1David M. Stresser, 1Reena Patel, 1Robert J. Clark, 1Thuy Ho, 1Sarah K. 1Trisdale, Ye Fang2, and 1J. George Zhang

Comparison of CYP2B6, 2C8, 2C9, 2C19, 3A4, and 3A5 Induction in 3 Donors of Plated Human Hepatocytes by 15 Compounds

1David M. Stresser, 1Reena Patel, 1Robert J. Clark, 1Thuy Ho, 1Sarah K. 1Trisdale, Ye Fang2, and 1J. George Zhang 1Corning Incorporated, Life Sciences, Corning GentestSM Contract Research Services, 6 Henshaw Street, Woburn, MA 01801 USA 2Corning Incorporated, Biochemical Technologies, Science and Technology Division, Corning, NY 14831 USA

Abstract Table 1. EC50 of CYP mRNA Induction by Model Test Compounds Figure 2. Emax of mRNA Induction by Model Compounds in Three We previously reported that calibration curve-based approaches generated Test Hepatocyte Lots from CYP3A4 mRNA induction data in human hepatocytes can be used to CYP3A4 CYP3A5 CYP2B6 CYP2C8 CYP2C9 Test Drug Concentration predict CYP3A4 induction in humans. Here, the same set of samples was Sulfinpyrazone Phenobarbital Range (µM) Mean SD Mean SD Mean SD Mean SD Mean SD 12.93 20.49 Sulfinpyrazone 5.73 analyzed to evaluate induction of CYP2B6, CYP2C8, CYP2C9, CYP2C19, Rifampicin 10.23 Troglitazone 14.13 Troglitazone 5.04 and CYP3A5 mRNAs. Hepatocytes from three lots were treated with a set of Rifampicin 0.01-50 0.65 0.67 nd - 0.60 0.28 0.21 -* 0.59 -* Carbamazepine 8.27 Sulfinpyrazone 13.98 Nifedipine 4.90 Phenytoin 0.23-500 12 2.4 35 -** 3.2 1.9 8.7 7.1 9.4 -** Phenobarbital 7.20 Nifedipine 12.78 Dexamethasone 4.34 15 CYP3A4 in vitro inducers for 2 days, typically over 8 concentration points Probenecid 6.80 Carbamazepine 10.16 Rosiglitazone 3.78 Dexamethasone 6.53 to enable calculation of EC50 and Emax. After treatment, total RNA was Carbamazepine 0.23-500 16 11 142 51 22 9.7 22.0 18 30 -* Rosiglitazone 9.68 Carbamazepine 3.49 Clotrimazole 6.47 isolated and CYP mRNA expression was determined by real-time Real-time Clotrimazole 8.70 Phenobarbital 3.09 Phenobarbital 0.91-2000 153 78 592 211 247 157 205 43 228 11 Rosiglitazone 6.30 Phenytoin Pioglitazone 2.82 PCR analysis. Based on E values, the overall rank order of induction 8.53 max Troglitazone 0.03-20 3.7 2.4 17 12 15 7.5 12 4.6 12 -* Troglitazone 6.27 Terbinafine 8.27 Clotrimazole 2.74 response was 2B6>3A4>2C8>3A5>2C9>2C19, which held for all 3 donors. Nifedipine 5.57 Rifampicin Probenecid 2.74 Terbinafine 0.05-100 4.5 3.6 16 8.6 6.0 3.5 8.1 3.7 5.3 5.2 7.40 Rank order and absolute differences often varied by compounds. For Phenytoin 4.10 Omeprazole 6.93 Terbinafine 2.10 Pleconaril 0.05-100 4.2 1.9 12 2.7 1.1 0.27 2.2 0.25 nd - Terbinafine 3.10 Probenecid 6.52 Rifampicin 2.07 example, rifampicin (RIF) induced CYP3A4 by a mean of 10-fold, but was Pioglitazone 2.83 Pleconaril Dexamethasone 0.11-250 26 4.6 29 4.9 34 1.6 34 2.8 55 21 Pleconaril 5.49 1.99 essentially unable to induce CYP3A5. In contrast, phenobarbital (PB) Omeprazole 2.30 CYP3A4 Dexamethasone 4.18 CYP2B6 Phenytoin 1.68 CYP2C8 induced CYP3A4 by 7.2-fold, and CYP3A5 by 4.9-fold. Nifedipine induced Sulfinpyrazone 0.09-200 16 10 30 8.0 34 9.4 29 7.5 31 7.2 Pleconaril 2.10 Pioglitazone 1.66 Omeprazole 0.94 CYP3A4 by 5.6-fold, and induced CYP2C9 by 3.9-fold, whereas RIF and PB Probenecid 0.05-300 80 42 50 33 64 49 56 19 63 -* 0 5 10 15 0 5 10 15 20 25 0 2 4 6 induced CYP2C9 by ≤2.5-fold. Interestingly, EC values for CYP3A5 mRNA Troglitazone 50 Nifedipine 0.05-100 9.1 3.5 23 17 8.8 1.5 22 15 