Comprehensive Evaluation for Substrate Selectivity of Cynomolgus Monkey Cytochrome P450 2C9, a New Efavirenz Oxidase

Abstract

Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans.

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

“…Interspecies differences regarding caffeine oxidation exist, but the predominant C-8-hydroxylation of caffeine in rodents and rabbits have been shown to be broadly similar [31]. Although efavirenz is known as a probe substrate for human P450 2B6, efavirenz was oxidized in a limited manner by cynomolgus monkey P450 2B6 but efficiently by cynomolgus monkey P450 2C9 [13] (Table 3). Liquid chromatography-mass spectrometry analysis revealed that both cynomolgus monkey P450 2C9 and human P450 2B6 formed the same 8-hydroxy-and 8,14-dihydroxy-efavirenz [13].…”

“…Although efavirenz is known as a probe substrate for human P450 2B6, efavirenz was oxidized in a limited manner by cynomolgus monkey P450 2B6 but efficiently by cynomolgus monkey P450 2C9 [13] (Table 3). Liquid chromatography-mass spectrometry analysis revealed that both cynomolgus monkey P450 2C9 and human P450 2B6 formed the same 8-hydroxy-and 8,14-dihydroxy-efavirenz [13]. An anti-histaminic drug ebastine has been recognized as a human P450 2J2 probe, but also partly Table 3 Typical substrates for marmoset, cynomolgus monkey, and human P450 enzymes.…”

“…Cynomolgus monkey P450 2C19 metabolized 34 of the 89 compounds, including representative human P450 2C19 substrates such as diazepam and omeprazole and human P450 2C9 substrates such as diclofenac, warfarin, and flurbiprofen [15]. Cynomolgus monkey P450 2C9 metabolized 20 of the 89 compounds, and 17 of those 20 drugs have been reported as substrates or inhibitors of human P450 2C9 and P450 2C19, but also metabolized efavirenz which is a marker substrate for P450 2B6 in humans [13]. Cynomolgus monkey P450 2C76 metabolized 19 of the 89 compounds with a wide variety of diverse structures from small to large molecules and formed a unique nifedipine metabolite which was not reported in humans [14].…”

“…On the other hand, cynomolgus monkey P450 2C76, which has no ortholog in humans, shows lower amino acid sequence identity to any human P450 2C forms [12]. Because a broad evaluation of potential substrates for cynomolgus monkey P450 2C enzymes was not conducted, to obtain the comprehensive information of substrate selectivity for major cynomolgus monkey P450 2C enzymes, 89 commercially available drugs were investigated [13][14][15][16]. Cynomolgus monkey P450 2C19 metabolized 34 of the 89 compounds, including representative human P450 2C19 substrates such as diazepam and omeprazole and human P450 2C9 substrates such as diclofenac, warfarin, and flurbiprofen [15].…”

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

“…Marmoset P450 2C8, 2C18, 2C19, 2C58, and 2C76 also catalyzes 7-pentoxyresorufin O-deethylation. Similarly, cynomolgus monkey P450 2C9 and 2C19 metabolize many human P450 2C substrates, but also show some limited differences in drug oxidations of human P450 2C substrates (Hosaka et al, 2015b). Interestingly, marmoset P450 2C58 had the highest catalytic activities among five marmoset P450s tested toward non-P450 2C substrates, such as ethoxyresorufin, 7-ethoxycoumarin, coumarin, 7-pentoxyresorufin, and chlorzoxazone, whereas cynomolgus monkey P450 2C58 and human P450 2C58 were both pseudogenes.…”