ECOLE
NATIONALE
VETERINAIRE
TOULOUSE
PK/PD approach for antibiotics:
tissue or blood drug level to predict antibiotic efficacy
PL Toutain
National Veterinary school; Toulouse
Leipzig 2009 1
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 ?
•
3.
Where is the biophase?
•
4.
6.
Interstitial space fluid vs. intracellular cytosol vs. intracellular
organelles
How to assess the biophase antibiotic concentration
•
5.
Extracellular vs. intracellular
Total tissular concentration vs. ISF concentration.
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
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
Aminoglycosides
24h
AUC/MIC
ratio
Optimize the
quantity of
administered
AB
125 H
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:
AUC Dose / Clearance

MIC
MIC90
PD:
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Time > MIC
T1
concentrations
Dose
100
2
%Time  MIC  Ln


Vd  MIC Ln2 
Half-life
MIC
t1
t2
24
Time (h)
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Cmax / MIC
PK
C max
MIC90
• Bioavailability (%)
• clearance
• Rate of absorptione Rate
of elimination
• Accumulation factor
PD
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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
ISF
Most pathogens of
clinical interest
•S. Pneumoniae, E. Coli,Klebsiella
Cell
(most often in phagocytic cell)
•
•
•
•
•Mannhemia ; Pasteurella
•
•
• Actinobacillius pleuropneumoniae •
•Mycoplasma hyopneumoniae
•
Mycoplasma (some)
Chlamydiae
Brucella
Cryptosporidiosis
Listeria monocytogene
Salmonella
Mycobacteria
Rhodococcus equi
•Bordetalla bronchiseptia
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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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Total tissular concentration
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
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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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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
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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
Phagolysosome
Cytosol
pH=7.4
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
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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 cage
in 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 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, UFsample
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
Microdialysis :
a fluid is pumped
through a membrane;
The analyte cross the
membrane by diffusion
The driving force is a
concentration gradient
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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
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What we learnt with animal and
human microdialysis studies
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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)
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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
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 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
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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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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.
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Leipzig 2009 49
•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
ELF
AB
Capillary
wall
AB
Alveolar
Epithelium
Thigh junctions
The alveolar epithelial cells would not be
expected to permit passive diffusion of
antibiotics between cells, the cells being
linked by tight junctions
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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
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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.
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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
Leipzig 2009 53
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
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
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Intracellular location of antibiotics
Phagolysosome
Cytosol
pH=7.4
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
Leipzig 2009 57
What are the antibiotic intracellular
expressions of activity
Phagolysosome
Cytosol
pH=7.2
Fluoroquinolones
beta-lactams
Rifampicin
Aminoglycosides
Good
Macrolides
Aminoglycosides
Low or nul
Leipzig 2009 58
The free plasma level is the most
meaningful concentration
In acute infections in nonspecialized tissues, where there
is no abscess formation, free
plasma levels of antibiotics are
good predictors of free levels in
interstitial fluid
Leipzig 2009 59
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
Leipzig 2009 60
Tissue concentrations
According to EMEA
"unreliable information is generated
from assays of drug concentrations
in whole tissues (e.g. homogenates)"
EMEA 2000
Leipzig 2009 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)
Leipzig 2009 62