Chief Scientific Officer, CellzDirect, Inc. Mechanisms of Hepatic Enzyme Induction in Humans and How to Assess It In Vitro Edward L. LeCluyse, Ph.D. Chief Scientific Officer, CellzDirect, Inc. November 2004

Pertinent Questions If a NME’s induction effect on CYP3A4 in vitro is NEGATIVE, then is it acceptable to NOT recommend any in vivo studies with substrates of CYP3A, CYP2C9, CYP2B6 and CYP2C19. YES or NO? If the in vitro induction (increase in enzyme activity) is more than 40% of the positive control (e.g., rifampin), then there IS a need to recommend an in vivo induction study.

Enzyme Induction in Humans Induced Plasma Drug CYPs Conc (µm) NR Drugs affected in vivo Carbamazepine 3A, 2B 20-40 CAR Praziquantel, Itraconazole, Cyclosporin A SJW 3A, 2C 0.2 AhR, PXR Indinavir, Digoxin, Cyclosporin, Theophylline Phenytoin 2C, 3A, 2B >10 CAR Warfarin, Praziquantel, Quinidine, Cyclosporin A Phenobarbital 2C, 3A, 2B 40-130 CAR, PXR Warfarin, Cyclosporin A Rifampin 2C, 3A, 2B 14 PXR Tolbutamide, Warfarin Cyclosporin, Oral contraceptives Troglitazone 3A, 2B 7 PXR Cyclosporin A, Terfenadine Avasimibe 2C, 3A, 2B 1-6 PXR Midazolam, Warfarin, Digoxin

Inducible P450 Enzymes in Human Liver P450 Enzyme Inducers CYP1A2 Aromatic hydrocarbons, cigarette smoke, cruciferous vegetables, charbroiled meat CYP2A6 Anticonvulsants, DEX CYP2B6 phenytoin, PB, RIF CYP2C8 Anticonvulsants, rifampin CYP2C9 Rifampin, Anticonvulsants CYP2C19 Rifampin, Anticonvulsants CYP3A4 CLZ, Rifampin, Anticonvulsants CYP2E1 Isoniazid, alcohol CYP2D6 Non-inducible CYP4A11 Non-inducible (?)

CAR RXR XREM / PBREM AhR ARNT XRE / DRE PXR XREM / PXRE CYP1A, UGT1A1, SULT1A1 CYP2B, CYP3A, CYP1A, CYP2A, CYP2Cs, OATP2 UGT1A1, MRP2, SULT1A1 CYP3A, CYP2B, CYP2Cs, CYP7A, MDR1, MRP2, OATP2, GSTA-2, UGT1A1, AldHs, Carboxyesterase 2, 3

Faucette S et al. Drug Metab Dispos 2004 Strong Mod Weak CLZ CMZ DTBA RIF SPZ RIT PHN PB Strong Mod Weak CLZ PB SDM RIF SPZ DEX RIT* CMZ PHN DTBA Faucette S et al. Drug Metab Dispos 2004

Co-regulation of CYP2C9 and CYP3A4 by Avasimibe

Mechanism-based Screening Strategy Goal is to screen for efficacious activators of AhR, PXR, and CAR Propose screening protocol using sensitive endpoint for each nuclear receptor Potent activators of individual NR’s will induce a number of target genes, but differentially: Potent hPXR activators will induce CYP3A4, CYP2B6, CYP2C8/9/19, UGT1A1, MDR1 etc., but CYP3A4 is the most sensitive. Potent hCAR activators will induce CYP2B6, CYP2C8/9/19, UGT1A1 etc., but CYP2B6 is the most sensitive. Potent AhR agonists will induce CYP1A2, UGT1A1, GST’s etc., but CYP1A2 is the most sensitive.

Treat with NME at 3-4 conc. for 1-3 days In Vitro Protocol for Enzyme Induction Treat with NME at 3-4 conc. for 1-3 days mRNA Content RT/PCR (TaqMan) Include Positive Controls: 3-MC/BNF Phenobarbital/Phenytoin Rifampin Cultured Hepatocytes Enzyme Activity LC-MS/MS assay HPLC assay Radiometric assay Protein Content Western Immunoblotting Major CYP target gene for each nuclear receptor: CYP1A2 (AhR), 2B6 (CAR), 3A4 (PXR)

CYP3A4 Induction in Human Hepatocytes

Effects of Inducers on CYP3A4 mRNA Expression

Important Factors to Consider Inter-donor differences in control/basal activity Relevant concentration range (plasma vs. tissue) Appropriate choice and concentration of positive controls Major species differences exist (e.g., RIF vs. PCN) Expression of data and relevant endpoints Exposure time important (# days) Solvent effects on CYP450 expression and activity

Summary of Key Points Our mechanistic understanding of enzyme induction in human liver has increased markedly in the past decade. Most inducible human P450’s, UGT’s and transporters involved in DDI’s are regulated by a few receptors (i.e., PXR, AhR and CAR). Screening for potential inducers during drug development can be achieved using a single selective and sensitive target gene for each NR. Activity data from in vitro induction studies for a NME should be: normalized to a negative control compared to an ‘appropriate’ positive control considered significant when they are 40% of the positive control complemented with protein or mRNA data