Transcript free concentration - Physiologie et Thérapeutique Ecole Véto

PK/PD approach for antibiotics:
tissue or blood drug levels to predict antibiotic efficacy
PL Toutain
National Veterinary school; Toulouse
Wuhan 8 October 2015
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
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
MIC
Cmax=0.5µg/mL ≤ to MIC90
• Good clinical
efficacy and
bacteriological
cure with
macrolides is
achievable with
plasma
concentrations
(much) lower,
than the in vitro
MICs for major
lung pathogens
MIC in MHB vs. calf serum
25%,50%,75% and 100%
MIC in MHB
MIC in serum
25%
50%
75%
100 %
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
ISF
Most pathogens of
clinical interest
•S. Pneumoniae, E. Coli,Klebsiella
Cell
(most often in phagocytic cell)
•
•
•
•
•Mannhemia ; Pasteurella
•
•
• Actinobacillius pleuropneumoniae •
•Mycoplasma hyopneumoniae
•
•Bordetalla bronchiseptia
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?
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 17
3: why a total tissular
concentration has no meaning
Leipzig 2009 19
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
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.
Intracellular location of antibiotics
Phagolysosome
volume 1 to 5% of cell volume
Cytosol
pH=7.4
Fluoroquinolones(x2-8)
beta-lactams (x0.2-0.6)
Rifampicin (x2)
Aminoglycosides (slow
pH=5.0
Macrolides (x10-50)
Aminoglycosides (x2-4)
Ion trapping for weak base
with high pKa value
Leipzig 2009 22
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
PET images following administration of
18f-trovafloxacine
Muller & al AAC 2004
Prague 2008 25
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
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
Leipzig 2009 28
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
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%)
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
• Excessive (in vivo)
calibration
procedures are
required for accurate
monitoring
• Unlike MD, UFsample
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
Microdialysis :
a fluid is pumped
through a membrane;
The analyte cross the
membrane by diffusion
The driving force is a
concentration gradient
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
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,
42
Enrofloxacin (SQ, 7.5mg/kg)
AUC (enro+cipro)
• Plasma=19 (total)
• ISF=25 (free)
• Ileaum=21 (free)
• Spiral colon =36 (free)
43
Ceftiofur SQ (2.2mg/kg)
AUC
• Plasma=137 (total)
• ISF=15 (free)
• Ileum=40 (free)
• Spiral Colon =34 free)
44
6-What we learnt with animal and
human microdialysis studies
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
Time (h)
12 20
30
40
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
• 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 wholetissue 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)
6
MIC (µg/mL)
5
4
MIC Broth
MIC Serum
Cf for MIC Serum
3
2
1
0
Ampi
Methy
fb 0.22 0.37
Benz
NAF
Oxa
0.65
0.90 0.93
Cloxa
0.95
The free concentration paradigm in
pharmacokinetics is supported by MD
findings
Blood/Plasma
Plasma bound
Total
Measured by
analytical
technique
Interstitial fluid Tissular space
ISF bound
Tissue bound
BUG
Free
Elimination
Free
Free
What we learnt with MD studies:
Inflammation
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
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)?
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.
Possible confounding factors in
interpreting ELF concentrations of
antibiotics measured by BAL
• ELF: Epithelial lining Fluid
• BAL:bronchoalveolar lavage
•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
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)
• 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.
• .
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
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
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 68
Which antibiotics do accumulate in
cells ?
•
•
•
•
•
•
beta-lactams :
aminoglycosides:
ansamycins:
tetracyclines:
fluoroquinolones:
macrolides:
1x
<1 to 2 x
2-3 x
2-4 x
10-20 x
4 to > 100 x
What are the antibiotic intracellular
activity
Phagolysosome
Cytosol
pH=7.2
Fluoroquinolones
beta-lactams
Rifampicin
Aminoglycosides
Good
Macrolides
Aminoglycosides
Low or nul
Leipzig 2009 71
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 nonspecialized 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 dose
According to EMEA
"unreliable information is
generated from assays of drug
concentrations in whole tissues
(e.g. homogenates)"
EMEA 2000