 329

[Resistance]interpretive breakpoints 343methods for detection 334

aminoglycoside 335beta-lactam 335cat 339chloramphenicol acetyltransferase

339direct detection of beta-lactamases

337gene products 337glycopeptide 335

mutational 350qualitative assays 347

disk diffusion 347ESBL disk diffusion 347

quantitative assays 341agar dilution 342e-test 342macrobroth dilution 341microbroth dilutions 342

pharmacodynamics and resistance349 352

ceftazidime 354levofloxacin 353ticarcillin 354

screening assays 344ESBL 346high level aminoglycoside screen

346MRSA screen 346vancomycin screen 346

susceptibility methods phenotypic339

in vivo correlation 339growth in or on artificial media

340inoculum size 340pharmacodynamic correlations

341protein binding 340

Serum bactericidal titers 226clindamycin 229metronidazole 226

Serum inhibitory titer 229

414 Index

Streptogramin 231absorption 232animal models 236antimicrobial activity 231bactericidal effect 234chemistry 231distribution 232excretion 234experimental infection models 235in vitro models 236mechanism of action 231metabolism 233PAE 235pharmacodynamics 234pharmacokinetics 232synergy 137 238

Sub-MIC effects 4Susceptibility breakpoint determinations

15

Teicoplanin 196Tetracyclines 247

doxycycline 249glycylcyclines 250minoclycline 249pharmacokinetics 248pharmacodynamics 251

Thigh infection model 83Time-dependent killing 158

beta-lactams 99macrolides 205

[Time dependent killing]tetracyclines 247vancomycin 178

Tissue distributionazalides 208macrolides 208

Tissue fluid 28Tissue penetration 109Total drug 163Toxicodynamics aminoglycosides

138Transitional therapy 156Two-compartment in vitro model

48

Vancomycin 178adverse drug reactions 193animal studies 188antimicrobial spectrum 178clinical application 189clinical uses 189dosing 193history 178human studies 188inappropriate uses 192in vitro studies 185pharmacokinetics 180pharmacology 179resistance 181therapeutic monitoring 193toxicity 193

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