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Utility of CYP3A4 and PXR-CAR-CYP3A4/3A7 transgenic mouse models to assess the magnitude of CYP3A4 mediated drug-drug interactions Justin Q. Ly, Kirsten Messick, Ann Qin, Ryan H. Takahashi, and Edna F. Choo Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00006 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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Molecular Pharmaceutics

Utility of CYP3A4 and PXR-CAR-CYP3A4/3A7 transgenic mouse models to assess the magnitude of CYP3A4 mediated drug-drug interactions

Justin Q Ly, Kirsten Messick, Ann Qina Ryan H Takahashi, Edna F Choo* Genentech Inc., South San Francisco, CA 94080 at Gilead Sciences, Foster City, CA 94404

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*Corresponding Author Edna F Choo, Ph.D. Genentech, Inc. 1 DNA Way South San Francisco, CA 94080 Phone: 650-467-3861 Fax: 650-225-6452 Email: [email protected]

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TABLE OF CONTENTS GRAPHIC

CYP3A4 expression in liver microsomes from human, Cyp3a-/-Tg-3A4Hep/Int mice, wildtype mice, and PXR-CAR-3A4Hep/Int mice (saline or rifampin treated)

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ABSTRACT Species differences in the expression, activity, regulation and substrate specificity of metabolizing enzymes preclude the use of animal models to predict clinical drugdrug interactions (DDI). The objective of this work is to determine if the transgenic (Tg) Cyp3a-/-Tg-3A4Hep/Int and Nr1i2/Nr1i3-/--Cyp3a-/-Tg-PXR-CAR-3A4/3A7Hep/Int (PXR-CAR-CYP3A4/3A7) mouse models could be used to predict in vivo DDI of 10 drugs; alprazolam, bosutinib, crizotinib, dasatinib, gefitinib, ibrutinib, regorafenib, sorafenib, triazolam, and vandetinib (as victims); with varying magnitudes of reported CYP3A4 clinical DDI. As an assessment of the effect of CYP3A4 inhibition, these drugs were co-administered to Cyp3a-/-Tg-3A4Hep/Int mice with the CYP3A inhibitor, itraconazole. For crizotinib, regorafenib, sorafenib, and vandetanib, there was no significant increase of AUC observed; with alprazolam, bosutinib, ibrutinib, dasatinib, and triazolam, pre-treatment with itraconazole resulted in a 2-, 4-, 17-, 7-, and 15-fold increase in AUC, respectively. With the exception of gefinitib for which the DDI effect was over-predicted (12-fold in Tg-mice vs. 2-fold in the clinic), the magnitude of AUC increase observed in this study was consistent (within 2-fold) with the clinical DDI observed following administration with itraconazole/ketoconazole. As an assessment of CYP3A4 induction, following rifampin pre-treatment to PXR-CAR-3A4Hep/Int mice, an 8% decrease in vandetanib mean AUC was observed; 39-52% reduction in AUC were observed for dasatinib, ibrutinib, regorafenib, and sorafenib compared to vehicle treated mice. The greatest effect of rifampin induction was observed with alprazolam, bosutinib, crizotinib, gefitinib, and triazolam where 72-91% decrease in AUC were observed. With the 3



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exception of vandetanib for which rifampin induction was under-predicted, the magnitude of induction observed in this study was consistent (within 2-fold) with clinical observations. These data set suggests that with 2 exceptions, these transgenic mice models were able to exclude or capture the magnitude of CYP3A4 clinical inhibition and induction. Data generated in transgenic mice may be used to gain confidence and complement in vitro and in silico methods for assessing DDI potential/liability.

