Transcript Diapositive 1 - Physiologie et Thérapeutique Ecole Véto Toulouse

ECOLE
NATIONALE
VETERINAIRE
TOULOUSE
Plasma vs. tissue concentration to
predict antibiotic efficacy
PL Toutain
UMR 181 Physiopathologie et Toxicologie Experimentales
INRA/ENVT
Fourth International conference on AAVM
Prague, Czech republic 24-28, 2008
Prague 2008 1
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
Prague 2008 2
The inadequate tissue penetration
hypothesis: Schentag 1990
•
Two false assumptions
1. tissue is homogenous
2. bacteria are evenly distributed through
tissue
 spurious interpretation of all
important tissue/serum ratios in
predicting the antibacterial effect of
AB
Schentag, 1990
Prague 2008 3
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
Prague 2008 4
Objectives of the presentation:
To address some basic questions
1.
Where are located the bugs ?
•
2.
Extracellular vs. intracellular
Where is the biophase?
•
Interstitial space fluid vs. intracellular cytosol vs. intracellular
organelles
3.
What is a tissue and what is a tissue concentration
4.
How to assess the biophase antibiotic concentration
•
5.
6.
Total tissular concentration vs. ISF concentration.
The issue of lung penetration
1.
Epithelial lining fluid (ELF):????
2.
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
Prague 2008 5
Q1: Where are located the
pathogens
Prague 2008 6
Where are located the pathogens
Cell
ISF
Most bacteria of clinical
interest
(most often in phagocytic cell)
•S. pneumoniae
•E. coli
•Klebsiella
•Mannhemia ; pasteurella
•Actinobacillius pleuropneumoniae
•Mycoplasma hyopneumoniae
•Bordetala bronchiseptia
•
•
•
•
•
•
•
•
mycoplasma (some)
Chlamydiae
Brucella
Cryptosporidiosis
Listeria monocytogene
Salmonella
Mycobacteria
Rhodococcus equi
Prague 2008 7
Q2: Where is the biophase
Prague 2008 8
The interstitial space fluid is the biophase
1. Most bacterial infections are located in the
extracellular compartment.
2. Except few cases, In acute infections in nonspecialized tissues, where there is no abscess
formation, interstitial space fluid (ISF) must be
considered as the actual target space for antiinfective agents
3. ISF concentrations are of primary interest
Muller et al. AAC , 2004, 48: 1441-1453
Prague 2008 9
Q3: what is a tissue & what is a
tissular concentration
Prague 2008 10
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
Prague 2008 11
Why a total drug tissue concentrations
may be misleading?
1. 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 underestimation of its concentrations at the site
of infection.
2. 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.
Prague 2008 12
Methods for studies of target site
drug distribution in antimicrobial
chemotherapy
Prague 2008 13
“Tissue concentrations”
• Total tissue
– homogenates
– biopsies
• Extracellular fluids
– implanted cages
– implanted threads
– wound fluid
– blister fluid
– ISF (Microdialysis, Ultrafiltration)
Prague 2008 14
The tissue cage model for in
vivo and ex vivo investigations
Prague 2008 15
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
Prague 2008 16
The tissue cage model :
PK limitations
• A foreign body
– Not a physiological space
– Clinical counterparts?
• Ascitic fluid, effusions ( pericardial, pleural…)
• Interpretation may be difficult because 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
Prague 2008 17
The tissue cage model
Drug administration
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
Slow
equilibration
inoculation
(C)
(C)
Time
Time
Prague 2008 18
Microdialysis & ultrafiltration
Techniques
Prague 2008 19
What is microdialysis (MD)?
• Microdialysis, a tool to monitors free
antibiotic concentrations in the fluid which
directly surrounds the infective agent
Prague 2008 20
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.
Prague 2008 21
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
Prague 2008 22
Microdialysis materials
CMA60 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.
Prague 2008 23
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%)
Prague 2008 24
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
Prague 2008 25
Ultrafiltration
• Excessive (in vivo)
calibration
procedures are
required for accurate
monitoring
• Unlike MD, UFsample
concentrations are
independent on
probe diffusion
characteristics
Prague 2008 26
Microdialysis vs. Ultrafiltration
Ultrafiltration
Vacuum
The driving force is a pressure
differential (a vacuum) applied
across the semipermeable
membrane
Microdialysis :
a fluid is pumped
through a membrane;
The analyte cross the
membrane by diffusion
The driving force is a
concentration gradient
Prague 2008 27
Marbofloxacin : plasma vs.ISF
In 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
Prague 2008 28
What we learnt with animal and
human microdialysis studies
Prague 2008 29
Concentration (ng/mL)
Plasma (total, free) concentration vs
interstitial concentration (muscle, adipose
tissue) (Moxifloxacin)
Total (plasma, muscle)
free (plasma)
interstitial muscle
interstitial adipose tissue
1000
100
2
Muller AAC, 1999
6
10
12 20
30
40
Time (h)
Prague 2008 30
Plasma (total, free) concentration
vs muscle (free) concentration
cefpodoxine
Total (plasma)
free (muscle)
free (plasma)
cefixime
Liu J.A.C. 2002
Prague 2008 31
What we learnt with animal and human
MD studies
•
MD studies showed that:
1. the concentrations in ISF of selected
antibiotics correspond to unbound
concentrations in plasma
2. They are generally much lower than total
concentrations reported from whole-tissue
biopsy specimens especially for macrolides
and quinolones
Prague 2008 32
What we learnt with MD studies:
Inflammation
Prague 2008 33
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
Inflammation
No inflammation
Bellmann & al Br J Clin Pharmacol 2004 57
Methods: Free Concentrations
measured in six patients 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
Prague 2008 34
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
Prague 2008 35
In acute infections in nonspecialized tissues, where there is
no abscess formation, free serum
levels of antibiotics are good
predictors of free levels in tissue
fluid
Prague 2008 36
The issue of lung penetration
Prague 2008 37
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)?
