Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2015/12/18/dmd.115.066985.DC1 1521-009X/44/3/466–475$25.00 http://dx.doi.org/10.1124/dmd.115.066985 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:466–475, March 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes s Rucha S. Sane, Diane Ramsden, John P. Sabo, Curtis Cooper, Lois Rowland, Naitee Ting, Andrea Whitcher-Johnstone, and Donald J. Tweedie Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut (R.S.S, D.R., J.P.S., L.R., N.T., A.W.J, D.J.T) and Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada (C.C.) Received September 4, 2015; accepted December 17, 2015 ABSTRACT The drug-drug interaction (DDI) potential of deleobuvir, an hepatitis note was more potent CYP2C8 inactivation by deleobuvir-AG than C virus (HCV) polymerase inhibitor, and its two major metabolites, deleobuvir and P450 induction by CD 6168 but not by deleobuvir. Downloaded from CD 6168 (formed via reduction by gut bacteria) and deleobuvir-acyl The predicted net AUC ratios for probe substrates were 2.92 glucuronide (AG), was assessed in vitro. Area-under-the-curve (CYP1A2), 0.45 (CYP2C9), and 0.97 (CYP3A4) compared with (AUC) ratios (AUCi/AUC) were predicted using a static model and clinically observed ratios of 1.64 (CYP1A2), 0.86 (CYP2C9), and compared with actual AUC ratios for probe substrates in a P450 1.23 (CYP3A4). Predictions of DDI using deleobuvir alone would cocktail of caffeine (CYP1A2), tolbutamide (CYP2C9), and midazo- have significantly over-predicted the DDI potential for CYP3A4 lam (CYP3A4), administered before and after 8 days of deleobuvir inhibition (AUC ratio of 6.15). Including metabolite data brought dmd.aspetjournals.org administration to HCV-infected patients. In vitro studies assessed the predicted net effect close to the observed DDI. However, inhibition,inactivationandinductionofP450s.Inductionwas the static model over-predicted the induction of CYP2C9 and assessed in a short-incubation (10 hours) hepatocyte assay, inhibition/inactivation of CYP1A2. This multiple-perpetrator DDI validated using positive controls, to circumvent cytotoxicity seen scenario highlights the application of the static model for with deleobuvir and its metabolites. Overall, P450 isoforms were predicting complex DDI for CYP3A4 and exemplifies the impor- differentially affected by deleobuvir and its two metabolites. Of tance of including key metabolites in an overall DDI assessment. at ASPET Journals on September 27, 2021 Introduction Agency (EMA, 2012), contribution of major metabolites should be Deleobuvir (BI 207127) is an inhibitor of hepatitis C virus (HCV) considered in assessing overall drug-drug interaction (DDI) liability . NS5B RNA polymerase and, in combination with faldaprevir and of the parent compound if a metabolite concentration is 25% of the . ribavirin, achieved high, sustained viral-load reduction in treatment- parent and/or if a metabolite concentration is 10% of total drug- naïve and treatment-experienced patients with chronic genotype 1b related material in circulation. Higher cut-off values have been infection (Zeuzem et al., 2013). In a disposition study in which 14C- proposed in the literature using the logic that polar metabolites deleobuvir was administered to healthy male human volunteers, two lacking structural alerts are typically less potent P450 inhibitors and major metabolites of deleobuvir were identified, an alkene reduction inactivators than the parent drug (Callegari et al., 2013; Yu and product (CD 6168) formed by gut bacteria and deleobuvir-acyl Tweedie, 2013; Yu et al., 2015). These approaches have largely glucuronide (deleobuvir-AG) (Fig. 1) (Chen et al., 2015; McCabe focused on DDI as a result of inhibition and inactivation. et al., 2015). Following a single oral dose of 800 mg deleobuvir to Recent evaluations to estimate clinical drug-drug interaction risk involving a combination of inhibition, inactivation, and induction, healthy volunteers, CD 6168 and deleobuvir-AG exposure (AUC0–‘) in the systemic circulation was 27 and 43%, respectively, of the using dynamic physiologically based pharmacokinetic models, as parent deleobuvir. Both CD 6168 and deleobuvir-AG are pharma- well as simpler static models, have shown promising results for cologically active against HCV replication, albeit 10- and 3-fold less CYP3A4 (Fahmi et al., 2008a,b; Zhao et al., 2011; Einolf et al., potent, respectively, than deleobuvir (data on file at Boehringer 2014). In this study, in vitro DDI assessment was performed for Ingelheim). parent drug deleobuvir and both of its major metabolites. In vitro, According to the drug-drug