Leipzig 2009 1

PK/PD approach for antibiotics: tissue or blood drug level to predict antibiotic efficacy

PL ToutainNational Veterinary school; Toulouse

ECOLENATIONALEVETERINAIRET O U L O U S E

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First (scientific) consensus:The goal of PK/PD indices

1. The goal of PK/PD indices is to predict, in vivo, clinical outcomes:

• Cure• prevention of resistance

2. Plasma free concentration is the relevant concentration for the establishment of a PKPD indice

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Second (marketing) consensus

• It is more easy to promote a macrolide showing its high lung concentrations than its low plasma concentrations

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PK/PD approach for antibiotics: tissue or blood drug level to predict antibiotic

efficacy

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Objectives of the presentation:

1. The three PK/PD indices

2. Where are located the bugs ?

• Extracellular vs. intracellular

3. Where is the biophase?

• Interstitial space fluid vs. intracellular cytosol vs. intracellular organelles

4. How to assess the biophase antibiotic concentration

• Total tissular concentration vs. ISF concentration.

5. The issue of lung penetration

1. Epithelial lining fluid (ELF):?

2. he hypothesis of targeted delivery of the active drug at the infection site by phagocytes

6. Plasma as the best surrogate of biophase concentration for PK/PD interpretation

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The three PK/PD indices

Antibiotics PK/PD indices

Goal Critical values

β Lactams Time>MIC Maximize exposure time

50-100% dosage interval

Quinolones

24h AUC/MIC

ratio

Optimize the quantity of

administered AB

125 H

Aminoglycosides Cmax/MIC

ratio

Optimize

the peak concentration

10

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By essence the three PK/PD indices are hybrid parameters PK & PD

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AUC/MIC

PK:

PD:

90

/

MIC

ClearanceDose

MIC

AUC

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Time > MIC

MIC

Half-life

con

cen

trat

ion

s

Time (h)24

t1 t2

100

221

%

Ln

T

MICVd

DoseLnMICTime

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Cmax / MIC

PK

PD

• Bioavailability (%)• clearance• Rate of absorptione Rate

of elimination• Accumulation factor90

max

MIC

C

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PK/PD indices are hybrid parameters

• For all indices:– the PD input is the MIC– The PK input is associated to plasma:

why?• And why not:

1. the actual concentration at the site of action (biophase)

2. the concentration of the tissue (organs) in which the infection develops

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What is an ideal concentration for a PK/PD indice

• A relevant concentration to serve as an input in a PK/PD model should be selective of the biophase i.e. of the fluid that bath the extracellular space namely the interstitial fluid (ISF).

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Q1: Where are located the pathogens and where is the

biophase

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Where are located the pathogens

ISFMost pathogens of

clinical interest•S. Pneumoniae, E. Coli,Klebsiella

•Mannhemia ; Pasteurella

• Actinobacillius pleuropneumoniae •Mycoplasma hyopneumoniae

•Bordetalla bronchiseptia

Cell(most often in phagocytic cell)

• Mycoplasma (some)• Chlamydiae• Brucella• Cryptosporidiosis• Listeria monocytogene• Salmonella• Mycobacteria• Rhodococcus equi

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What are Antibiotic concentrations that are considered in the veterinary

literature to explain antibiotic efficacy?

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Antibiotic concentrations vs. efficacy

1. Total tissue concentrations – homogenates– biopsies

2. Extracellular fluids concentrations– implanted cages– implanted threads– wound fluid– blister fluid

– ISF (Microdialysis, Ultrafiltration)

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In veterinary medicine, there are many publications on tissular concentrations to promote the idea that some antibiotics having a high tissular concentration accumulate in biophase (quinolones, macrolides) and are more efficacious as suggested by their low or undetectable plasma concentrations

Total tissular concentration

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Statements such as ‘concentrations in tissue x h after dosing are much higher than the MICs for

common pathogens that cause disease’ are meaningless

Mouton & al JAC 2007

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Q3: why a total tissular concentration has no meaning

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• Two false assumptions1. tissue is homogenous2. bacteria are evenly distributed through

tissue

spurious interpretation of all important tissue/serum ratios in predicting the antibacterial effect of AB

The inadequate tissue penetration hypothesis: Schentag 1990

Schentag, 1990

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Total tissular concentration for betalactams and aminoglycosides

• if a compound is distributed mainly extra-cellularly (betalactams and aminoglycosides), a total tissular concentration will underestimate the active concentration at the biophase by diluting the ISF with intracellular fluids.

