Substrate Selectivities and Catalytic Activities of Marmoset Liver Cytochrome P450 2A6 Differed from Those of Human P450 2A6

Abstract

The common marmoset (Callithrix jacchus), a New World primate species, is potentially a useful animal model for preclinical studies in drug development. However, cytochrome P450 (P450) enzymes have not been fully identified and characterized in marmosets. In this study, we identified P450 2A6 cDNA with the sequence highly identical (91-94%) to human P450 2A6, 2A7, and 2A13 cDNA and cynomolgus monkey P450 2A23, 2A24, and 2A26 cDNA. Among the tissue types examined, marmoset P450 2A6 mRNA was most abundantly expressed in livers where P450 2A6 protein was also detected by immunoblotting. Phylogenetic analysis showed that marmoset P450 2A6 was more closely clustered with human and cynomolgus monkey P450 2As than P450 2As of dog, rat, and mouse (the species also used in drug metabolism). Marmoset P450 2A6 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation, similar to human P450 2A6 and 2A13 and cynomolgus monkey P450 2A23, 2A24, and 2A26, but much less effectively coumarin 7-hydroxylation, showing some difference as well. Interestingly, marmoset P450 2A6 and cynomolgus monkey P450 2A23 catalyzed phenacetin O-deethylation, which is catalyzed by human P450 1A2 and 2A13, but not by P450 2A6. Marmoset P450 2A6 also exhibited catalytic activity toward testosterone by the multiple sites, but not rat P450 2A-specific testosterone 7a-hydroxylation activity. These results indicated that marmoset P450 2A6 had functional characteristics different from those of human and cynomolgus monkey P450 2As in terms of partially different substrate specificities and catalytic activities, indicating its importance of further studies for P450 2A-dependent drug metabolism in marmosets.

Molecular Cloning, Tissue Distribution, and Functional Characterization of Marmoset Cytochrome P450 1A1, 1A2, and 1B1

“…This study was approved by the Institutional Animal Care and Use Committee. Pooled marmoset tissue microsomes were prepared as described previously (Uehara et al, 2015d). Recombinant rat P450 1B1 was purchased from Nosan (Yokohama, Japan).…”

“…Recombinant marmoset P450 1A1, 1A2, and 1B1, and cynomolgus monkey P450 1B1 were prepared in E. coli using pCW vector (coexpressed with NADPH-P450 reductase) as described previously (Uehara et al, 2015d). The cDNAs were modified N terminus by PCR amplification using primers containing the restriction sites of the NdeI and XbaI sites (underlined).…”

Hepatic expression of cytochrome P450 enzymes in non‐human primate species

“…In contrast, CYP2A bands were detectable (Figure 1), but coumarin 7-hydroxylation was negligible (Table 1), likely due to lack of activity for this reaction by marmoset CYP2A. 18 In gibbons, two separate CYP2B bands were clearly observed (Figure 1), possibly indicating the two different CYP2B isoforms expressed in liver. Chimpanzees showed two separate CYP2C9 bands, and the size of the upper band was similar to that of the band detected in humans and one of the gorillas (Figure 1).…”

“…Low or undetectable CYP2A expression coincided with low or unappreciable coumarin 7‐hydroxylation, a typical marker reaction of human CYP2A6, in gorillas and orangutans (Table ). In contrast, CYP2A bands were detectable (Figure ), but coumarin 7‐hydroxylation was negligible (Table ), likely due to lack of activity for this reaction by marmoset CYP2A . In gibbons, two separate CYP2B bands were clearly observed (Figure ), possibly indicating the two different CYP2B isoforms expressed in liver.…”

Individual differences in in vitro and in vivo metabolic clearances of the antipsychotic drug olanzapine from non‐smoking and smoking Japanese subjects genotyped for cytochrome P4502D6 and flavincontaining monooxygenase 3

“…Activities for the N ‐deethylation of phenacetin, the O ‐demethylation and N ‐demethylation of dextromethorphan, and the N ‐oxygenation of benzydamine were assayed according to previously described methods (Taniguchi‐Takizawa et al , ; Uehara et al , ; Uno et al , ). Briefly, standard incubation mixtures consisted of 100 mM potassium phosphate buffer (pH 7.4–8.4), the NADPH‐generating system, a substrate (50 μM phenacetin, 400 μM dextromethorphan, or 50 μM benzydamine), and liver microsomes (0.125–0.300 mg protein/mL) in a final volume of 0.20–0.25 mL.…”