Evaluation of 89 Compounds for Identification of Substrates for Cynomolgus Monkey CYP2C76, a New Bupropion/Nifedipine Oxidase

Abstract

Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical ( ‡90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.

Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors

“…Loratadine, cleared to 78.9% at a substrate concentration of 1.0 μ m , yielded an almost apparent borderline level of 0.28 nmol/min per nmol CYP2C8. It was already confirmed that amodiaquine and montelukast were hardly depleted by other monkey CYP2C enzymes, CYP2C9, CYP2C19 and CYP2C76 (˂ 0.27 nmol/min per nmol P450) under similar experimental conditions. Therefore, these drugs could be selective marker substrates of monkey CYP2C8.…”

“…In conclusion, the four monkey CYP2C8 substrates identified under the present conditions were all substrates and/or competitive inhibitors of human CYP2C8, indicating that monkey CYP2C8 most likely has similar substrate specificity to human CYP2C8 with slightly different characteristics. Among the newly identified substrates, amodiaquine and montelukast showed high selectivity to CYP2C8 among monkey CYP2C enzymes tested , and could possibly be selective marker substrate candidates of monkey CYP2C8. Our findings on substrate specificity of monkey CYP2C8, in combination with those of monkey CYP2C9, CYP2C19 and CYP2C76, should help to understand more about species differences in drug metabolism between monkeys and humans and to extrapolate the preclinical study data obtained using monkeys to humans.…”

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

“…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]. These findings collectively suggest that cynomolgus monkey P450 2C enzymes have similar substrate recognition functionality as human P450 2C forms, but there would be possibly limited specific differences in drug oxidative metabolism between the two species.…”

“…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

“…These findings might account for some limited species differences in drug-metabolizing enzymes between marmosets and cynomolgus monkeys or humans. Although monkey-specific P450 2C76 had a high impact on drug oxidations (Hosaka et al, 2015a), marmoset P450 2C76 showed few unique characteristics of human P450 2C substrates under the present conditions. These findings suggested that the substrate selectivity of marmoset P450 2C19 may reflect the functional similarity of the P450 2C enzyme between marmosets and humans.…”