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Keywords: CYP3A4, Transgenic mouse model, drug-drug interaction

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Abbreviations: Pregnane X receptor, PXR; Constitutive androstane receptor, CAR; Cytochrome P450, CYP; Drug-drug interactions, DDI; Knockout, KO; Liquid chromatographymass spectrometry/mass spectrometry, LC-MS/MS

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INTRODUCTION Many drugs undergo biotransformation so that they may be more readily eliminated from the body. Cytochrome P450s (CYP) are the major enzymes responsible for the metabolism of approximately 50% of drugs 1. Of the different isoforms of CYP, CYP3A4 is the most abundant CYP in human and the most important enzyme in terms of drug metabolism. CYP3A are expressed in many tissues, however, their expressions in the gut and liver is of most interest as they play a key role in firstpass metabolism and clearance of many drugs 2. Alterations to the activity of CYP3A4 enzymes can potentially lead to a pronounced effect on the exposure of drugs; inhibition of CYP3A4 enzymes by strong inhibitors (e.g. ketoconazole, itraconzazole, etc.) could lead to an increase in concentrations of CYP3A4 substrates potentially resulting in toxicities. On the other hand, induction of CYP3A4 enzymes by strong inducers (e.g. rifampin, ritonavir, etc.) could result in loss of efficacy.

The assessment of a compound’s DDI liability begins in the early drug discovery process with in vitro evaluations using human reagents. Based on this data, static and/or mechanistic models as well as PBPK modeling are then employed to predict the magnitude of clinical DDI. Due to species differences in the expression, activity, regulation and substrate specificity of metabolizing enzymes preclinical in vivo studies have not been used to predict clinical DDI; however, more recently, genetically modified animal models, for instance where the mouse CYP ortholog(s) are replaced with specific human CYPs have been developed in an effort to provide in vivo animal models that can be used to predict human drug disposition. For 7



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example, the disposition of triazolam was studied using Cyp3a-/-Tg-3A4Hep/Int, and it was confirmed that after oral administration, intestinal metabolism would be more important than hepatic metabolism in oral disposition of triazolam 3. In another study using the transgenic mouse model expressing human PXR, CAR, CYP3A4/CYP3A7 and CYP2D6 (Tg-composite model), the effect of induction mediated by rifampin on the pharmacokinetics of tamoxifen and its metabolites was investigated. The Tg-composite model was able to describe the extent of rifampinmediated induction for tamoxifen, suggesting the applicability of this model in describing the DDI of drugs/compounds whose metabolism is mediated by CYP3A4 and/or CYP2D6 4. More recently we have shown using Cyp3a-/-Tg-3A4Hep, Cyp3a-/Tg-3A4Int and Cyp3a-/-Tg-3A4Hep/Int, with differential expression of CYP3A4 in the liver and intestine or both organs, respectively, the role or contribution of intestinal CYP3A4 to the oral disposition of cobimetinib could be elucidated 5. Furthermore, using Cyp3a-/-Tg-3A4Hep/Int and Nr1i2/Nr1i3-/--Cyp3a-/-Tg-PXR-CAR-3A4/3A7Hep/Int (PXR-CAR-3A4/3A7Hep/Int) transgenic mice, the clinical magnitude of CYP3A4 mediated DDI (with cobimetinib as the victim) appeared to be well predicted 5.

The objective of the present study was to further investigate the utility of the Cyp3a/-Tg-3A4Hep/Int

and PXR-CAR-3A4/3A7Hep/Int mouse models in predicting the

magnitude of CYP3A4 mediated DDI for drugs with varying magnitudes reported clinical and thus dependence on the CY3A4 pathway for clearance. In using mice that that express humanized CYP3A4 in the liver and intestine this best reflects DDI evaluation in human, where the net effect of CYP3A4 inhibition and induction 8


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includes contributions from both intestinal and liver CYP. The use of PXR-CAR3A4/3A7Hep/Int would have been adequate to test the effect of both inhibition and induction. However, because Cyp3a-/-Tg-3A4 with hepatic and intestinal only expression of CYP3A4 are available, we tested the effect of CYP3A4 inhibition in these mice to give us the option (at a latter point) of separately evaluating the effect of the liver vs. intestine to the overall DDI effect. In our evaluation, the effect of itraconazole inhibition or induction by rifampin on alprazolam, bosutinib, crizotinib, dasatinib, gefitinib, ibrutinib, regorafenib, sorafenib, triazolam, and vandetinib oral exposure was investigated.