6.8 5.9 Nifedipine 3.93 1.87 Phenobarb… 4.85 induction were generally much higher than CYP3A4 (5.5- ± 6.1-fold for 14 Sulfinpyrazone 3.03 Phenobarbital 1.86 Troglitazone 3.89 Pioglitazone 0.05-100 3.4 0.50 3.4 -* 3.8 0.21 2.2 1.0 2.8 -* Troglitazone 2.92 Nifedipine 1.68 Rosiglitazo… 3.70 compounds that induced CYP3A5). In contrast, EC50 values for CYP2B6, Probenecid 1.51 Nifedipine 3.51 CYP2C8, and CYP2C9 were modestly higher (1.7- ± 1.3- to 2.9- ± 4.4-fold Rosiglitazone 0.05-100 8.8 1.1 28 12 16 6.0 11 2.1 9.2 -* Phenobarbital 2.51 Probenecid 2.32 Sulfinpyrazone 1.35 Pleconaril 3.09 greater). Our results demonstrate that induction profiles of CYP2B, 2C, and Omeprazole 0.05-100 6.4 -* 26 13 20 13 nd - nd - Pioglitazone 2.03 Pioglitazone 1.27 Carbamaze… 2.95 3A enzymes, that are mediated largely or in part by PXR, can vary Clotrimazole 0.005-10 3.3 0.75 4.7 3.0 3.4 0.64 4.1 0.50 4.0 3.4 Dexamethasone 1.98 Carbamazepine 1.24 Sulfinpyraz… 2.95 Rosiglitazone 1.22 substantially by compound and the response is not simply a scaled value of Data are the mean ±standard deviation from three hepatocyte lots except where indicated [* n = 2 lots, ** n = 1 lot, nd = not determined Carbamazepine 1.83 Omeprazole 2.88 Rifampicin Clotrimazole 1.19 (EC50 calculation was not achievable for all lots)]. 1.79 Dexameth… 2.47 the CYP3A4 induction response. Finally, these results underscore that Rosiglitazone 1.78 Terbinafine 1.14 Terbinafine . 2.25 induction response of certain enzymes not typically considered (e.g., Clotrimazole 1.73 Dexamethasone 1.11 Probenecid 1.94 CYP2C8 and CYP3A5) in drug development can be significant. Figure 1. Paired-data Analysis of EC Values for CYP3A4 vs. Terbinafine 1.38 Omeprazole 1.07 Clotrimazole 1.91 50 Pleconaril 1.24 Rifampicin 1.07 Pioglitazone 1.51 CYP3A5, and CYP3A4 vs. CYP2C8 Phenytoin 1.23 Pleconaril 0.99 Rifampicin 1.16 CYP2C9 CYP2C19 CYP3A5 Introduction Omeprazole 1.12 Phenytoin 0.96 Phenytoin 1.14 -3 -3 0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 0 2 4 Efficacy loss and altered pharmacokinetics of co-medications due to Lot 295 Lot 312 -3 Lot 318 E (fold of vehicle control) E (fold of vehicle control) Emax (fold of vehicle control) cytochrome P450 (CYP) induction is a significant concern during drug max max -4 ) -4 ) -4 ± development. CYP3A4 is highly inducible and is involved in the ) Data are the mean standard deviation from three hepatocyte lots. 50 50 biotransformation of about half of all drugs that undergo oxidative 50 metabolism. It is well-established that CYP3A4 inducers also induce -5 -5 -5 CYP2B6 and CYP2Cs via overlap in activation and cross-talk between Figure 3. Distribution Analysis of Emax Log (EC Log (EC nuclear receptors such as pregnane X receptor (PXR) and constitutive -6 -6 Log (EC -6 androstane receptor (CAR)1. Therefore, the FDA recommends evaluation of 2 -7 -7 30 CYP2C enzyme induction should CYP3A induction be observed in vitro . We -7 30 Lot 318 30 Lot 312 previously reported that calibration curve-based approaches generated from Lot 295 3A4 3A5 3A4 3A5 CYP3A4 mRNA induction data in human hepatocytes can be used to predict 3A4 3A5 20 3 -3 -3 -3 CYP3A4 induction in humans . Here, the same set of samples treated with Lot 295 Lot 312 Lot 318 20 20 known CYP3A4 inducers was analyzed to evaluate induction of CYP2B6, Emax

-4 Emax )

-4 Emax -4 ) CYP2C8, CYP2C9, CYP2C19, and CYP3A5 mRNA. ) 10 50 50 50 10 10 Materials and Methods -5 -5 -5 0