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Intracellular location of antibiotics

Phagolysosomevolume 1 to 5% of cell volume

pH=5.0

Macrolides (x10-50)Aminoglycosides (x2-4)

CytosolpH=7.4

Fluoroquinolones(x2-8)beta-lactams (x0.2-0.6)

Rifampicin (x2)Aminoglycosides (slow

Ion trapping for weak base with high pKa value

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Total tissular concentration for macrolides & quinolones

• if a drug is accumulated in cells (the case for fluoroquinolones and macrolides), assays of total tissue levels will lead to gross overestimation of the extracellular biophase concentration.

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Methods for studies of target site drug distribution in antimicrobial

chemotherapy

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Methods considered of limited interest for studies of target site drug distribution

• Tools developed to determine antibiotic concentrations in various surrogates for the ISF and having no pathophysiologic counterpart in humans .– in vitro models,– fibrin clots,– tissue chambers, – skin chambers(blister) – wound exudates, – surface fluids, – implanted fibrin clots, – peripheral lymph.

Muller & al AAC 2004

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The tissue cage model for in vivo and ex vivo investigations

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Methods for studies of target site drug distribution in antimicrobial

chemotherapy

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The tissue cage model

• Perforated hollow devices• Subcutaneous

implantation• development of a highly

vascularized tissue• fill up with a fluid with half

protein content of serum (delay 8 weeks)

•C.R. Clarke. J. Vet. Pharmacol. Ther. 1989, 12: 349-368

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PK in tissue cagein situ administration

• PK determined by the cage geometry (SA/V ratio is

the major determinant of peak and trough drug

level)

• T1/2 varies with the surface area / volume ratio of the tissue cage– Penicillin 5 to 20 h– Danofloxacin 3 to 30 h

Greko, 2003, PhD Thesis

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The Tissue cage model: veterinary application

• To describe PK at site of infection (calves, dogs, horses…): NO

• To investigate PK/PD relationship: YES– ex vivo : Shojaee AliAbadi & Lees

(exudate/transudate)– in vivo : Greko (inoculation of the tissue

cage)

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Microdialysis & ultrafiltration Techniques

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What is microdialysis (MD)?

• Microdialysis, a tool to monitors free antibiotic concentrations in the fluid which directly surrounds the infective agent

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Microdialysis: The Principle

• The MD Probe mimics a "blood capillary".

•There is an exchange of substances via extracellular fluid

•Diffusion of drugs is across a semipermeable membrane at the tip of an MD probe implanted into the ISF of the tissue of interest.

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Microdialysis Technique

CMA60 MicrodialysisCMA60 Microdialysis CMA60 MicrodialysisCMA60 Microdialysis

1. Introducer with CMA 60 Microdialysis Catheter

2. Outlet tube

3. Vial holder

4. Microvial

5. Inlet tube

6. Luer lock connection

7. Puncture needle.

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Microdialysis Pump

• Perfusion fluid is pumped from the Microdialysis Pump through the Microdialysis Catheter into the Microvial.

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Microdialysis : Limits

• MD need to be calibrated• Retrodialysis method

– Assumption: the diffusion process is quantitatively equal in both directions through the semipermeable membrane.

– The study drugs are added to the perfusion medium and the rate of disappearance through the membrane equals in vivo recovery.

– The in vivo percent recovery is calculated (CV of about 10-20%)

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Ultrafiltration

• Excessive (in vivo) calibration procedures are required for accurate monitoring

• Unlike MD, UF-

sample concentrations are independent on probe diffusion characteristics

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Microdialysis vs. Ultrafiltration

Ultrafiltration

Vacuum

The driving force is a pressure differential (a

vacuum) applied across the semipermeable membrane

The analyte cross the membrane by diffusion

The driving force is a concentration gradient

Microdialysis :a fluid is pumped

through a membrane;

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Marbofloxacin : plasma vs.ISFIn vivo filtration

Bidgood & Papich JVPT 2005 28 329

Microdialysis•Not suitable for long term in vivo studies

Ultrafiltration•Suitable for long term sampling (in larger animals, the UF permits complete freedom of movement by using vacutainer collection method)

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What we learnt with animal and human microdialysis studies

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Plasma (total, free) concentration vs interstitial concentration (muscle, adipose

tissue) (Moxifloxacin)

Muller AAC, 1999 Time (h)

Total (plasma, muscle)free (plasma)interstitial muscleinterstitial adipose tissue

2 6 10 12 30 4020

100

1000

Co

nce

ntr

ati

on

(n

g/m

L)

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What we learnt with MD studies: Inflammation

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Tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissue

Methods: Free Concentrations measured by microdialysis after

administration of a single intravenous dose of 500 mg.

