CYP2C8, 2C9 and 2C19 Induction by Model P450 …

1 CYP2C8, 2C9 and 2C19 Induction by Model P450 Inducers in Human Hepatocytes: A Correlative Study with CYP1A2, 2B6 and 3A4 Induction in Hepatocytes from Multiple Donors Albert P. Li, Ph. D. In Vitro ADMET Laboratories Inc. Columbia, MD and Malden, MA U. S. FDA requires CYP3A4 inducers to be evaluated for CYP2C Induction If CYP3A Induction results are positive, then Induction of CYP2C should be studied either in vitro or in vivo. CYP2C Induction in Human Hepatocytes Role of CYP in drug toxicity and DDI Current understanding of the mechanism of CYP2C Induction Human hepatocyte-derived individual differences in P450 Induction Correlation of CYP2C with CYP1A, 2B, and CYP3A Induction with human hepatocytes from multiple donors Conclusion and future directions CYP2C9 and Drug Toxicity Sulfonylureas Association between CYP2C9 slow metabolizer genotypes and severe hypoglycaemia on medication with sulphonylurea hypoglycaemic agents (Holstein A , Plaschke A , Ptak M et al.)

2 Br J Clin Pharmacol 2005 ; 60 : 103-106) CYP2C9*2 Allele Increases Risk for Hypoglycemia in POR*1/*1 Type 2 Diabetic Patients Treated with Sulfonylureas (Ragia G et al. Exp Clin Endocrinol Diabetes 2014; 122: 60 63) Bosentan Association of CYP2C9*2 With Bosentan-Induced Liver Injury (Markova et al, Clinical Pharmacology & Therapeutics (2013); 94 6, 678 686. ) CYP2C and Drug-Drug Interactions Human CYP2C8, CYP2C9, CYP2C18, and CYP2C19 represent approx. 20% of hepatic P450 and metabolize more than 20% of all therapeutic drugs as well as a number of endogenous compounds. Review: Chen and Golstein, Current Drug Metab (2016) Clinically significant CYP2C9 DDI Victims: Sulfonylureas (glibenclamide, glimepiride, and glipizide) Nonsteroidal antiinflammatory drugs (NSAIDs): ibuprofen, diclofenac, naproxen, piroxicam, and tenoxicam, Hypoglycemic Agents: tolbutamide, glipizide Angiotensin II Blockers: losartan, irbesartan Perpetrators: Inducers: rifampin, phenobarbital, hyperforin Inhibitors: fluconazole, amiodarone Mechanism of CYP2C Induction Xenobiotic-mediated Induction : Nuclear receptors CAR, PXR, VDR, and GR Constitutive expression: HNF4 , HNF3 , C/EBP and RORs Maximal transcriptional Induction of CYP2C genes: coordinative cross-talk between drug responsive nuclear receptors, hepatic factors, and coactivators.

3 Nuclear Receptors and CYP2C9 Induction Ferguson, Stephen S., et al. "Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4 ." Molecular pharmacology (2005): 747-757. Chen, Yuping, et al. "The nuclear receptors constitutive androstane receptor and pregnane X receptor cross-talk with hepatic nuclear factor 4 to synergistically activate the human CYP2C9 promoter." Journal of Pharmacology and Experimental Therapeutics (2005): 1125-1133. Chen, Yuping, Stephen S. Ferguson, Masahiko Negishi, and Joyce A. Goldstein. "Identification of constitutive androstane receptor and glucocorticoid receptor binding sites in the CYP2C19 promoter." Molecular pharmacology 64, no. 2 (2003): 316-324.

4 CYP2C9 Induction Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping (Makia, Goldstein et al; Molecular Pharmacology August 2014 vol. 86 no. 2 125-137) oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes Protein-Protein Interaction Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes (Ramsden, Tweedy et al, DMD November 2014 vol. 42 no. 11 1940-1946) siRNA silencing and recovery evaluation in long-term cultured human hepatocyes show inverse correlation of CYP2C9 with CYP3A4 activity CYP2C8, 2C9 and 2C19 Induction by Model P450 Inducers in Human Hepatocytes: A Correlative Study with CYP1A2, 2B6 and 3A4 Induction Cryopreserved Plateable Human Hepatocytes: Gold Standard for P450 Induction Studies IVAL Plateable Cryopreserved Hepatocytes Human Cynomolgus Monkey Beagle Dog CD-1 Mouse SD Rat Wistar Rat OnDemand Human Hepatocytes 14 Objectives Overall evaluation of inducibility of CYP2C8, CYP2C9 and CYP2C19 in human hepatocytes from 9 donors Inducers: Omeprazole (AHR); phenobarbital (CAR).

5 Rifampin (PXR) Identify donors with highest sensitivity to CYP2C Induction Correlate CYP2C Induction with CYP1A, CYP2B and CYP3A Induction to provide insight towards relative roles of AHR, CAR, and PXR in CYP2C Induction Procedures Plateable human hepatocytes from 9 donors 96-well matrigel-collagen sandwich culture Day 1: Thaw, plate and matrigel overlay Day 2: Initiation of treatment with omeprazole, phenobarbital and rifampin Days 3, 4: repeat treatment (72 h treatment) Day 5: RNA extraction RT-PCR quantification of CYP1A2, 2B6, 2C8, 2C9, 2C19 and 3A4 gene expression Delta-delta Ct calculation of fold Induction Donor Demorgraphics Lot Ethnicity Sex Age BMI Plateable/ Suspension HH1032 C F 68 Plateable HH1051 C M 23 Plateable HH1052 C M 44 Plateable HH1053 C F 33 Plateable HH1083 C F 45 Plateable HH1089 C F 57 Plateable HH1085 H F 77 Plateable HH1103 C/H F 44 Plateable Results CYP1A2, 2B6, and 3A4 Induction in Human Hepatocytes (9 Donors)

