1. Atelier PK sur articles

2. 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

3. Article 5: In vitro/in vivo extrapolation of metabolic clearance 3

4. Why precociously predict in vivo metabolic (hepatic) clearance ? To evaluate oral bioavailability 4 Can I administer my drug by the oral route ?

5. Can a new drug be developed for oral route ? Gut Lumen Gut Wall Portal vein 1 : f abs 2 : F gut 3 : F H Liver 5

6. Can a new drug be developed for oral route ? Components of oral bioavailability Hepatic first-pass effect 6

7. Bioavailability and interindividual variability 7

8. Dose 15 Mean Exposure Threshold For toxicity Threshold for efficacy Underexposure (therapeutic failure, resistance) p.o. Overexposure (adverse effects) AUC or concentrations I.V. Dose x 5 8

9. Two solutions for early evaluation of hepatic clearance (CL H ) during development Interspecies extrapolation (from preclinical species to human) In vitro to in vivo extrapolation 9

10. Availability of in vitro systems Purified enzymes Subcellular fractions S9, microsomes Hepatocytes Suspensions, primary cultures Liver slices 10

11. Strategy for in vitro/in vivo extrapolation In vitro metabolism CL int, in vitro Microsomes Hepatocytes Scaling factors Clearance model CL int, in vivo CL H f u, Q H ° 11

12. Strategy for in vitro/in vivo extrapolation In vitro metabolism CL int, in vitro Microsomes Hepatocytes Scaling factors CL int, in vivo Clearance model CL H f u, Q H ° 12

13. In vitro metabolism E E E Free drug (free concentration) No limited diffusion to enzymes (E) Drug (concentration C 0 ) 13

14. In vitro intrinsic clearance Quantification of metabolism by CL int Rate : M.T -1, C: M.V -1 => CL int expressed in V.T -1 (flow units) “Intrinsic clearance (CL int ) 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

15. concentration Initial rate V Vmax Vmax/2 KMKM V = V max. C K M + C Michaelis-Menten kinetics Vmax : maximum rate of metabolism (related to enzyme quantity) K M : Michaelis constant (related to affinity between enzyme and analyte)

16. conc Initial rate Intrinsic clearance Michaelis-Menten kinetics 16

17.  When C << K M Clearance is constant Michaelis-Menten kinetics 17 First-order / linear kinetics

18. conc Michaelis-Menten kinetics Initial rate C << K M The highest intrinsic clearance is obtained for C << K M Intrinsic clearance  When C << K M : 18 Graphic : slope of tangent

19. Strategy for in vitro/in vivo extrapolation CL int, in vitro Microsomes Hepatocytes Scaling factors CL int, in vivo In vitro metabolism Clearance model CL H f u, Q H °

20. Microsomes CL int is expressed in µL/min/mg microsomal protein Hepatocytes CL int is expressed in µL/min/10 6 hepatic cells Scaling factors (SF) 20

21. Scaling factors (SF) From test tube to liver : quantitative relationship 21 Ex: (mL/min)(µL/min/10 6 hepatic cells)

22. Scaling factors : rat liver 22 Relevance of these scaling factors ?

23. 23 SF = 1.5 x 10 9 cells SF = 500 mg microsomal proteins hepatocytesmicrosomes Underestimation of in vivo clearance

24. Issue of experimental conditions (not taken into account) Documented species Human, rat Veterinary species ? Other species : to establish scaling factors Experimental determination Allometric scaling Scaling factors (SF) 24

25. Strategy for in vitro/in vivo extrapolation CL int, in vitro Microsomes Hepatocytes Clearance model CL int, in vivo CL H f u, Q H ° In vitro metabolism Scaling factors

26. In vitro vs. vivo situation Metabolism ORGAN (ex : liver) cell drug C u input Organ blood flow Q drug C u output site of metabolism metabolism organ C Rate CL  Q  E  (E=extraction coefficient) 26

27. Models of hepatic clearance CL H = f (Q H ; fu ; CL int ) 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 Example: well-stirred model (= venous equilibration model) ° = E 27

28. Models of hepatic clearance CL H = f (Q H ; fu ; CL int ) Model Complexity Differences between models Low E H : minimal differences between models When E H ≥ 0.7: obvious differences between models, which become considerable when E H ≥ 0.9 ° Well-stirred model Sinusoidal perfusion model Dispersion model 28

29. 29

30. Validation of in vitro/in vivo extrapolation Clearance model In vivo PK In vitro metabolism V max KMKM CL int, in vitro CL tot CL H CL int, in vivo Scaling factors CL int, in vivo 30

31. In vivo pharmacokinetic studies Intravenous administration Plasma concentration - time profile Urinary excretion of unchanged drug (X u ) Validation of in vitro/in vivo extrapolation 31

32. In vivo pharmacokinetic studies In vivo intrinsic clearance (homogeneous model) 32 Validation of in vitro/in vivo extrapolation

33. Cl int,in vitro (mL/min/g liver) Iwatsubo et al. Pharmacol Ther, 73, 147-171, 1997 lidocaïne warfarin Correct prediction Important underestimation Validation of in vitro/in vivo extrapolation Cl int,in vivo (mL/min/g liver)

34. Reasons for discrepancies between Cl int,in vitro and Cl int,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

35. Cl int,in vitro (mL/min/10 6 cells) E H : classification of compounds EARLY PHARMACOKINETIC SCREENING LOWINTERMEDIATEHIGH Hepatic extraction ratios ORAL BIOAVAILABILITY LOWINTERMEDIATEHIGH high low Lavé et al. Clin Pharmacokinet, 36, 1999 Validation of in vitro/in vivo extrapolation

36. Houston Biochem Pharmacol, 47, 1994