1521-009X/43/7/969–976$25.00 http://dx.doi.org/10.1124/dmd.115.063909 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:969–976, July 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Substrate Selectivities and Catalytic Activities of Marmoset Liver Cytochrome P450 2A6 Differed from Those of Human P450 2A6 Shotaro Uehara, Yasuhiro Uno, Takashi Inoue, Erika Sasaki, and Hiroshi Yamazaki Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo, Japan (S.U., H.Y.); Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Kainan, Wakayama, Japan (Y.U.); Department of Applied Developmental Biology, Central Institute for Experimental Animals, Kawasaki, Japan (T.I., E.S.); and Keio Advanced Research Center, Keio University, Minato-ku, Tokyo, Japan (E.S.) Received February 18, 2015; accepted April 9, 2015 ABSTRACT The common marmoset (Callithrix jacchus), a New World primate species, is potentially a useful animal model for preclinical studies in drug development. However, cytochrome P450 (P450) enzymes have not been fully identified and characterized in marmosets. In this study, we identified P450 2A6 cDNA with the sequence highly identical (91–94%) to human P450 2A6, 2A7, and 2A13 cDNA and cynomolgus monkey P450 2A23, 2A24, and 2A26 cDNA. Among the tissue types examined, marmoset P450 2A6 mRNA was most abundantly expressed in livers where P450 2A6 protein was also detected by immunoblotting. Phylogenetic analysis showed that marmoset P450 2A6 was more closely clustered with human and cynomolgus monkey P450 2As than P450 2As of dog, rat, and mouse (the species also used in drug metabolism). Marmoset P450 2A6 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation, similar to human P450 2A6 and 2A13 and cynomolgus monkey P450 2A23, 2A24, and 2A26, but much less effectively coumarin 7-hydroxylation, showing some difference as well. Interestingly, marmoset P450 2A6 and cynomolgus monkey P450 2A23 catalyzed phenacetin O-deethylation, which is catalyzed by human P450 1A2 and 2A13, but not by P450 2A6. Marmoset P450 2A6 also exhibited catalytic activity toward testosterone by the multiple sites, but not rat P450 2A-specific testosterone 7a-hydroxylation activity. These results indicated that marmoset P450 2A6 had functional characteristics different from those of human and cynomolgus monkey P450 2As in terms of partially different substrate specificities and catalytic activities, indicating its importance of further studies for P450 2A-dependent drug metabolism in marmosets. Introduction Nonhuman primate species, especially the Old World monkey such as cynomolgus monkeys (Macaca fascicularis) and rhesus monkeys (Macaca mulatta), are widely used for preclinical assessment of metabolism, pharmacokinetics, and toxicity of drugs because of their genetic closeness and phenotypic similarities to humans. The common marmoset (Callithrix jacchus), a New World monkey, has benefits such as small body size, ease handling, high fertility, and early sexual maturity (Orsi et al., 2011). Therefore, marmosets have attracted considerable attention as a useful animal model in various fields such as neuroscience, stem cell research, drug metabolism, toxicology, and immune and autoimmune diseases (Sasaki et al., 2005; Orsi et al., 2011; Carrion and Patterson, 2012; Jagessar et al., 2013; Kishi et al., 2014) Cytochrome P450s (P450s) are the major enzymes that play a role in the metabolism of various endogenous and exogenous compounds, such as steroids, environmental carcinogens, and drugs. Human P450s contains 57 functional genes and 58 pseudogenes, including those of the P450 1–3 subfamilies important for drug metabolism. The human P450 2A subfamily comprises P450 2A6, 2A7, and 2A13 (Fernandez- Salguero and Gonzalez, 1995) with their amino acid sequences being highly homologous to each other (92–94%). Human P450 2A6, mainly expressed in the liver, is a major catalyst of coumarin and precarcinogens, including many N-nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone, and aflatoxin B 1 (Yamazaki et al., 1992; Koskela et al., 1999; Su et al., 2000; He et al., 2006). Human P450 2A7 mRNA expression has been shown in the liver, but heterologously expressed P450 2A7 showed no catalytic activity (Ding et al., 1995). Human P450 2A13 mRNA has been shown to be highly expressed in the lungs, and the respiratory tract such as nasal mucosa, and shares several substrates with P450 2A6 (Su et al., 2000). In macaques, more than 20 P450s were identified and characterized (Uno et al., 2011). The cynomolgus monkey P450 2A subfamily consists of P450 2A23, 2A24, and 2A26, which have high sequence identities (89–95%) to human P450 2As at amino acid level. As with human P450 2A6, cynomolgus monkey P450 2A23, 2A24, and 2A26 mRNAs were expressed predominantly in the liver; their proteins catalyzed coumarin 7-hydroxylation (Uno et al., 2007; Uehara et al., 2010). Species differences for drug metabolism are an important issue for drug development. Marmosets and rats are reportedly poor coumarin 7-hydroxylators, unlike mice, cynomolgus monkeys, and humans (Lake et al., 1989; Steensma et al., 1994), indicating the differences of P450 2A enzymes in marmosets as compared with cynomolgus monkeys and humans. Marmoset P450 2A enzymes expressed in liver could account This work was supported in part by Grant-in-Aid for Scientific Research and also resulted from “Construction of System for Spread of Primate Model Animals” under the Strategic Research Program for Brain Sciences of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. S.U. and Y.U. equally contributed to this work. dx.doi.org/10.1124/dmd.115.063909. ABBREVIATIONS: PCR, polymerase chain reaction; RT, reverse transcription. 969 at ASPET Journals on July 8, 2017 dmd.aspetjournals.org Downloaded from