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

Abstract

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 characterized 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'-hydroxylation. 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 most important drug-metabolizing enzymes and consists of a large number of subfamilies [14]. In humans, the CYP2C subfamilies contain important enzymes that metabolize approximately 20% of all prescribed drugs [3]. In the CYP2C subfamily, CYP2C8, CYP2C9 and CYP2C19 are highly expressed in the liver, whereas protein expression of human CYP2C18 has not been seen at a detectable level in the liver [3]. CYP2C8, CYP2C9 and CYP2C19 exhibit metabolic activities toward clinically important drugs; the anticancer drug paclitaxel is metabolized by CYP2C8; the hypoglycemic agent tolbutamide, nonsteroidal anti-inflammatory drug diclofenac, anticonvulsant phenytoin and anticoagulant warfarin are metabolized by CYP2C9; and the antiulcer drug omeprazole and the anticonvulsant drug mephenytoin are metabolized by CYP2C19 [3]. 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. 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. To investigate this possibility, in this study, we isolated cynomolgus CYP2C18 cDNA encoding an ortholog of human C...

Newly Identified CYP2C93 Is a Functional Enzyme in Rhesus Monkey, but Not in Cynomolgus Monkey

“…To determine the expression level of CYP2C93 mRNA relative to other CYP2C mRNAs, CYP2C93 mRNA, along with CYP2C8, CYP2C43, CYP2C75, and CYP2C76 mRNAs, was measured in the livers of two rhesus monkeys expressing CYP2C93 SV1. CYP2C18 mRNA was excluded from the analysis due to its hepatic expression level substantially lower than other CYP2C mRNAs [16]. The analysis indicated that the expression level of CYP2C93 mRNA was lower than that of any other CYP2C mRNA (Fig.…”

Immunochemical Detection of Cytochrome P450 Enzymes in Liver Microsomes of 27 Cynomolgus Monkeys

“…The recombinant proteins of 19 cynomolgus P450s (CYP1A1, CYP1A2, CYP1D1, CYP2A23, CYP2A24, CYP2A26, CYP2B6, CYP2C18, CYP2C8, CYP2C9, CYP2C19, CYP2C76, CYP2D17, CYP2D44, CYP2E1, CYP2J2, CYP3A4, CYP3A5, or CYP3A43) were expressed in E. coli, and membrane preparations were performed as described previously (Uno et al, 2006(Uno et al, , 2007a(Uno et al, , 2009b(Uno et al, , 2010b(Uno et al, , 2011dUehara et al, 2010). For expression of cynomolgus CYP2J2 recombinant protein, the N-terminus modification was conducted by polymerase chain reaction with the forward and reverse primers, 5Ј-GGAATTC-CATATGGCTCTGTTATTAGCAGTTTTTGCGGCTGCCC TCTGGG-3Ј and 5Ј-GCTCTAGAGCAAAATCACACCCGAGGAAC-3Ј, respectively.…”

Macaque cytochromes P450: nomenclature, transcript, gene, genomic structure, and function

“…Cynomolgus CYP2C18 mRNA is expressed in the liver at a much lower level than other cynomolgus CYP2C mRNAs (Uno et al, 2010b). Cynomolgus CYP2C18 catalyzes S-mephenytoin 4′-hydroxylation (much less efficiently than human CYP2C19) (Table 3), but did not metabolize other human CYP2C substrates, such as paclitaxel or diclofenac (Uno et al, 2010b). Similarly, human CYP2C18 protein is a functional enzyme, that metabolizes human CYP2C substrates, but is not expressed, or is expressed at an undetectable level, in the liver (Gerbal-Chaloin et al, 2001).…”

“…Cynomolgus CYP2C18 cDNA, 96% identical to human CYP2C18 cDNA, was recently identified (Uno et al, 2010b) (Table 1). Rhesus CYP2C18 cDNA, nearly identical to cynomolgus CYP2C18 cDNA, was found in GenBank (Table 2).…”

“…Rhesus CYP2C18 cDNA, nearly identical to cynomolgus CYP2C18 cDNA, was found in GenBank (Table 2). Cynomolgus CYP2C18 mRNA is expressed in the liver at a much lower level than other cynomolgus CYP2C mRNAs (Uno et al, 2010b). Cynomolgus CYP2C18 catalyzes S-mephenytoin 4′-hydroxylation (much less efficiently than human CYP2C19) (Table 3), but did not metabolize other human CYP2C substrates, such as paclitaxel or diclofenac (Uno et al, 2010b).…”