Atelier PK sur articles

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

5. Can a new drug be developed fororal route ? Gut Lumen Portal vein Gut Wall 3 : FH 1 : fabs 2 : Fgut Liver 5

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

7. 100 75 CV (%) 50 25 0 0 25 50 75 100 150 125 F% Hellriegel et al, 1996 Clin. Pharmacol. Ther Bioavailability and interindividual variability 7

8. p.o. Bioavailability and interindividual variability AUC or concentrations Overexposure (adverse effects) Threshold For toxicity I.V. Mean Exposure Threshold for efficacy p.o. Underexposure (therapeutic failure, resistance) 1 5 Dose x 5 Dose 8

9. 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

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

11. Scaling factors Clearance model CLint, in vivo CLH ° fu, QH Strategy for in vitro/in vivo extrapolation In vitro metabolism CLint, in vitro Microsomes Hepatocytes 11

12. Scaling factors Clearance model CLint, in vivo CLH ° fu, QH Strategy for in vitro/in vivo extrapolation In vitro metabolism CLint, in vitro Microsomes Hepatocytes 12

13. In vitrometabolism Drug (concentration C0) E E E Free drug (free concentration) No limited diffusion to enzymes (E) 13

14. In vitro intrinsic clearance • Quantification of metabolism by CLint • 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

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

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

17. Michaelis-Menten kinetics • When C << KM Clearance is constant First-order / linear kinetics 17

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

19. Scaling factors Clearance model CLint, in vivo CLH ° fu, QH Strategy for in vitro/in vivo extrapolation In vitro metabolism CLint, in vitro Microsomes Hepatocytes

20. Scaling factors (SF) • Microsomes • CLint is expressed in µL/min/mg microsomal protein • Hepatocytes • CLint is expressed in µL/min/106 hepatic cells 20

21. Scaling factors (SF) • From test tube to liver : quantitative relationship Ex: (mL/min) (µL/min/106 hepatic cells) 21

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

23. Underestimation of in vivo clearance SF = 1.5 x 109 cells SF = 500 mg microsomal proteins hepatocytes microsomes 23

24. 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

25. Scaling factors Clearance model CLint, in vivo CLH fu, QH Strategy for in vitro/in vivo extrapolation In vitro metabolism CLint, in vitro Microsomes Hepatocytes °

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

27. CLH = f (QH ; fu ; CLint) 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 Models of hepatic clearance ° 27

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

29. 29

30. Validation of in vitro/in vivo extrapolation Scaling factors In vitro metabolism Clearance model In vivo PK Vmax CLint, in vitro CLint, in vivo KM CLtot CLH CLint, in vivo 30

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

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

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

34. 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

35. LOW INTERMEDIATE HIGH Hepatic extraction ratios LOW INTERMEDIATE HIGH ORAL BIOAVAILABILITY Validation of in vitro/in vivo extrapolation EH : classification of compounds high low Clint,in vitro (mL/min/106 cells) Lavé et al. Clin Pharmacokinet, 36, 1999 EARLY PHARMACOKINETIC SCREENING

36. Validation of in vitro/in vivo extrapolation Houston Biochem Pharmacol, 47, 1994