Novel Marmoset Cytochrome P450 2C19 in Livers Efficiently Metabolizes Human P450 2C9 and 2C19 Substrates,S-Warfarin, Tolbutamide, Flurbiprofen, and Omeprazole

Abstract

The common marmoset (Callithrix jacchus), a small New World monkey, has the potential for use in human drug development due to its evolutionary closeness to humans. Four novel cDNAs, encoding cytochrome P450 (P450) 2C18, 2C19, 2C58, and 2C76, were cloned from marmoset livers to characterize P450 2C molecular properties, including previously reported P450 2C8. The deduced amino acid sequence showed high sequence identities (>86%) with those of human P450 2Cs, except for marmoset P450 2C76, which has a low sequence identity (∼70%) with any human P450 2Cs. Phylogenetic analysis showed that marmoset P450 2Cs were more closely clustered with those of humans and macaques than other species investigated. Quantitative polymerase chain reaction analysis showed that all of the marmoset P450 2C mRNAs were predominantly expressed in liver as opposed to the other tissues tested. Marmoset P450 2C proteins were detected in liver by immunoblotting using antibodies against human P450 2Cs. Among marmoset P450 2Cs heterologously expressed in Escherichia coli, marmoset P450 2C19 efficiently catalyzed human P450 2C substrates, S-warfarin, diclofenac, tolbutamide, flurbiprofen, and omeprazole. Marmoset P450 2C19 had high V max and low K m values for S-warfarin 7-hydroxylation that were comparable to those in human liver microsomes, indicating warfarin stereoselectivity similar to findings in humans. Faster in vivo S-warfarin clearance than R-warfarin after intravenous administration of racemic warfarin (0.2 mg/kg) to marmosets was consistent with the in vitro kinetic parameters. These results indicated that marmoset P450 2C enzymes had functional characteristics similar to those of humans, and that P450 2C-dependent metabolic properties are likewise similar between marmosets and humans.

Assessment of multiple cytochrome P450 activities in metabolically inactivated human liver microsomes and roles of P450 2C isoforms in reaction phenotyping studies

“…Drug oxidation activities were determined as described previously (Ohyama, Murayama, Shimizu, & Yamazaki, ; Uehara et al, ; Yamazaki et al, , ; Yamazaki, Shaw, Guengerich, & Shimada, ). Briefly, substrates (1.0–200 μ m ) were incubated with liver microsomes (0.50 mg/ml) and an NADPH‐generating system in 50 m m phosphate buffer (pH 7.4) at 37°C for 15 min.…”

Utility of non-human primates in drug development: Comparison of non-human primate and human drug-metabolizing cytochrome P450 enzymes

“…Newly identified marmoset P450 2C19, highly homologous to human P450 2C19, catalyzed S-warfarin 7-hydroxylation [27]. Similarly, in vivo pharmacokinetics of racemic warfarin and its metabolites at a dose of 1.0 mg/kg were studied after oral and intravenous administrations to fasted male marmosets (n = 6, >2 years of age, 0.3 kg of body weight), which had been genotyped for P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)] [28].…”

A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine

“…As a result, 20-hydroxyleukotriene B 4 and leukotriene B 4 were detected with retention times of 8.3 and 20.1 minutes, respectively. 7-Ethoxyresorufin O-deethylation, S-warfarin 7-hydroxylation, bufuralol 19-hydroxylation, and midazolam 19-hydroxylation by recombinant proteins were measured as described previously (Uehara et al, 2015b). Rates of metabolite formation were determined from standard curves prepared using reference standards.…”