NOTE Toxicology Identification and Characterization of CYP2C18 in the Cynomolgus Macaque (Macaca fascicularis) Yasuhiro UNO1)*, Kiyomi MATSUNO1), Chika NAKAMURA1), Masahiro UTOH1) and Hiroshi YAMAZAKI2) 1)Pharmacokinetics and Bioanalysis Center (PBC), Shin Nippon Biomedical Laboratories (SNBL), Kainan, Wakayama 642–0017 and 2)Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194–8543, Japan (Received 3 August 2009/Accepted 15 September 2009/Published online in J-STAGE 25 November 2009) ABSTRACT. The macaque is widely used for investigation of drug metabolism due to its evolutionary closeness to the human. However, the genetic backgrounds of drug-metabolizing enzymes have not been fully investigated; therefore, identification and characterization of drug-metabolizing enzyme genes are important for understanding drug metabolism in this species. In this study, we isolated and char- acterized a novel cytochrome P450 2C18 (CYP2C18) cDNA in cynomolgus macaques. This cDNA was highly homologous (96%) to human CYP2C18 cDNA. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney. Moreover, a metabolic assay using cynomolgus CYP2C18 protein heterologously expressed in Escherichia coli revealed its activity toward S-mephenytoin 4’-hydrox- ylation. These results suggest that cynomolgus CYP2C18 could function as a drug-metabolizing enzyme in the liver. KEY WORDS: cloning, CYP2C18, cytochrome P450, liver, monkey. J. Vet. Med. Sci. 72(2): 225–228, 2010 Cytochrome P450 (CYP) is a superfamily of some of the Such species differences also could be explained by func- most important drug-metabolizing enzymes and consists of tional disparity of the orthologous CYPs between the two a large number of subfamilies [14]. In humans, the CYP2C species, such as, if an ortholog to human CYP with low (or subfamilies contain important enzymes that metabolize no) expression and drug-metabolizing activity, for example approximately 20% of all prescribed drugs [3]. In the CYP2C18, shows abundant expression and substantial CYP2C subfamily, CYP2C8, CYP2C9 and CYP2C19 are activity in cynomolgus macaques. To investigate this possi- highly expressed in the liver, whereas protein expression of bility, in this study, we isolated cynomolgus CYP2C18 human CYP2C18 has not been seen at a detectable level in cDNA encoding an ortholog of human CYP2C18 and char- the liver [3]. CYP2C8, CYP2C9 and CYP2C19 exhibit acterized it by sequence and phylogenetic analyses, tissue metabolic activities toward clinically important drugs; the expression pattern and drug-metabolizing activity toward anticancer drug paclitaxel is metabolized by CYP2C8; the human CYP2C substrates. Human CYP2C18 catalyzes hypoglycemic agent tolbutamide, nonsteroidal anti-inflam- phenytoin hydroxylation more efficiently than CYP2C9 or matory drug diclofenac, anticonvulsant phenytoin and anti- CYP2C19 [8], making CYP2C18 a suitable candidate for coagulant warfarin are metabolized by CYP2C9; and the our investigation. antiulcer drug omeprazole and the anticonvulsant drug To clone cynomolgus CYP2C18 cDNA, liver samples mephenytoin are metabolized by CYP2C19 [3]. All human were collected from six cynomolgus macaques (three males CYP2Cs exhibit genetic polymorphisms. In particular, and three females, 4–5 years of age) of Indochinese origin some genetic polymorphisms of CYP2C9 and CYP2C19 weighing 3–5 kg, which were kept under established guide- have been shown to result in toxicity or altered efficacy of lines and procedures (Shin Nippon Biomedical Laborato- drugs in humans. ries, Tokyo, Japan). The six liver samples were pooled and In cynomolgus macaques, which are used to evaluate used for extraction of total RNA, which was carried out as drug effects and toxicities in preclinical studies of drug described previously [20]. This study has been reviewed development, cDNAs for CYP2C20, CYP2C43, CYP2C75 and approved by the Institutional Animal Care and Use and CYP2C76 have been identified [9, 20]. CYP2C20 Committee. Reverse transcription (RT) reaction was per- cDNA is highly homologous (95%) to human CYP2C8 formed with the liver RNA, and a subsequent polymerase cDNA, while CYP2C43 and CYP2C75 cDNAs are highly chain reaction (PCR) with the generated RT product was homologous (93–95%) to both human CYP2C9 and carried out as described previously [20]. The primers were CYP2C19 cDNAs. CYP2C76 cDNA shows only 75–76% designed based on the rhesus macaque genome sequence, sequence identity to human CYP2Cs. CYP2C76 is not which is highly homologous to the human CYP2C18 cDNA orthologous to any human CYPs [20] and is partly responsi- sequence, as determined by the BLAT program (UCSC ble for the difference of drug metabolism between cynomol- Genome Bioinformatics). The PCR primers were 5’- gus macaques and humans [21]. GTGAAAGCCCACAGTTTTCTTAC-3’ and 5’-AGG- GAATGGGAAGATGTGG-3’. The PCR products were *CORRESPONDENCE TO: UNO, Y., Pharmacokinetics and Bioanalysis Center (PBC), Shin Nippon Biomedical Laboratories (SNBL), cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA, 16–1 Minami Akasaka, Kainan, Wakayama 642–0017, Japan. U.S.A.) according to the manufacturer’s protocol, e-mail: [email protected] sequenced using an ABI PRISM BigDye Terminator v3.0 226 Y. UNO ET AL. Fig. 1. Amino acids of cynomolgus and human CYP2C18s. CYP2C18 sequences were compared between cynomolgus macaques (mf) and humans (h). Above the sequences, the broken and solid lines indicate a heme-binding region and six substrate recognition sites (SRSs), respectively. Under the sequences, asterisks indicate identical amino acids. Ready Reaction Cycle Sequencing Kit (Applied Biosys- Table 1. Sequence comparisons between cynomolgus CYP2C18 tems, Foster City, CA, U.S.A.) and then subjected to elec- and other CYP2Cs trophoresis with an ABI PRISM 3730 DNA Analyzer cDNA (%) Amino acids (%) (Applied Biosystems). Sequence analysis was carried out with DNASIS Pro (Hitachi Software Engineering Co., Ltd., Human: CYP2C8 87 77 Tokyo, Japan). The nucleotide sequence of the cynomolgus CYP2C9 88 81 CYP2C18 96 96 CYP2C18 cDNA identified (GenBank accession number CYP2C19 87 81 DQ297685) contained an open reading frame of 490 amino Cynomolgus: CYP2C20 86 77 acids with primary sequence structures characteristic of CYP2C43 86 80 CYP proteins, including a heme-binding region and six CYP2C75 88 81 potential substrate recognition sites (SRSs) [4] (Fig. 1). CYP2C76 79 75 Among human and other cynomolgus CYP2Cs, the deduced amino acids of cynomolgus CYP2C18 cDNA showed the highest sequence identity to human CYP2C18 amino acids (Table 1). A phylogenetic analysis based on the amino acid sequences for cynomolgus and human CYP2C proteins indicated that cynomolgus CYP2C18 was grouped into the same clade with human CYP2C18 (Fig. 2), indicating the evolutionary closeness of CYP2C18 between cynomolgus macaques and humans. 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. These tissues were collected and used to prepare total RNA as described earlier for the liver tissue. The Taq- Man MGB probe (5’-TCTGCAATAATTTCCCAGCTC- 3’) was labeled at the 5’ end with FAM fluorescence reporter dye. The primers were 5’-AAAATTCAATGAAA ACCTCAGGATTC-3’ and 5’-GTTATGACTTCCTGGGA GATAATCG-3’. The probe and primers were used at 250 nM and 300 nM, respectively. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney (Fig. 3), but Fig. 2. Phylogenetic analysis of CYP2C amino acid sequences its hepatic expression was low, with the level being approx- from the cynomolgus macaque and human. The phylogenetic imately 1/950–1/200 of that of cynomolgus CYP2C20, tree was created with the ClustalW program. The CYP2C amino acid sequences used are from the cynomolgus macaque (mf; CYP2C43, CYP2C75 and CYP2C76 based on a comparison Macaca fascicularis) and human (h). with our published data of the latter genes [20]. Quantifica- CHARACTERIZATION OF CYNOMOLGUS CYP2C18 227 the partially purified recombinant CYP2C18 at 37C for 5 min. This was followed by addition of an NADPH-generat- ing system (0.25 mM NADP+, 2.5 mM glucose 6-phosphate and 0.25 units/ml glucose 6-phosphate dehydrogenase), so that the metabolic reaction was initiated at a final concentra- tion of 100 pmol CYP/ml. The reactions were incubated at 37C for 30 min and quenched by addition of a half volume of ice-cold acetonitrile. The resultant mixtures were centri- fuged (1,500 g, 4C, 10 min), and the metabolite, 4- hydroxymephenytoin, in the supernatant was analyzed by high-performance liquid chromatography. Measurement of S-mephenytoin 4’-hydroxylation using Fig. 3. Gene expression of CYP2C18 in cynomolgus macaque cynomolgus CYP2C18 recombinant protein showed that the tissues. Real-time RT-PCR was performed with a gene-specific activity of cynomolgus CYP2C18 was 0.019 nmol/min/ probe and primer set. Gene expression was measured in the nmol CYP, which was lower than that generally observed brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, with human CYP2C19, for which S-mephenytoin 4’- ovary and uterus. For each tissue, total RNAs from six animals hydroxylation is a marker reaction. Activity was not were pooled and used for real-time RT-PCR. The expression level of cynomolgus CYP2C18 was normalized to the 18S detected with paclitaxel or diclofenac, typical substrates for rRNA level.