Download Atelier PK sur articles

Atelier PK sur articles
5 thèmes

Absorption / biodisponibilité

Métabolisme hépatique et effet de premier passage



Les modèles in vitro d’étude de l’absorption :
pertinence par rapport aux modèles in vivo
Métabolisme intestinal et effet de premier passage
Présentation de la stratégie d’extrapolation décrite
dans l’article 5, en insistant sur les fondements
théoriques et les limites de cette approche.
2
Article 5: In vitro/in vivo extrapolation of
metabolic clearance
3
Why precociously predict in vivo
metabolic (hepatic) clearance ?

To evaluate oral bioavailability
Can I administer my drug by the oral route ?
4
Can a new drug be developed for
oral route ?
Gut
Lumen
Portal
vein
Gut Wall
1 : fabs
2 : Fgut
Liver
3 : FH
Foral  f abs  F G  F H
5
Can a new drug be developed for
oral route ?

Components of oral bioavailability

Hepatic first-pass effect
Foral  f abs  F G  F H
Cl H
Foral, max  1  
QH
6
Bioavailability and interindividual
variability
CV (%)
100
75
50
25
0
0
25
50
75
100
125
150
F%
Hellriegel et al, 1996 Clin. Pharmacol. Ther
7
Bioavailability and interindividual
variability
Overexposure
(adverse effects)
Mean Exposure
AUC or
concentrations
I.V.
Threshold
For toxicity
p.o.
Threshold
for efficacy
p.o.
Dose
1
Underexposure
(therapeutic failure, resistance)
5
Dose x 5
8
Two solutions for early evaluation of hepatic
clearance (CLH) during development


Interspecies extrapolation (from preclinical
species to human)
In vitro to in vivo extrapolation
9
Availability of in vitro systems

Purified enzymes

Subcellular fractions


Hepatocytes


S9, microsomes
Suspensions, primary cultures
Liver slices
10
Strategy for in vitro/in vivo
extrapolation
metabolism
CLint, in vitro
Microsomes
Hepatocytes
Scaling
factors
Clearance
model
CLint, in vivo
°
In vitro
fu, QH
CLH
Cl H
Foral, max  1  
QH
11
Strategy for in vitro/in vivo
extrapolation
metabolism
CLint, in vitro
Microsomes
Hepatocytes
Scaling
factors
Clearance
model
CLint, in vivo
°
In vitro
fu, QH
CLH
Cl H
Foral, max  1  
QH
12
In vitro metabolism
Drug
(concentration C0)
E
E
E
Free drug (free concentration)
No limited diffusion to enzymes (E)
13
In vitro intrinsic clearance

Quantification of metabolism by CLint
Rate of metabolism
CLint 
C


Rate : M.T-1 , C: M.V-1 => CLint expressed in V.T-1 (flow units)
“Intrinsic clearance (CLint) is a pure measure of
enzyme activity towards a drug and is not influenced
by other physiological determinants such as hepatic
blood flow or drug binding within the blood matrix”
14
Michaelis-Menten kinetics
Initial rate V
Vmax
Vmax/2
V=
KM
Vmax . C
KM + C
concentration
Vmax : maximum rate of metabolism (related to enzyme quantity)
KM : Michaelis constant (related to affinity between enzyme and analyte)
Michaelis-Menten kinetics
Rate  CLint  C
Initial rate
 Vmax 
Initial rate  
C

 KM  C 
Intrinsic clearance
conc
16
Michaelis-Menten kinetics
 Vmax 
Rate  
C

 KM  C 
V
max
CLint 
KM  C
 When C << KM
First-order / linear kinetics
 Vmax 
Rate  
C
 KM 
Clearance is constant
V
max
CLint 
KM
17
Michaelis-Menten kinetics
 When C << KM :
Initial rate
 Vmax 
Rate  
C
 KM 
Intrinsic clearance
Graphic : slope of tangent
conc
The highest intrinsic clearance is obtained for C << KM
18
Strategy for in vitro/in vivo
extrapolation
metabolism
CLint, in vitro
Microsomes
Hepatocytes
Scaling
factors
Clearance
model
CLint, in vivo
°
In vitro
fu, QH
CLH
Cl H
Foral, max  1  
QH
Scaling factors (SF)

