Individual Differences in Metabolic Clearance of S-Warfarin Efficiently Mediated by Polymorphic Marmoset Cytochrome P450 2C19 in Livers

Abstract

Marmoset cytochrome P450 2C19, highly homologous to human P450 2C9 and 2C19, has been identified in common marmosets (Callithrix jacchus), a nonhuman primate species used in drug metabolism studies. Although genetic variants in human and macaque P450 2C genes account for the interindividual variability in drug metabolism, genetic variants have not been investigated in the marmoset P450 2C19. In this study, sequencing of P450 2C19 in 24 marmosets identified three variants p.[(Phe7Leu; Ser254Leu; Ile469Thr)], which showed substantially reduced metabolic capacity of S-warfarin compared with the wild-type group in vivo and in vitro. Although mean plasma concentrations of R-warfarin in marmosets determined after chiral separation were similar between the homozygous mutant and wild-type groups up to 24 hours after the intravenous and oral administrations of racemic warfarin, S-warfarin depletion from plasma was significantly faster in the three wild-type marmosets compared with the three homozygous mutant marmosets. These variants, cosegregating in the marmosets analyzed, influenced metabolic activities in 18 marmoset liver microsomes because the homozygotes and heterozygotes showed significantly reduced catalytic activities in liver microsomes toward S-warfarin 7-hydroxylation compared with the wild-type group. Kinetic analysis for S-warfarin 7-hydroxylation indicated that the recombinant P450 2C19 Ser254Leu variant would change the metabolic capacity. These results indicated that the interindividual variability of P450 2C-dependent drug metabolism such as S-warfarin clearance is at least partly accounted for by P450 2C19 variants in marmosets, suggesting that polymorphic P450 2C-dependent catalytic functions are relatively similar between marmosets and humans.

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

“…Although mean plasma concentrations of R-warfarin in marmosets determined after chiral separation were similar between the homozygous mutant and wild-type groups up to 24 h after the intravenous and oral administrations of racemic warfarin, S-warfarin clearances from plasma were significantly faster in the three wild-type marmosets compared to the three homozygous mutant marmosets. These marmoset P450 2C19 polymorphisms influenced metabolic activities of S-warfarin 7-hydroxylation in 18 marmoset livers in vitro because the homozygotes and heterozygotes showed significantly reduced catalytic activities in liver microsomes toward S-warfarin 7-hydroxylation compared to the wild-type group [28]. Because inter-individual variability of P450 2C-dependent drug clearances in cynomolgus monkeys and marmosets is partly accounted for by polymorphic P450 2C19 variants, similar to humans, genotyping of drug-metabolizing enzyme genes would be beneficial before and after drug metabolism testing and evaluations in cynomolgus monkeys and marmosets.…”

“…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]. Although mean plasma concentrations of R-warfarin in marmosets determined after chiral separation were similar between the homozygous mutant and wild-type groups up to 24 h after the intravenous and oral administrations of racemic warfarin, S-warfarin clearances from plasma were significantly faster in the three wild-type marmosets compared to the three homozygous mutant marmosets.…”

Molecular and Functional Characterization of N-Acetyltransferases in Common Marmosets and Pigs

“…In humans, genetic variants are responsible for inter-individual variability of NAT activities toward such substrates as isoniazid, hydralazine, and sulfamethazine, which are acetylated by NAT2 (Sim et al, 2014). Similarly, for marmosets and pigs, genetic variants have been found in drug-metabolizing enzyme genes such as cytochromes P450 (Puccinelli et al, 2011;Uehara et al, 2016a), although information on such genetic variants is currently limited. Investigation of genetic variants might help us to understand the potential inter-animal variability of NAT activities in marmosets and pigs.…”

Different Roles of Human Cytochrome P450 2C9 and 3A Enzymes in Diclofenac 4′- and 5-Hydroxylations Mediated by Metabolically Inactivated Human Hepatocytes in Previously Transplanted Chimeric Mice

“…S -Warfarin and 7-hydoxywarfarin were determined by a liquid chromatography (LC) system consisting of a fluorescence detector and pump (Shimadzu, Kyoto, Japan) with an analytical octadecylsilane (C 18 ) reversed-phase column (5 μm, 4.6 × 250 mm, Mightysil RP-18 GP 2, Kanto Chemicals, Tokyo, Japan) according to previously reported methods with minor modifications. − Diclofenac, 4′-hydroxydiclofenac, 5-hydroxydiclofenac, and diclofenac acyl-β- d -glucuronide were determined according to previously reported methods with modifications using LC–tandem mass spectrometry. An API 4000 tandem mass spectrometer (AB Sciex, Framingham, MA) was operated in electrospray positive ionization mode and was directly coupled to a 20A System liquid chromatograph (Shimadzu, Kyoto, Japan) equipped with a C 18 column (4.6 × 250 mm, Kanto Chemicals).…”