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Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation. The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys. 3. In vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4′-hydroxylation, and S-mephenytoin 4′-hydroxylation showed low activities in MIM and MLM. IC₅₀ values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4′-hydroxylation, respectively. These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.
[Show abstract][Hide abstract] ABSTRACT:
Cynomolgus monkeys (Macaca fascicularis) are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage because the monkeys are closer to humans than other experimental animals in terms of their sequence identity. Some studies have revealed the properties of monkey cytochrome P450s (P450s, CYPs) such as the expression profiles and enzymatic functions; however, these are not enough to understand the species difference. Therefore, characterization of the substrate specificity and inhibitor selectivity of cynomolgus P450s was conducted using typical substrates and inhibitors for human P450s to clarify the species difference between monkeys and humans. Kinetic parameters were determined in pooled monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine maker reactions for human P450s. Some kinetic parameters were unable to be determined because of the low activities. Similar to human P450 reactions, testosterone 6β-hydroxylation in MIM and MLM showed sigmoidal rate-substrate concentration profiles known as the CYP3A-specific reaction pattern, where Hill coefficient were 1.40 and 1.54, respectively. Intrinsic clearance (CLint) for coumarin 7-hydroxylation and bufuralol 1’-hydroxylation were higher than those of other reactions in MLM, with the values of 238 and 158 μL/min/mg, respectively. On the other hand, paclitaxel 6α-hydroxylation, diclofenac 4’-hydroxylation, and S-mephenytoin 4’-hydroxylation showed low activities in MIM and MLM with CLint values of less than 0.125 and 5.65 μL/min/mg, respectively. IC50 values of selective inhibitors for human P450s were determined in MIM and MLM. Ketoconazole strongly inhibited intestinal and hepatic testosterone 6β-hydroxylation with IC50 values of less than 0.0124 μM. Tranylcypromine showed inhibition on hepatic chlorzoxazone 6-hydroxylation but did not show on intestinal chlorzoxazone 6-hydroxylation. On the other hand, the inhibitory effect of ketoconazole on intestinal chlorzoxazone 6-hydroxylation was more potent than that on hepatic chlorzoxazone 6-hydroxylation. Furafylline and sulfaphenazole showed little inhibition in monkey microsomes on phenacetin O-deethylation and diclofenac 4’-hydroxylation, respectively. Because furafylline is also known as a mechanism-based inhibitor for human CYP1A2, time-dependent inhibition assay was carried out with MLM. The inhibitory effect of furafylline on hepatic phenacetin O-deethylation was weak even when furafylline was preincubated with MLM in the presence of NADPH/NADH (43.8% inhibition at 100 μM). Our studies provide comprehensive enzymatic profiles of cynomolgus P450s in the intestine and liver. These contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.
19th MDO and 12th European Regional International society for the study of xenobiotics Meeting;