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Plasma vs. tissue concentration to predict antibiotic efficacy. ECOLE NATIONALE VETERINAIRE T O U L O U S E. PL Toutain UMR 181 Physiopathologie et Toxicologie Experimentales INRA/ENVT. Fourth International conference on AAVM Prague, Czech republic 24-28, 2008.

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plasma vs tissue concentration to predict antibiotic efficacy

Plasma vs. tissue concentration to predict antibiotic efficacy

ECOLE

NATIONALE

VETERINAIRE

T O U L O U S E

PL Toutain

UMR 181 Physiopathologie et Toxicologie Experimentales

INRA/ENVT

Fourth International conference on AAVM

Prague, Czech republic 24-28, 2008

the inadequate tissue penetration hypothesis
The inadequate tissue penetration hypothesis

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

the inadequate tissue penetration hypothesis schentag 1990
The inadequate tissue penetration hypothesis: Schentag 1990
  • Two false assumptions
    • tissue is homogenous
    • bacteria are evenly distributed through tissue
  • spurious interpretation of all important tissue/serum ratios in predicting the antibacterial effect of AB

Schentag, 1990

slide4
Statements such as ‘concentrations in tissue x h after dosing are much higher than the MICs for common pathogens that cause disease’ aremeaningless

Mouton & al JAC 2007

objectives of the presentation to address some basic questions
Objectives of the presentation:To address some basic questions
  • Where are located the bugs ?
    • Extracellular vs. intracellular
  • Where is the biophase?
    • Interstitial space fluid vs. intracellular cytosol vs. intracellular organelles
  • What is a tissue and what is a tissue concentration
  • How to assess the biophase antibiotic concentration
    • Total tissular concentration vs. ISF concentration.
  • The issue of lung penetration
    • Epithelial lining fluid (ELF):????
    • The hypothesis of targeted delivery of the active drug at the infection site by phagocytes
  • Plasma as the best surrogate of biophase concentration for PK/PD interpretation
where are located the pathogens
Where are located the pathogens

ISF

Most bacteria of clinical interest

  • S. pneumoniae
  • E. coli
  • Klebsiella
  • Mannhemia ; pasteurella
  • Actinobacillius pleuropneumoniae
  • Mycoplasma hyopneumoniae
  • Bordetala bronchiseptia

Cell

(most often in phagocytic cell)

  • mycoplasma (some)
  • Chlamydiae
  • Brucella
  • Cryptosporidiosis
  • Listeria monocytogene
  • Salmonella
  • Mycobacteria
  • Rhodococcus equi
the interstitial space fluid is the biophase
The interstitial space fluid is the biophase
  • Most bacterial infections are located in the extracellular compartment.
  • Except few cases, In acute infections in non-specialized tissues, where there is no abscess formation, interstitial space fluid (ISF) must be considered as the actual target space for anti-infective agents
  • ISF concentrations are of primary interest

Muller et al. AAC , 2004, 48: 1441-1453

historical definition of a tissue drug concentration
Historical definition of a tissue drug concentration
  • In the past, it was used to characterize the total concentration in a homogenized biopsy sample
    • For vet medicine: a by-product of regulatory residue studies
  • It was assumed that:
    • tissue is homogenous
    • that antibiotics is evenly repartited in tissue
    • That bacteria are evenly repartited in tissue
    • Each of these assumptions is false and can be very misleading
why a total drug tissue concentrations may be misleading
Why a total drug tissue concentrations may be misleading?
  • Drug distributed mainly extracellularly
    • β-lactams and aminoglycosides,
    • grinding up the tissue means dilution of the drug by mixing intracellular and extracellular fluids, resulting in underestimationof its concentrations at the site of infection.
  • Drug accumulated by cells
    • fluoroquinolones or macrolides
    • assay of total tissue levels will lead to gross overestimation of the extracellular concentration.
    • The opposite is true for intracellular infections.
tissue concentrations
“Tissue concentrations”
  • Total tissue
    • homogenates
    • biopsies
  • Extracellular fluids
    • implanted cages
    • implanted threads
    • wound fluid
    • blister fluid
    • ISF (Microdialysis, Ultrafiltration)
the tissue cage model
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
the tissue cage model pk limitations
The tissue cage model : PK limitations
  • A foreign body
    • Not a physiological space
    • Clinical counterparts?
      • Ascitic fluid, effusions ( pericardial, pleural…)
  • Interpretation may be difficultbecause PK determined by:
    • diffusion capacity across the TC
    • TC size and geometry
      • surface area/volume ratio is the major determinant of peak and trough drug level
the tissue cage model18
(C)

Time

The tissue cage model
  • 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

Drug administration

Slow equilibration

inoculation

(C)

Time

what is microdialysis md
What is microdialysis (MD)?
  • Microdialysis, a tool to monitors free antibiotic concentrations in the fluid which directly surrounds the infective agent
microdialysis the principle
Microdialysis: The Principle
  • The MD Probe mimics a "blood capillary".
  • Diffusion of drugs is across a semipermeable membrane at the tip of an MD probe implanted into the ISF of the tissue of interest.
  • There is an exchange of substances via extracellular fluid
microdialysis the principle22
Microdialysis: The Principle
  • the implanted MD probe is perfused with the perfusate, ie, a physiologic liquid at a very slow rate.
  • Substances present in the interstitial space fluid of the investigated site can diffuse into the perfusate through a semipermeable membrane at the tip of the MD probe and appear in the dialysate.
  • Afterward the concentration in the dialysate is chemically analyzed and the true concentration in the interstitial space fluid can be calculated.

