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

PL ToutainNational Veterinary school; Toulouse

Wuhan 8 October 2015

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

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

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

For pulmonary infection, plasma free antibiotic concentration, not the epithelial Lining Fluid (ELF), is the best surrogate of

biophase concentration

Second (marketing) consensus

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

MIC distribution for M haemolytica & P multocida (2004-20010) for tulathromycin

The PK/PD issue for macrolides (triamilides): plasma concentration lower

than MICs

• Good clinical efficacy and bacteriological cure with macrolides is achievable with plasma concentrations (much) lower, than the in vitro MICs for major lung pathogens

• Good clinical efficacy and bacteriological cure with macrolides is achievable with plasma concentrations (much) lower, than the in vitro MICs for major lung pathogens

Cmax=0.5µg/mL ≤ to MIC90 Cmax=0.5µg/mL ≤ to MIC90

MICMIC

MIC in MHB vs. calf serum25%,50%,75% and 100%

25% 50% 75% 100 %

MIC in MHBMIC in MHB

MIC in serumMIC in serum

9

The case of tulathromycin

• See presentation entitled “ how to establish a dosage regimen for a sustainable use of antibiotics

10

1: Where are located the pathogens and where is the

biophase

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

2: What are Antibiotic concentrations that are considered in the veterinary

literature to explain antibiotic efficacy?

Leipzig 2009 17

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)

Leipzig 2009 19

3: why a total tissular concentration has no meaning

• 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

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.

Leipzig 2009 22

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

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.

4: what are the methods for studies of target site drug distribution in

antimicrobial chemotherapy

Prague 2008 25Muller & al AAC 2004

PET images following administration of 18f-trovafloxacine

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

The tissue cage model for in vivo and ex vivo investigations

Leipzig 2009 28

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

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

The Tissue cage model: veterinary application

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

• To assess the influence pf inflammation by comparing exudate and transudate concentration

• To investigate PK/PD relationship: YES– ex vivo : killing curves (exudate/transudate)– in vivo : Greko (inoculation of the tissue cage)

5-Microdialysis & ultrafiltration Techniques

What is microdialysis (MD)?

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

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.

Microdialysis : Limits

• MD need to be calibrated• Retrodialysis method

– tedious.– The in vivo percent recovery is calculated

(CV of about 10-20%)

A small experimental error in recovery estimate results in a relatively larger error in

drug concentration estimates which is probably responsible for the greater

interanimal variability observed in lung tissue than in the other media

Marchand & al AAC June 2005

MD need to be calibrated:

Ultrafiltration

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

• Unlike MD, UF-

sample concentrations are independent on probe diffusion characteristics

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;

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)

42

This study’s objectives were to determine intestinal antimicrobial concentrations in calves administered enrofloxacin or ceftiofur sodium subcutaneously, and their impact on representative enteric bacteria

Ultrafiltration devices were implanted in the ileum and colon of 12 steers,

Enrofloxacin (SQ, 7.5mg/kg)

43

AUC (enro+cipro)•Plasma=19 (total)

•ISF=25 (free)•Ileaum=21 (free)

•Spiral colon =36 (free)

AUC (enro+cipro)•Plasma=19 (total)

•ISF=25 (free)•Ileaum=21 (free)

•Spiral colon =36 (free)

Ceftiofur SQ (2.2mg/kg)

44

AUC•Plasma=137 (total)

•ISF=15 (free)•Ileum=40 (free)

•Spiral Colon =34 free)

AUC•Plasma=137 (total)

•ISF=15 (free)•Ileum=40 (free)

•Spiral Colon =34 free)

6-What we learnt with animal and human microdialysis studies

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)

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

Total (plasma)free (muscle)free (plasma)

Liu J.A.C. 2002

cefpodoxine

cefixime

What we learnt with animal and human MD studies

• MD studies showed that:– the concentrations in ISF of selected antibiotics

correspond to unbound concentrations in plasma and are much lower than concentrations reported from whole-tissue biopsy specimens.

– Concentrations of beta –lactams and aminoglycosides in ISF are mostly in the range of free concentrations in serum

– Concentration of quinolones and macrolides at their target site are considerably lower than those predicted from tissue biopsy specimens

What we learnt with animal and human microdialysis studies

• Free plasma concentration is a good surrogate of most interstial fluid (ISF) concentration

MIC measured in MHB is homogeneous to a “free

concentration”

Effect of protein binding on antimicrobial activity

MICs of Staphylococcus aureus (Data from Kunin et al 1973)

0

1

2

3

4

5

6

Ampi Methy Benz NAF Oxa Cloxa

MIC Broth

MIC Serum

Cf for MIC Serum

MIC

(µ

g/m

L)

fb 0.22 0.37 0.65 0.90 0.93 0.95

The free concentration paradigm in pharmacokinetics is supported by MD

findings

Blood/Plasma Interstitial fluid

Total

Measured by analytical technique

Plasma bound

Free

ISF bound

Free Free

Tissue bound

BUG

Elimination

Tissular space

What we learnt with MD studies: Inflammation

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

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

7-The issue of lung penetration

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)?

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

Possible confounding factors in interpreting ELF concentrations of

antibiotics measured by BAL

• ELF: Epithelial lining Fluid

• BAL:bronchoalveolar lavage

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

• 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)

• 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.

• .

Kiem & Schentag’ Conclusions (2)

8-The site of infection: Intracellular pathogens

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.

Key questions for intracellular antibiotherapy

• Where are the bacteria ?• Which antibiotics accumulate in cells ?• Where are antibiotics located ?• What is the intracellular expression of activity ?• What is the bacterial responsiveness ?• Cooperation with the cell own defenses and

cytokines ?

Tulkens - Bangalore

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

Leipzig 2009 68

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

Which antibiotics do accumulate in cells ?

• beta-lactams : 1 x

• aminoglycosides: <1 to 2 x

• ansamycins: 2-3 x

• tetracyclines: 2-4 x

• fluoroquinolones: 10-20 x

• macrolides: 4 to > 100 x

Leipzig 2009 71

What are the antibiotic intracellular activity

Phagolysosome

Macrolides Aminoglycosides

CytosolpH=7.2

Fluoroquinolonesbeta-lactamsRifampicin

Aminoglycosides

Good Low or nul

Conclusions

PK/PD indices and tissular concentrations

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

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

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

Never use tissue concentrations to determine

an antibiotic doseAccording to EMEA

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

EMEA 2000