Microsomes


CLint is expressed in µL/min/mg microsomal protein
Hepatocytes

CLint is expressed in µL/min/106 hepatic cells
20
Scaling factors (SF)

From test tube to liver : quantitative relationship
CLint,in vivo  SF CLint,in vitro
Ex:
(mL/min)
(µL/min/106 hepatic cells)
21
Scaling factors : rat liver
Relevance of these scaling factors ?
22
CLint,in vivo  CLint,in vitro SF
log(CL int,in vivo)  log(CL int,in vitro)  log(SF)
SF = 1.5 x 109 cells
SF = 500 mg microsomal proteins
Underestimation of
in vivo clearance
hepatocytes
microsomes
23
Scaling factors (SF)



Issue of experimental conditions (not taken into
account)
Documented species

Human, rat

Veterinary species ?
Other species : to establish scaling factors

Experimental determination

Allometric scaling
24
Strategy for in vitro/in vivo
extrapolation
metabolism
CLint, in vitro
Microsomes
Hepatocytes
Clearance
model
Scaling
factors
CLint, in vivo
°
In vitro
fu, QH
CLH
Cl H
Foral, max  1  
QH
In vitro vs. vivo situation
ORGAN (ex : liver)
cell
drug
cell
Cu input
CL organ
Cu output
cell
Organ blood
flow Q
drug
Metabolism
Rate
metabolism 

QE
C site of metabolism
(E=extraction coefficient)
26
Models of hepatic clearance

Assumptions :

no active transport

only free drug crosses plasma membranes
good mixing of hepatic arterial blood and hepatic portal blood

homogenous distribution of enzymes within the liver


f (QH ; fu ; CLint)
°
CLH =
=E
Example: well-stirred model CL  Q  f u  CLint
H
H
  f u  CLint
Q
(= venous equilibration model)
H
27
Models of hepatic clearance

f (QH ; fu ; CLint)
Model Complexity
Well-stirred
model

°
CLH =
Sinusoidal perfusion
model
Dispersion model
Differences between models


Low EH: minimal differences between models
When EH ≥ 0.7: obvious differences between models, which
become considerable when EH ≥ 0.9
28
29
Validation of in vitro/in vivo
extrapolation
In vitro
metabolism
Scaling
factors
Clearance
model
In vivo PK
Vmax
CLint, in vitro
CLint, in vivo
KM
CLint, in vivo
CLH
CLtot
30
Validation of in vitro/in vivo
extrapolation

In vivo pharmacokinetic studies
Intravenous administration
 Plasma concentration - time profile
 Urinary excretion of unchanged drug (Xu)

CLTOT  CLH  CLR  ε
CLH 
Dose  X u
AUC plasma
31
Validation of in vitro/in vivo
extrapolation

In vivo pharmacokinetic studies

In vivo intrinsic clearance (homogeneous model)
1
Q H  D IV  X u 

CL int,in vivo 
f u AUC IV  Q H  D IV  X u 
32
Validation of in vitro/in vivo
extrapolation
Clint,in vivo (mL/min/g liver)
lidocaïne
Correct prediction
warfarin
Important underestimation
Clint,in vitro (mL/min/g liver)
Iwatsubo et al.
Pharmacol Ther, 73, 147-171, 1997
Reasons for discrepancies between
Clint,in vitro and Clint,in vivo

Extra-hepatic metabolism

Drug transport through membranes



Slow equilibrium between blood and hepatocytes
Presence of active transport
Interindividual variability


Intrinsic : genetic polymorphism / P450 identification
Extrinsic : liver sample handling / scaling factors
Validation of in vitro/in vivo
extrapolation
EH : classification of compounds
LOW
INTERMEDIATE
HIGH
Hepatic extraction ratios
high
LOW
low
INTERMEDIATE
HIGH
ORAL BIOAVAILABILITY
Clint,in vitro (mL/min/106 cells)
Lavé et al.
Clin Pharmacokinet, 36, 1999
EARLY PHARMACOKINETIC SCREENING
Validation of in vitro/in vivo
extrapolation
Houston
Biochem Pharmacol, 47, 1994