MATERIALS AND METHODS Animals CYP3A4 transgenic mice with mouse Cyp3a deleted and replaced with human CYP3A4 in liver and intestine (Cyp3a-/-Tg-3A4Hep/Int) were obtained from Taconic, New York. Nr1i2/Nr1i3-/--Cyp3a-/-Tg-PXR-CAR-3A4/3A7Hep/Int (PXR-CARCYP3A4/3A7) mice with mouse Nr1i2/Nr1i3 and Cyp3a deleted and replaced with human PXR, CAR, and CYP3A4 were obtained from Taconic, Artemis, Germany. All animals were female, 10-12 weeks old at the time of study and weighed between 20 to 25 g. In all studies, serial blood samples (15 µL) were collected by tail nick at 0.25, 0.5, 1, 3, 6, 8, and 24 h post-dose and diluted with 60 µL water containing 1.7 mg/mL EDTA. All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S.

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National Institute of Health, and were approved by the Institution’s Animal Care and Use Committee.

Effect of CYP3A4 Inhibition or Induction on CYP3A4 Substrate Exposure in Mice The effect of CYP3A4 inhibition was determined in Cyp3a-/-Tg-3A4Hep/Int mice (n = 34 female mice/group). All animals were divided into two groups, control and treated groups. Animals in the control group were administered test drug (5 mg/kg PO; 5 mL/kg in 0.5% methylcellulose with 0.2% Tween 80 in water (MCT)). DDI were assessed based on the oral route of administration as this reflects the route of clinical administration of these drugs. In addition, to mimic clinical DDI study designs, animals in the treated group were administered itraconazole solution (SporanoxR) 100 mg/kg PO daily for 4 days prior to CYP3A4 substrate administration. On the 4th day, test drug (5 mg/kg PO) was administered 30 min after the last dose of itraconazole. The systemic exposure from a 100 mg/kg dose of itraconazole was previously shown to reflect clinical exposures from a 200 mg dose of itraconazole 5. Induction of CYP3A4 was tested in PXR-CAR-3A4/3A7Hep/Int mice (n = 3-4), where rifampin (10 mg/kg PO) or vehicle (saline) were administered daily for 5 days. On the 5th day, test drug (5 mg/kg PO) was administered at 5 mL/kg in MCT 1 h after the last dose of rifampin. The systemic exposure from 10 mg/kg rifampin was previously shown to be consistent with clinical concentrations observed from a dose of 600 mg 5. PK samples were collected as described above and concentrations of test drugs were determined by LC-MS/MS.

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Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis Concentrations of test drugs were determined by a non-validated LC-MS/MS assay. The diluted blood samples were prepared for analysis by placing a 25 µL aliquot into a 96-well plate followed by the addition of 5 µL of internal standard (100 ng/mL labetalol, 100 ng/mL indomethacin, and 10 ng/mL loperamide in acetonitrile) and 200 µL acetonitrile. The samples were vortexed and centrifuged at 1600 g for 15 min at room temperature; 50 µL of the supernatant was diluted with 150 µL of water and 5 µL of the solution was injected onto a Kinetex XB C-18 (30 x 2.1 mm, 2.6 µm) analytical column (Phenomenix, Torrance, CA). A SIL-30ACMP autosampler system (Shimadzu, Columbia, MD) was linked to LC-30AD pumps (Shimadzu), coupled with an API 5500 Qtrap mass spectrometer (AB Sciex, Foster City, CA) for sample analysis. The aqueous mobile phase was water with 0.1% formic acid (A) and the organic mobile phase was acetonitrile with 0.1% formic acid (B). The gradient was as follows: starting at 25% B and increased to 95% B for 0.6 min, maintained at 95% B for 0.1 min, then decreased to 25% B within 0.1 min. The total flow rate was 1.4 mL/min with a total run time of 0.8 min. Data were acquired using multiple reactions monitoring in positive ion electrospray mode with an operating source temperature of 600ºC.

Pharmacokinetic Analysis Pharmacokinetic parameters were calculated by non-compartmental methods as described in Gibaldi and Perrier (1982) using Phoenix™ WinNonlin®, version 6.3.0

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(Pharsight Corporation, Mountain View, CA). All PK parameters are presented as mean ± standard deviation (SD).

Statistical Analysis Data in all experiments were represented as mean ± SD. Comparisons between two groups were made using an unpaired t test. GraphPad Prism was used for all statistical analysis (version 5.00 for Mac OS X, GraphPad Software, San Diego, CA), P

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