Antibiotic

microdialysis materials
Microdialysis materials
  • Introducer with CMA 60 Microdialysis Catheter
  • Outlet tube
  • Vial holder
  • Microvial
  • Inlet tube
  • Luer lock connection
  • Puncture needle.

CMA60 Microdialysis

microdialysis limits
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%)
md need to be calibrated
MD need to be calibrated:

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

ultrafiltration
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
Microdialysis vs. Ultrafiltration

Microdialysis:

a fluid is pumped through a membrane;

    • 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
marbofloxacin plasma vs isf in vivo filtration
Marbofloxacin : plasma vs.ISFIn vivo filtration
  • 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)

Bidgood & Papich JVPT 2005 28 329

plasma total free concentration vs interstitial concentration muscle adipose tissue moxifloxacin
Total (plasma, muscle)

free (plasma)

interstitial muscle

interstitial adipose tissue

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

1000

Concentration (ng/mL)

100

2

6

10

12

40

20

30

Time (h)

Muller AAC, 1999

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

cefpodoxine

Total (plasma)

free (muscle)

free (plasma)

cefixime

Liu J.A.C. 2002

what we learnt with animal and human md studies
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
    • They are generally much lower than total concentrations reported from whole-tissue biopsy specimens especially for macrolides and quinolones
tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissue
Tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissue

Hypothesis: Accumulation of fibrin and other proteins, oedema, changed pH and altered capillary permeability may result in local penetration barriers for drugs

Methods: Free Concentrations measured in six patients by microdialysis after administration of a single intravenous dose of 500 mg.

Inflammation

No inflammation

Results:The penetration of levofloxacin into tissue appears to be unaffected by local inflammation.

Same results obtained with others quinolones

Bellmann & al Br J Clin Pharmacol 2004 57

what we learnt with md studies inflammation35
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
slide36
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
animal and human studies md 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)?
slide39
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

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

Plasma

Free plasma

Lung

Muscle

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

the issue of lung penetration imipem
The issue of lung penetration:Imipem
  • The major finding of this study was the observation of virtually superimposed free IPM concentration-versus-time profiles in the three media investigated,
  • This result not only is in agreement with theory but also is consistent with most of the data in the literature.

imipenem distribution in muscle and lung interstitial fluids

Marchand & al AAC June 2005

lung infections
Lung infections
  • Uncertainty of the relevant actual location of proliferating bacteria
    • Alveoli, pulmonary interstitium, bronchioles, blood??
  • What is the biophase??
    • Epithelium lining fluid (ELF)
    • Lung IF, alveolar macrophages, tisue biopsies, blood, bronchial secretion, sputum??
      • ELF seems the most relevant specimen but potential sources of error: dilution, release of AB from alveolar macrophage in the sample
the blood alveolar barrier
Alveolar

Alveolar

Alveolar

macrophage

macrophage

macrophage

Alveolar

Alveolar

Alveolar

space

space

space

ISF

ISF

ISF

AB

AB

AB

AB

AB

AB

Alveolar

Alveolar

Alveolar

Epithelium

Epithelium

Epithelium

Capillary

Capillary

Capillary

Thigh junctions

Thigh junctions

Thigh junctions

wall

wall

wall

The blood-alveolar barrier
  • Fenestrated pulmonary capillary bed
  • expected to permit passive diffusion of antibiotics with a molecular weight 1,000

Epithelial lining fluid

ELF

(protein:<10%)

The alveolar epithelial cells would not be expected to permit passive diffusion of antibiotics between cells, the cells being linked by tight junctions

drug passage in the lung
Drug passage in the lung

ELF

Drug passage through the alveolar epithelial cells will depend on the lipophilicity and diffusibility of the antibiotics, similar to the drug entry into the central nervous system.

Kiem & Schentag AAC 2008 Jan 24-36

elf concentration possible biais
ELF concentration: possible biais

ELF

  • Measurement problems may confound the interpretation of the ELF concentrations of antibiotics.
  • Cells, especially AM cells (that constitute 3.8 to 10.0% of ELF volume) are included in the composition of ELF
  • The cells may be lysed during the measurement of antibiotic concentration in BAL-derived fluids.

