1521-009X/43/11/1679–1690$25.00 http://dx.doi.org/10.1124/dmd.115.065656 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1679–1690, November 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Defining Human Pathways of Drug Metabolism In Vivo through the Development of a Multiple Humanized Mouse Model s Nico Scheer, 1 Yury Kapelyukh, Anja Rode, Stefan Oswald, Diana Busch, Lesley A. McLaughlin, De Lin, Colin. J. Henderson, and C. Roland Wolf Taconic Biosciences GmbH, Köln, Germany (N.S., A.R.); University Medicine of Greifswald, Center of Drug Absorption and Transport (C_DAT), Department of Clinical Pharmacology, Greifswald, Germany (S.O., D.B); and Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom (Y.K., L.A.M., D.L., C.H., C.R.W) Received May 22, 2015; accepted August 10, 2015 ABSTRACT Variability in drug pharmacokinetics is a major factor in defining drug efficacy and side effects. There remains an urgent need, particularly with the growing use of polypharmacy, to obtain more informative experimental data predicting clinical outcomes. Major species dif- ferences in multiplicity, substrate specificity, and regulation of enzymes from the cytochrome P450–dependent mono-oxygenase system play a critical role in drug metabolism. To develop an in vivo model for predicting human responses to drugs, we generated a mouse, where 31 P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene families were exchanged for their relevant human counterparts. The model has been improved through additional humanization for the nuclear receptors consti- tutive androgen receptor and pregnane X receptor that control the expression of key drug metabolizing enzymes and transporters. In this most complex humanized mouse model reported to date, the cytochromes P450 function as predicted and we illustrate how these mice can be applied to predict drug-drug interactions in humans. Introduction Cytochrome P450-dependent mono-oxygenases (CYPs) play a major role in the metabolism and disposition of most therapeutic drugs, with .80% of currently used drugs metabolized by these enzymes (Williams et al., 2004; Guengerich, 2008). The P450 system comprises a number of multigene families, with individual members exhibiting a distinct pattern of substrate specificity (Nelson et al., 2004), and provides an adaptive response, where on exposure to drugs or environmental chemicals, transcription factors are activated, which increase the expression of specific cytochromes P450 and drug transporters, resulting in increased rates of elimination (Omiecinski et al., 2011). Two transcription factors, the constitutive androgen receptor (CAR) and the pregnane X receptor (PXR), play a pivotal role in this process (Stanley et al., 2006). This regulatory network is complex. Both CAR and PXR can be activated by the same compounds and can activate an overlapping spectrum of detoxication genes. To define how drugs may be handled in humans, it is critical to obtain a detailed analysis of these pathways. A number of in vitro and in vivo approaches have been developed to predict how drugs interact with the human P450 system and the consequences for drug therapy involving in vitro screens using recombinant P450 enzymes, hepatic microsomal fractions, or isolated hepatocytes (Gebhardt et al., 2003) and in vivo pharmacokinetic studies in animals (Tang et al., 2007). These data are then extrapolated to the human situation using in silico algorithms (Rostami-Hodjegan, 2012; Tang et al., 2007). Although valuable, these models have a number of limitations, including the challenge in predicting complex clinical outcomes from reductionist in vitro results and profound species differences between the pathways of drug disposition. In mice, for example, there are 34 cytochromes P450 in the major gene families involved in drug metabolism, i.e., the Cyp1a, Cyp2c, Cyp2d, and Cyp3a gene clusters, whereas in humans, there are eight (Nelson et al., 2004). Interestingly three human enzymes, CYP2C9, CYP2D6, and CYP3A4, account for ;75% of all reactions, with CYP3A4 being the single most important human P450 accounting for ;45% of phase 1 drug metabolism (Guengerich, 2008). Differences in the number of gene-duplication events during the past ;65 million years since the human and mouse genomes have diverged prohibit the assignments of orthologous genes between humans and mice, and unsurprisingly, the substrate specificity of proteins within gene families varies greatly across species (Martignoni et al., 2006). Furthermore, regulation of cytochromes P450 and indeed This work was supported in part by a Cancer Research UK programme grant [C4639/A10822] awarded to C.R.W. and by the InnoProfile-Transfer grant [Grant 03IPT612X] awarded to S.O. Part of this work was supported by ITI Life Sciences, Scotland. N.S. and Y.K. contributed equally to this work. 1 Current affiliation: Independent Consultants, Cologne, Germany. dx.doi.org/10.1124/dmd.115.065656. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: AUC, area under the curve; CAR, constitutive androgen receptor; CYP, cytochrome P450; DDI, drug-drug interaction; EMC, erythromycin; HLM, human liver microsome; hPXR/CAR/CYP3A4/7, humanized mice for PXR, CAR, and CYP3A4/7; hPXR/CAR/CYP3A4/7/2D6/ 2C9, humanized mice for PXR, CAR, CYP3A4/7, CYP2D6, and CYP2C9; K I , enzyme-inhibitor complex dissociation constant; k inact , rate constant of inactive enzyme formation; KO, knockout; KTZ, ketoconazole; MDZ, midazolam; PB, phenobarbital; PEG, polyethylene glycol; PO, by mouth; PXR, pregnane X receptor; RIF, rifampicin; WT, wild type. 1679 http://dmd.aspetjournals.org/content/suppl/2015/08/11/dmd.115.065656.DC1 Supplemental material to this article can be found at: at ASPET Journals on June 11, 2020 dmd.aspetjournals.org Downloaded from