CYP2C76, a Novel Cytochrome P450 in Cynomolgus Monkey, Is a Major CYP2C in Liver, Metabolizing Tolbutamide and Testosterone

Abstract

Monkeys are widely used as a primate model to study drug metabolism because they generally show a metabolic pattern similar to humans. However, the paucity of information on cytochrome P450 (P450) genes has hampered a deep understanding of drug metabolism in the monkey. In this study, we report identification of the CYP2C76 cDNA newly identified in cynomolgus monkey and characterization of this CYP2C along with cynomolgus CYP2C20, CYP2C43, and CYP2C75. The CYP2C76 cDNA contains the open reading frame encoding a protein of 489 amino acids that are only approximately 80% identical to any human or monkey P450 cDNAs. Gene and protein expression of CYP2C76 was confirmed in the liver of cynomolgus and rhesus monkeys but not in humans or the great apes. Moreover, CYP2C76 is located at the end of the CYP2C gene cluster in the monkey genome, the region of which corresponds to the intergenic region adjacent to the CYP2C cluster in the human genome, strongly indicating that this gene does not have the ortholog in humans. Among the four CYP2C genes expressing predominantly in the liver, the expression level of CYP2C76 was the greatest, suggesting that CYP2C76 is a major CYP2C in the monkey liver. Assays for the capacity of CYP2C76 to metabolize drugs using several substrates typical for human CYP2Cs revealed that CYP2C76 showed unique metabolic activity. These results suggest that CYP2C76 contributes to overall drug-metabolizing activity in the monkey liver and might account for species difference occasionally seen in drug metabolism between monkeys and humans.Cytochrome P450s (P450s) are one of the most important drug-metabolizing enzymes and form a superfamily consisting of a large number of subfamilies (Nelson et al., 1996(Nelson et al., , 2004. The cDNA sequences encoding P450s have been reported for many species of not only mammals but also birds, insects, plants, bacteria, and others (see http://drnelson. utmem.edu/CytochromeP450.html). In humans, 57 functional genes have been identified to date (Nelson et al., 2004). The human CYP2C subfamily, comprising CYP2C8, CYP2C9, CYP2C18, and CYP2C19, is essential in metabolizing approximately 20% of all prescribed drugs, including tolbutamide, phenytoin, warfarin, and ibuprofen (Goldstein, 2001). The CYP2C subfamily consists of multiple members in each mammalian species, including 15 in mice, 12 in rats, and 9 in rabbits (for the latest information, see http://drnelson.utmem.edu/CytochromeP450.html). Between humans and rodents, the number of the subfamily members is different, and none of the CYP2Cs seems to show a clear orthologous relationship between the two species, suggesting that the data from rodents must be cautiously interpreted and extrapolated to humans (Nelson et al., 2004).For monkeys, which generally mean Old or New World monkeys, three CYP2C cDNAs have been identified in the macaque and cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) monkeys. Two sequences have been published including cynomolgus CYP2C20 (Komori et al., 1992) and rhesus CYP...

Identification and Characterization of CYP2C18 in the Cynomolgus Macaque (Macaca fascicularis)

“…CYP2C76 cDNA shows only 75-76% sequence identity to human CYP2Cs. CYP2C76 is not orthologous to any human CYPs [20] and is partly responsible for the difference of drug metabolism between cynomolgus macaques and humans [21]. Such species differences also could be explained by functional disparity of the orthologous CYPs between the two species, such as, if an ortholog to human CYP with low (or no) expression and drug-metabolizing activity, for example CYP2C18, shows abundant expression and substantial activity in cynomolgus macaques.…”

“…All human CYP2Cs exhibit genetic polymorphisms. In particular, some genetic polymorphisms of CYP2C9 and CYP2C19 have been shown to result in toxicity or altered efficacy of drugs in humans.In cynomolgus macaques, which are used to evaluate drug effects and toxicities in preclinical studies of drug development, cDNAs for CYP2C20, CYP2C43, CYP2C75 and CYP2C76 have been identified [9,20]. CYP2C20 cDNA is highly homologous (95%) to human CYP2C8 cDNA, while CYP2C43 and CYP2C75 cDNAs are highly homologous (93-95%) to both human CYP2C9 and CYP2C19 cDNAs.…”

“…The six liver samples were pooled and used for extraction of total RNA, which was carried out as described previously [20]. This study has been reviewed and approved by the Institutional Animal Care and Use Committee.…”

“…To analyze the tissue expression pattern of CYP2C18, real-time RT-PCR was performed as described previously [20]. The expression level was measured in the brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary and uterus.…”

“…This study has been reviewed and approved by the Institutional Animal Care and Use Committee. Reverse transcription (RT) reaction was performed with the liver RNA, and a subsequent polymerase chain reaction (PCR) with the generated RT product was carried out as described previously [20]. The primers were designed based on the rhesus macaque genome sequence, which is highly homologous to the human CYP2C18 cDNA sequence, as determined by the BLAT program (UCSC Genome Bioinformatics).…”