Effect of Fifteen CYP3A4 in Vitro Inducers on the Induction of P119 CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A5 in Plated Human Hepatocytes: a Trend Analysis J

Effect of Fifteen CYP3A4 in vitro Inducers on the Induction of P119 CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A5 in Plated Human Hepatocytes: A Trend Analysis J. George Zhang, Reena Patel, Robert J. Clark, Thuy Ho, Sarah K. Trisdale, Ye Fang1 and David M. Stresser Corning GentestSM Contract Research Services, 6 Henshaw Street, Woburn, MA 01801, USA 1Biochemical Technologies, Science and Technology Division, Corning Incorporated, NY 14831, USA

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

) -4 ) -4 ) 0 0 0 5

5 Data are the mean ±standard deviation from three hepatocyte lots. 5 C C C E

-5 E ( -5 E (

-5 ( Introduction

g g g o o Figure 3. Distribution Analysis of Emax o L L

Efficacy loss and altered pharmacokinetics of co-medications due to cytochrome P450 -6 L -6 -6 (CYP) induction is a significant concern during drug development. CYP3A4 is highly inducible and is involved in the biotransformation of about half of all drugs that undergo -7 30 -7 -7 30 Lot 318 oxidative metabolism. It is well-established that CYP3A4 inducers also induce CYP2B6 4 5 30 Lot 312 A A 4 5 4 5 Lot 295 3 3 A A A A and CYP2Cs via overlap in activation and cross talk between nuclear receptors such as 3 3 3 3 20 pregnane X receptor (PXR) and constitutive androstane receptor (CAR) (1). Therefore, -3 -3 -3 20 Lot 295 Lot 312 Lot 318 20 x a x x

the FDA recommends evaluation of CYP2C enzyme induction should CYP3A induction a m a m E m

be observed in vitro (2). We previously reported that calibration curve-based -4 E -4 ) -4 ) ) E 0

0 0 10 5 5 approaches generated from CYP3A4 mRNA induction data in human hepatocytes can 5 10 10 C C C E E -5 E be used to predict CYP3A4 induction in humans (3). Here, the same set of samples -5 ( -5 ( (

g g treated with known CYP3A4 inducers was analyzed to evaluate induction of CYP2B6, g o 0 o o

L 0 0 L -6 -6 L -6 4 6 8 9 9 5 CYP2C8, CYP2C9, CYP2C19 and CYP3A5 mRNA. 4 6 8 9 9 5 4 6 8 9 9 5 A B C C 1 A A B C C 1 A A B C C 1 A 3 2 2 2 C 3 3 2 2 2 C 3 3 2 2 2 C 3 2 2 2 CYP CYP Materials and Methods -7 -7 -7 CYP 4 8 4 8 4 8 A C A C A C Materials. All model test drugs were obtained from Sigma-Aldrich. Cryopreserved 3 2 3 2 3 2 human hepatocytes (Lots 295, 312, and 318) were obtained from Corning Life Sciences Conclusion (Tewksbury, MA). Notable Trendsand Observations ¾ Our results demonstrate that induction profiles of CYP2B, 2C and 3A enzymes can vary substantially Hepatocyte plating and treatment. Cryopreserved hepatocytes were thawed using ¾ EC values for CYP3A5 mRNA induction were on average much higher than CYP3A4 (5.5-fold) by compound. Therefore, an assumption that induction response may be simply a scaled value of a Corning® Gentest™ High Viability CryoHepatocyte Recovery Kit and plated in Corning 50 “sentinel” P450 induction response (such as CYP3A4 for PXR-mediated induction) would appear risky. Collagen I-coated 96-well plates. Cell cultures were maintained in Corning Hepatocyte (Table 1 and Fig.1). By comparison, EC50 values for CYP2B6, CYP2C8 (Fig.1) and CYP2C9 were defined medium supplemented with glutamine, gentamicin and fungizone, overnight on average (1.7- to 2.9-fold higher). ¾ Our results underscore that maximal induction response of certain enzymes not typically considered prior to treatment with model drugs at eight concentrations for 48 h (Table1). After ¾ The overall rank order of maximum fold-induction response was 2B6>3A4>2C8>3A5>2C9>2C19, a (e.g. CYP2C8 and CYP3A5) in drug development can be significant. treatment, total RNA was isolated using Qiagen RNeasy® 96 Kit. The mRNA expression pattern which held for each of the three hepatocyte lots (Fig.3), despite some notable interindividual ¾ The EC50 values (which are a measure of induction potency) for CYP3A5 are notably higher than those for each isoform was determined under the two-step protocol using a model 7300 or differences (e.g. CYP2B6 with lot 318) for CYP3A4. ViiA 7 Real-Time PCR System (Taqman®, Applied Biosystems). ¾ Within compounds (Fig.2), notable exceptions to the rank order included: ¾ These data provide a useful data-mining set. For example, several compounds were identified that are ‰ Pioglitazone: CYP3A4 = 2C8 > 2B6 > others Data analysis. Mean fold induction over solvent vehicle control for mRNA expression likely more robust positive controls for CYP2Cs and CYP3A5 (e.g. troglitazone for CYP2C8, 2C9 and from triplicate treatment samples was used to calculate EC50 and Emax. The calculation ‰ Pleconaril and omeprazole: CYP2B6 > 3A5 > 3A4 > others 3A5) than rifampicin, a compound typically selected for such analyses. was conducted with XLfit™ (IDBS) curve-fitting software using a 4 Parameter Logistic ‰ Rifampicin: CYP3A4 > 2B6 > 2C8 > 2C9 > 3A5 (no induction) Model, no. 205. Acceptance criteria for the fits are described previously (3).Whena ‰ Phenobarbital: CYP2B6 > 3A4 > 3A5 (4.9-fold) > others References discernible plateau was not evident or no induction response was found, the maximal (1) Fahmi O et al. (2010) Drug Metabolism and Disposition 38:1605. observed fold over control was used for analysis. Inducibility of CYP isoforms was ¾ Troglitazone and sulfinpyrazone gave the highest response for CYP2C8 (5.0 and 5.7-fold, respectively); rifampicin (2.1-fold) and phenobarbital (3.1-fold) were much less. These data have (2) FDA guidance for Drug Interaction Studies (2012) determined by frequency of a >2 fold induction of each enzyme for these compounds in implications for selection of a robust positive control for CYP2C8 induction response. (3) Zhang JG et al (2014) Drug Metabolism and Disposition 42:1379. the three hepatocyte lots. Emax distribution, and a paired EC50 graph between CYP3A4 ¾ Relative to other compounds, rifampicin, exhibited high selectivity for CYP3A4 maximal induction; 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. vs. CYP3A5 or CYP3A4 vs. CYP2C8 of all compounds were used to visualize distinct Corning Life Sciences makes no claims regarding the performance of these products for clinical or diagnostic applications. Based on its EC value, it was by far the most potent inducer for all CYPs, except for CYP2C19 and inducibility patterns of CYP isoforms where. The EC50 values were converted to molar 50 For a listing of trademarks, visit us at ww w.corning.com/lifesciences/trademarks. All other trademarks included in this document are the property of their respective ow ners. CYP3A5 (no induction) © 2015 Corning Incorporated units prior to log transformation comparison analysis of EC50.