interaction guidance documents from the US deleobuvir, CD 6168, and/or deleobuvir-AG affected activities of FoodandDrugAdministration (FDA, 2012) and the European Medicines several P450 isoforms by competitive inhibition, inactivation, and/or induction. Dynamic models incorporate temporal changes in drug and enzyme levels in a sophisticated manner, thereby minimizing dx.doi.org/10.1124/dmd.115.066985. overprediction of interaction. Static models usually incorporate s This article has supplemental material available at dmd.aspetjournals.org. only the maximal plasma concentrations or the portal vein inlet ABBREVIATIONS: AUC, area under the concentration time curve; Cmax, maximum plasma concentration; CAR, constitutive androstane receptor; DDI, drug-drug interaction; deleobuvir-AG, deleobuvir-acyl glucuronide; GI, gastrointestinal; HCV, hepatitis C virus; HLM, human liver microsomes; LC-MS/MS, high-performance liquid chromatography–tandem mass spectrometry; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide; OATP, organic anion-transporting polypeptide; P450, cytochromes P450; PEG, polyethylene glycol; PXR, pregnane X receptor; QD, every day (or daily); r, recombinant. 466 P450 DDI of Deleobuvir and Its Metabolites 467 for managing comedication use in larger scale clinical studies with deleobuvir are also discussed. Materials and Methods 13 13 Materials. Deleobuvir, CD 6168, deleobuvir-AG, C6-deleobuvir, C6-CD 6168 were synthesized at Boehringer Ingelheim Pharmaceuticals Inc. (Ridgefield, CT). Cryoplateable human hepatocytes were purchased from Invitrogen/ ThermoFisher Scientific (Grand Island, NY); donor information can be found in (Supplemental Table 1). Pooled human liver microsomes (HLM; (Supple- mental Table 2) and recombinant human cytochromes P450 (rP450; produced in baculovirus-infected insect cells) and control insect cell microsomes, rat type 1 collagen, L-glutamine, penicillin/streptomycin, ITS+ premix, and fetal bovine serum were purchased from Corning (Woburn, MA). NADPH, omeprazole (OME), phenobarbital (PB), rifampicin (RIF), trypan blue, ECM, William’s E media, LDH, and MTT cytotoxicity assay kits and dexamethasone were purchased from Sigma-Aldrich (St. Louis, MO). The mRNA catcher kits, reverse transcription polymerase chain reaction (RT-PCR), and TaqMan primer probes were purchased from Invitrogen/ThermoFisher Downloaded from Scientific. All other reagents and solvents were of analytical grade or higher purity and were obtained from commercial suppliers. Human liver microsomes and rP450s were stored at –80°C until used. Fig. 1. Structures of deleobuvir, CD 6168, and deleobuvir-AG. Cytochrome P450 Inhibition. Each isoform-specific cytochrome P450 (P450) probe substrate (CYP1A2, phenacetin; CYP2B6, bupropion; CYP2C8, amodia- quine; CYP2C9, diclofenac; CYP2C19, (S)-mephenytoin; CYP2D6, dextromethor- dmd.aspetjournals.org concentrations, which is not a realistic assumption throughout the phan; CYP3A4, midazolam) was incubated in the presence of various concentrations of deleobuvir, CD 6168, or deleobuvir-AG with pooled HLM time course of co-administration of interacting drugs. In the case of in potassium phosphate buffer (50 mM, pH 7.4) and respective isoform-specific deleobuvir, the complexity of incorporating three perpetrator com- metabolites (acetaminophen, hydroxybupropion, N-desmethylamodiaquine, 49- pounds, with mixed effects, made attempts at dynamic modeling very hydroxydiclofenac, 49 -hydroxymephenytoin, dextrorphan, 19-hydroxymidazolam) challenging, and consequently a simpler static model, modified to were monitored. HLM, inhibitor, and substrate were preincubated at 37°Cfor incorporate multiple perpetrators, was used to evaluate the accuracy of 5 minutes. Incubations with isoform-selective P450 inhibitors (CYP1A2, prediction of the effect of deleobuvir on CYP1A2, CYP2C9, and a-naphthoflavone; CYP2B6, ticlopidine; CYP2C8, montelukast; CYP2C9, CYP3A4activityinvivo,whichthenmadefeasibleapredictionofthe sulfaphenazole; CYP2C19, S-(+)-N-3-benzylnirvanol; CYP2D6, quinidine; at ASPET Journals on September 27, 2021 effects on other P450 isoforms. CYP3A4, ketoconazole) were included as positive controls and showed An in vivo study was conducted in HCV-infected patients to assess the expected results (data not shown). All experiments were performed under net effect of deleobuvir (and generated metabolites) on the pharmacoki- linear conditions with respect to time and protein concentration and were conducted at substrate concentrations equivalent to their K values determined netics of caffeine, tolbutamide, and midazolam, used in combination as an m in HLM