Prague 2008 38
Cefpodoxime at steady state:
plasma vs. ISF (muscle & Lung)
Plasma
Free plasma
Muscle
Lung
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.
Prague 2008 39
The issue of lung penetration:
Imipem
imipenem distribution in muscle and lung interstitial fluids
Marchand & al AAC
June 2005
• 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.
Prague 2008 40
The issue of lung penetration
Prague 2008 41
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
Prague 2008 42
Prague 2008 43
•Fenestrated pulmonary capillary bed
• expected to permit passive diffusion of
antibiotics with a molecular weight 1,000
The blood-alveolar barrier
Alveolar
macrophage
Alveolar
space
ISF
Epithelial lining fluid
AB
Capillary
wall
AB
Alveolar
Epithelium
Thigh junctions
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
Prague 2008 44
Drug passage in the lung
ELF
ISF
AB
Capillary
wall
AB
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.
Alveolar
Epithelium
Thigh junctions
Kiem & Schentag AAC 2008 Jan 24-36
Prague 2008 45
ELF concentration: possible biais
ELF
• Cells, especially AM cells (that
constitute 3.8 to 10.0% of ELF
volume) are included in the
composition of ELF
ISF
AB
Capillary
wall
• Measurement problems may
confound the interpretation of the
ELF concentrations of antibiotics.
AB
•
Alveolar
Epithelium
Thigh junctions
The cells may be lysed during the
measurement of antibiotic
concentration in BAL-derived
fluids.
Kiem & Schentag AAC 2008 Jan 24-36
Prague 2008 46
BETA-LACTAMS
ELF
ISF
AB
Capillary
wall
AB
Alveolar
Epithelium
Thigh junctions
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
Prague 2008 47
MACROLIDES AND KETOLIDES
ELF
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.
ISF
AB
Capillary
wall
Measured ELF concentrations of macrolides and
ketolides and their derived AUCs were consistently
higher than serum levels by as much as 10-fold
AB
Alveolar
Epithelium
Thigh junctions
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
Prague 2008 48
FLUOROQUINOLONES
Fluoroquinolones
achieved higher ELF
levels than their free
serum concentrations
ISF
AB
Capillary
wall
AB
Alveolar
Epithelium
Thigh junctions
Kiem & Schentag AAC 2008 Jan 24-36
Prague 2008 49
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
Prague 2008 50
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.
Prague 2008 51
The site of infection:
Intracellular pathogens
Prague 2008 52
Intracellular location of bacteria
Fusion
B
3
pH=7.4
Phagosome
1
B
Lysosome
4
B
2
B
B
Chlamydiae
No fusion with lysosome
Phagolysosome
B
S.aureaus
B Brucella B
Salmonella
Coxiella burneti
pH=5.0
Listeria
Cytosol
Prague 2008 53
Intracellular location of antibiotics
Phagolysosome
Cytosol
pH=7.2
Fluoroquinolones(x2-8)
beta-lactams (x0.2-0.6)
Rifampicin (x2)
Aminoglycosides (slow)
volume 1 to 5% of cell volume
pH=5.0
Macrolides (x10-50)
Aminoglycosides (x2-4)
Ion trapping for weak base
with high pKa value
Prague 2008 54
What are the antibiotic intracellular
expressions of activity
Phagolysosome
Cytosol
pH=7.2
Fluoroquinolones
beta-lactams
Rifampicin
Aminoglycosides
Good
Macrolides
Aminoglycosides
Low or nul
Prague 2008 55
The hypothesis of targeted
delivery of active drug at the
active site by the phagocytes
Prague 2008 56
Drug delivery by the phagocytes
• Transport by "non professional"
phagocytes (e.g. azithromycin by
fibroblast)
• Fibroblast acts as a reservoir for drug
and macrophages
Prague 2008 57
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)
Prague 2008 58
Macrolides: how to explain efficacy of low plasma
concentrations?
PMN
1.PK hypothesis
M
M
Sink or reservoir???
High local M concentration in the
vicinity of the bug
M
Nucleus
2-PD hypothesis
Prague 2008 59
PK model for exposure to macrolides: The limits
No In vivo data to support the hypothesis
2. Slow or very slow efflux
M
M+
M
M
M
Sink rather than reservoir
M
M
(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
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
Prague 2008 60
Macrolides: how to explain efficacy of low plasma
concentrations?
PMN
1.PK hypothesis
M
M
Sink or reservoir???
Cytokine modulation, ERK signaling
M
High local M concentration in the
vicinity of the bug
MIC in vivo < MIC in vitro
M
Nucleus
2-PD hypothesis
Prague 2008 61
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)
Prague 2008 62