Log (EC 0 Log (EC Log (EC 0 Materials. All model test drugs were obtained from Sigma-Aldrich. Corning® -6 -6 -6 3A4 2B6 2C8 2C9 3A5 3A4 2B6 2C8 2C9 3A5 3A4 2B6 2C8 2C9 3A5 2C19 Cryopreserved human hepatocytes (Lots 295, 312, and 318) were obtained 2C19 2C19 CYP CYP from Corning Life Sciences. -7 -7 -7 CYP

Hepatocyte plating and treatment. Cryopreserved hepatocytes were 3A4 2C8 3A4 2C8 3A4 2C8 thawed using Corning Gentest™ High Viability CryoHepatocyte Recovery Summary and Conclusions Kit and plated in Corning Collagen I-coated 96-well microplates. Cell cultures were maintained in Corning Hepatocyte defined medium Notable Trends and Observations  Our results demonstrate that induction profiles of CYP2B, 2C, and 3A enzymes can vary substantially by supplemented with glutamine, gentamicin, and fungizone, overnight prior to compound. Therefore, an assumption that induction response may be simply a scaled value of a “sentinel” • EC50 values for CYP3A5 mRNA induction were on average much higher than CYP3A4 (5.5-fold) (Table 1 P450 induction response (such as CYP3A4 for PXR-mediated induction) would appear risky. treatment with model drugs at eight concentrations for 48 hours (Table 1). and Figure 1). By comparison, EC50 values for CYP2B6, CYP2C8 (Figure 1), and CYP2C9 were on After treatment, total RNA was isolated using Qiagen RNeasy® 96 Kit. The average (1.7- to 2.9-fold higher).  Our results underscore that maximal induction response of certain enzymes not typically considered (e.g., CYP2C8 and CYP3A5) in drug development can be significant. mRNA expression for each isoform was determined under the two-step • The overall rank order of maximum fold-induction response was 2B6>3A4>2C8>3A5>2C9>2C19, a  protocol using a model 7300 or ViiA™ 7 Real-Time PCR System (Taqman®, pattern which held for each of the three hepatocyte lots (Figure 3), despite some notable interindividual The EC50 values (which are a measure of induction potency) for CYP3A5 are notably higher than those for Applied Biosystems). differences (e.g., CYP2B6 with lot 318). CYP3A4. • Within compounds (Figure 2), notable exceptions to the rank order included:  These data provide a useful data-mining set. For example, several compounds were identified that are likely Data analysis. Mean fold induction over solvent vehicle control for mRNA • Pioglitazone: CYP3A4 = 2C8 > 2B6 > others more robust positive controls for CYP2Cs and CYP3A5 (e.g., troglitazone for CYP2C8, 2C9, and 3A5) than expression from triplicate treatment samples was used to calculate EC50 and • Pleconaril and omeprazole: CYP2B6 > 3A5 > 3A4 > others rifampicin, a compound typically selected for such analyses. E . The calculation was conducted with XLfit™ (IDBS) curve-fitting max • Rifampicin: CYP3A4 > 2B6 > 2C8 > 2C9 > 3A5 (no induction) software using a 4 Parameter Logistic Model, No. 205. Acceptance criteria • Phenobarbital: CYP2B6 > 3A4 > 3A5 (4.9-fold) > others for the fits are described previously3. When a discernible plateau was not References • Troglitazone and sulfinpyrazone gave the highest response for CYP2C8 (5.0- and 5.7-fold, respectively); evident or no induction response was found, the maximal observed fold over rifampicin (2.1-fold) and phenobarbital (3.1-fold) were much less. These data have implications for 1. Fahmi O, et al. (2010) Drug Metabolism and Disposition 38:1605. control was used for analysis. Inducibility of CYP isoforms was determined selection of a robust positive control for CYP2C8 induction response. 2. FDA guidance for Drug Interaction Studies (2012) by frequency of a >2-fold induction of each enzyme for these compounds in • Relative to other compounds, rifampicin, exhibited high selectivity for CYP3A4 maximal induction; Based 3. Zhang JG, et al. (2014) Drug Metabolism and Disposition 42:1379. the three hepatocyte lots. Emax distribution, and a paired EC50 graph on its EC50 value, it was by far the most potent inducer for all CYPs, except for CYP2C19 and CYP3A5 between CYP3A4 vs. CYP3A5 or CYP3A4 vs. CYP2C8 of all compounds (no induction). were used to visualize distinct inducibility patterns of CYP isoforms were. Warranty/Disclaimer: Unless otherwise specified, all products are for research use only. Not intended for use in diagnostic or therapeutic procedures. Not for use in humans. Corning Life Sciences makes no claims regarding the performance of these products for clinical or diagnostic applications. The EC50 values were converted to molar units prior to log transformation For a listing of trademarks, visit www.corning.com/clstrademarks. All other trademarks are the property of their respective owners. CLS-GT-PST-034 3/16 © 2016 Corning Incorporated. All Rights reserved. comparison analysis of EC50.