6 CYP1A2 CYP2B6 CYP3A4 OMP PB RIF OMP PB RIF OMP PB RIF Donor mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd HH1032 HH1051 HH1052 HH1053 HH1072 HH1083 HH1085 HH1089 HH1103 OMP: Omeprazole (50 uM); PB: Phenobarbital (1000 uM); RIF: Rifampin (20 uM) CYP2C8, CYP2C9 and CYP2C19 Induction in Human Hepatocytes (9 Donors) CYP2C8 CYP2C9 CYP2C19 OMP PB RIF OMP PB RIF OMP PB RIF Donor mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd HH1032 HH1051 HH1052 HH1053 HH1072 HH1083 HH1085 HH1089 HH1103 OMP: Omeprazole (50 uM); PB: Phenobarbital (1000 uM); RIF: Rifampin (20 uM) Individual Difference Individual Difference in P450 Induction : CYP1A2, CYP2B6, CYP3A4 RIF (20 uM); PB (1000 uM).

7 OMP (50 uM) Induction Donors CYP1A2 Induction Donors CYP2B6 Induction Donors CYP3A4 RifampinPhenobarbitalOmeprazoleIndividua l Difference in P450 Induction : CYP2C8, CYP2C9, CYP2C19 RIF (20 uM); PB (1000 uM); OMP (50 uM) Induction Donors CYP2C8 Induction Donors CYP2C9 Induction Donors CYP2C19 HH1053: Dose-Response HH1053 inducer CYP1A2 CYP2B6 CYP3A4 CYP2C8 CYP2C9 CYP2C19 Omeprazole ( M) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 2 10 25 50 100 Phenobarbital ( M) 4 20 50 100 250 500 1 1000 Rifampin ( M) 2 10 20 40 HH1051.

8 Dose Response HH1053 inducer CYP1A2 CYP2B6 CYP3A4 CYP2C8 CYP2C9 CYP2C19 Omeprazole ( M) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1 2 10 25 50 100 Phenobarbital ( M) 4 20 50 100 250 500 1000 Rifampin ( M) 2 10 20 40 Correlation Analysis Pool results with 9 donors with the three Model inducers.

9 Omeprazole (50 uM), phenobarbital (1000 uM) and rifampin (50 uM) Plot fold Induction of one CYP vs another to identify isoforms with best correlations, thereby revealing similar mechanisms of Induction CYP3A4 Induction Fold CYP2B Induction CYP3A4 vs CYP2B6 PhenobarbitalRifampinOmeprazoleCYP3A4 (PXR) vs CYP2B6 (CAR) Fold Induction Correlation of CYP1A2, CYP2B6 and CYP3A4 Induction CYP1A2 Induction Fold CYP3A4 Induction CYP1A2 vs CYP3A4 y = + R = CYP1A2 Induction Fold CYP3A4 Induction CYP2B6 vs CYP3A4 CYP1A2 Induction Fold CYP2B6 Induction CYP1A2 vs CYP2B6 Correlation of CYP2C and CYP3A4 Induction y = + R = CYP2C8 Induction Fold CYP3A4 Induction CYP2C8 vs CYP3A4 y = + R = CYP2C9 Induction Fold CYP3A4 Induction CYP2C9 vs CYP3A4 y = + R = CYP2C19 Induction Fold CYP3A4 Induction CYP2C19 vs CYP3A4 Correlation of CYP2C and CYP2B6 Induction y = + R = CYP2C8 Induction Fold CYP2B6 Induction CYP2C8 vs CYP2B6 y = + R = CYP2C9 Induction Fold CYP2B6 Induction CYP2C9 vs CYP2B6 y = + R = CYP2C19 Induction Fold CYP2B6 Induction CYP2C19 vs CYP2B6 Correlation of CYP2C8.

10 CYP2C9 and CYP2C19 Induction y = - R = CYP2C8 Induction Fold CYP2C9 Induction CYP2C8 vs CYP2C9 y = + R = CYP2C19 Induction Fold CYP2C9 Induction CYP2C19 vs CYP2C8 y = + R = CYP2C19 Induction Fold CYP2C9 Induction CYP2C19 vs CYP2C9 Summary and Conclusions Summary CYP2C Induction was evaluated in human hepatocytes from 9 donors and compared to CYP1A2, CYP2B6 and CYP3A4 Isoform dependent fold Induction : CYP1A2>CYP3A4>CYP2B6>CYP2C8=CYP2C9>CYP2C 19 Individual variations was observed While all lots exhibited robust response to CYP1A2, CYP2B6, and CYP3A4 Induction , only 2 of the 9 lots were also inducible (>2 fold Induction ) for CYP2C8, CYP2C9, and CYP2C19 Phenobarbital was the best positive control for CYP2C Induction Correlation demonstrated that CYP2C Induction correlates best among CYP2C isoforms followed by CYP3A4 and CYP2B6, with no correlation with CYP1A2 Induction Conclusion Careful selection of human hepatocyte lots is important for the evaluation of CYP2C Induction Mechanism of CYP2C9 Induction is different from CYP1A Induction , confirming that AHR is not involved Correlation of CYP2C Induction with CYP2B6 and CYP3A4 Induction is consistent with the roles of CAR and PXR Best correlations are among CYP2C8, CYP2C9 and CYP2C19, suggesting that similar mechanism for the three isoforms which is different from CYP2B6 and CYP3A4 Induction U.