Kiem & Schentag AAC 2008 Jan 24-36

beta lactams
BETA-LACTAMS

ELF

Measured ELF concentrations of the beta-lactams are well below serum concentrations, and their respective concentrations in AM cells were negligible

The low measured ELF concentrations of betalactams in comparison to their corresponding serum levels could be the result of low capacity of their unbound free fractions for penetration through the alveolar epithelial cell barriers.

Kiem & Schentag AAC 2008 Jan 24-36

macrolides and ketolides
MACROLIDES AND KETOLIDES

ELF

Measured ELF concentrations of macrolides and ketolides and their derived AUCs were consistently higher than serum levels by as much as 10-fold

the high ratios of ELF concentration

to serum concentration for macrolides and ketolides could not be explained solely on the basis of good penetration across the alveolar epithelium.

The high concentrations of macrolides and ketolides in ELF might be explained by the possible contamination of intracellular antibiotics occurring during the process of BAL.

Kiem & Schentag AAC 2008 Jan 24-36

fluoroquinolones
FLUOROQUINOLONES

Fluoroquinolones achieved higher ELF levels than their free serum concentrations

Kiem & Schentag AAC 2008 Jan 24-36

kiem schentag conclusions 1
Kiem & Schentag’Conclusions (1)
  • 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 2
Kiem & Schentag’ Conclusions (2)
  • 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.
intracellular location of bacteria
Intracellular location of bacteria

Fusion

Lysosome

B

3

pH=7.4

Phagosome

Phagolysosome

B

B

4

1

B

S.aureaus

Brucella

Salmonella

Coxiella burneti

pH=5.0

B

B

B

2

Chlamydiae

B

No fusion with lysosome

Listeria

Cytosol

intracellular location of antibiotics
Intracellular location of antibiotics

Phagolysosome

volume 1 to 5% of cell volume

pH=5.0

Cytosol

pH=7.2

Fluoroquinolones(x2-8)

beta-lactams (x0.2-0.6)

Rifampicin (x2)

Aminoglycosides (slow)

Macrolides (x10-50)

Aminoglycosides (x2-4)

Ion trapping for weak base with high pKa value

what are the antibiotic intracellular expressions of activity
What are the antibiotic intracellular expressions of activity

Phagolysosome

Cytosol

pH=7.2

Fluoroquinolones

beta-lactams

Rifampicin

Aminoglycosides

Good

Macrolides

Aminoglycosides

Low or nul

drug delivery by the phagocytes
Drug delivery by the phagocytes
  • Transport by "non professional" phagocytes (e.g. azithromycin by fibroblast)
  • Fibroblast acts as a reservoir for drug and macrophages
neutrophils as antibiotic delivery system
Neutrophils as antibiotic delivery system

The ability of neutrophils to migrate preferentially to sites of infection makes them attractive as a delivery mechanism for antibiotics; theoretically , with the proper cellular PK, an antibiotic could be taken up by neutrophils, which would then transport the drug and later release it . This could provide a mechanism for achieving higher levels of antibiotic in tissues i.e. directly at the nidus of infection.

Scorneaux & Shryock tilmicosine in pigs; JVPT 1998, 21: 257-258

& tilmicosine in cattle in: J. Dairy Sci. 1999, 82: 1202-1212)

macrolides how to explain efficacy of low plasma concentrations
Macrolides: how to explain efficacy of low plasma concentrations?

M

PMN

  • PK hypothesis

M

Sink or reservoir???

High local M concentration in the vicinity of the bug

M

Nucleus

2-PD hypothesis

pk model for exposure to macrolides the limits
PK model for exposure to macrolides: The limits

No In vivo data to support the hypothesis

  • Slow or very slow efflux
  • (e.g. 20% within 3 h for azithromycin) more rapid (90% within 1 h) for erythromycin)
  • Transit time in a (normal) lung capillary: 26 sec

M

M

M

M+

M

M

M

M

M

Sink rather than reservoir

  • Mass balance considerations (Tylosine in piglet 10 Kg BW, dose: 10 mg/kg)
    • Total pool of PMN=2 mL/L of blood
    • Peak Plasma concentration: 1 µg/mL
    • Accumulation ratio: 50
    • Total amount of drug located in PMN :100µg in toto =1/1000 of the dose
macrolides how to explain efficacy of low plasma concentrations61
Macrolides: how to explain efficacy of low plasma concentrations?

M

PMN

  • PK hypothesis

M

Sink or reservoir???

High local M concentration in the vicinity of the bug

Cytokine modulation, ERK signaling

MIC in vivo < MIC in vitro

M

M

Nucleus

2-PD hypothesis

conclusions
Conclusions:
  • 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
  • PK/PD indices predictive of antibiotic efficacy should be based on free plasma concentration
  • People who truly understand tissue concentration work in corporate marketing departments (Apley, 1999)
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