Results:The penetration of levofloxacin into tissue appears to be unaffected by local inflammation.Same results obtained with others quinolones

Hypothesis: Accumulation of fibrin and other proteins, oedema, changed pH and altered capillary permeability

may result in local penetration barriers for drugs

Bellmann & al Br J Clin Pharmacol 2004 57

Inflammation

No inflammation

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What we learnt with MD studies: Inflammation

• Acute inflammatory events seem to have little influence on tissue penetration.

• “These observations are in clear contrast to reports on the increase in the target site availability of antibiotics by macrophage drug uptake and the preferential release of antibiotics at the target site a concept which is also used as a marketing strategy by the drug industry” Muller & al AAC May 2004

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The issue of lung penetration

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Animal and human studies MD: The issue of lung penetration

•Lung MD require maintenance under anesthesia, thoracotomy (patient undergoing lung surgery)..

•Does the unbound concentrations in muscle that are relatively accessible constitute reasonable predictors of the unbound concentrations in lung tissue (and other tissues)?

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Free muscle concentrations of cepodoxime were similar to free lung concentration and therefore provided a surrogate measure

of cefpodoxime concentraion at the pulmonary target site

Liu et al., JAC, 2002 50 Suppl: 19-22.

Cefpodoxime at steady state: plasma vs. ISF (muscle & Lung)

Plasma

Free plasma

Muscle Lung

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The blood-alveolar barrier

The alveolar epithelial cells would not be expected to permit passive diffusion of

antibiotics between cells, the cells being linked by tight junctions

•Fenestrated pulmonary capillary bed• expected to permit passive diffusion of antibiotics with a molecular weight 1,000

Epithelial lining fluid

ELF

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

space

Alveolarmacrophage

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

space

Alveolarmacrophage

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

space

Alveolarmacrophage

ABAB

AlveolarAlveolarAlveolar

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• The high ELF concentrations of some antibiotics, which were measured by the BAL technique, might be explained by possible contamination from high achieved intracellular concentrations and subsequent lysis of these cells during the measurement of ELF content.

• This effect is similar to the problem of measuring tissue content using homogenization

Kiem & Schentag’ Conclusions (1)

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• Fundamentally, ELF may not represent the lung site where antibiotics act against infection.

• In view of the technical and interpretive problems with conventional ELF and especially BAL, the lung microdialysis experiments may offer an overall better correlation with microbiological outcomes.

• We continue to express PK/PD parameters using serum concentration of total drug because these values do correlate with microbiological outcomes in patients.

Kiem & Schentag’ Conclusions (2)

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In acute infections in non-specialized tissues, where there is no abscess formation, free serum

levels of antibiotics are good predictors of free levels in tissue

fluid

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PK/PD indices and tissular concentrations

• Currently, no equivalent recommendation has been published with tissular concentration as PK input and that, for any tissue or any type of infection including intracellular infection.

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The site of infection: Intracellular pathogens

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Intracellular location of bacteria

Phagosome

Lysosome

Chlamydiae

Listeria

No fusion with lysosome

Phagolysosome

S.aureausBrucella

SalmonellaCoxiella burneti

pH=5.0

3

4

2

1

Fusion

pH=7.4

BB

B

B

B

B

B B

Cytosol

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Intracellular location of antibiotics

Phagolysosomevolume 1 to 5% of cell volume

pH=5.0

Macrolides (x10-50)Aminoglycosides (x2-4)

CytosolpH=7.4

Fluoroquinolones(x2-8)beta-lactams (x0.2-0.6)

Rifampicin (x2)Aminoglycosides (slow

Ion trapping for weak base with high pKa value

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What are the antibiotic intracellular expressions of activity

Phagolysosome

Macrolides Aminoglycosides

CytosolpH=7.2

Fluoroquinolonesbeta-lactamsRifampicin

Aminoglycosides

Good Low or nul

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The free plasma level is the most meaningful concentration

In acute infections in non-specialized tissues, where there is no abscess formation, free plasma levels of antibiotics are good predictors of

free levels in interstitial fluid

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Some statements on total tissular concentrations

• For veterinary medicine (Apley, 1999)– people who truly understand tissue

concentration work in corporate marketing departments

• For human medicine (Kneer, 1993)– tissular concentrations are inherently inaccurate – tissular concentrations studies little contribute to

the understanding of in vivo efficacy and optimal dosing

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Tissue concentrations

According to EMEA

"unreliable information is generated from assays of drug concentrations in whole tissues (e.g. homogenates)"

EMEA 2000

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Conclusions:

1. In acute infections in non-specialized tissues, where there is no abscess formation, free plasma levels of antibiotics are good predictors of free levels in interstitial fluid

2. PK/PD indices predictive of antibiotic efficacy should be based on free plasma concentration

3. People who truly understand tissue concentration work in corporate marketing departments (Apley, 1999)