| Summary: | Objective: To explore the glucuronic acid metabolism of acacetin in human liver and intestinal microsomes to better characterize human uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT) isoforms. In addition, interspecies comparisons were performed to identify the most appropriate experimental animal model for an in vivo study. Methods: Liquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) were used to confirm the successful biosynthesis of acacetin-7-O-glucuronide. Human isoforms of UGT and isozyme-specific chemical inhibitors were used for recombinant assays. Acacetin glucuronidation kinetics were assessed by combining acacetin with recombinant human UGT isoforms or with microsomes from humans or experimental animals. Kinetic differences between species were assessed in vitro using the same approach. Results: We identified multiple UGT isoforms that facilitated acacetin glucuronidation, and found that UGT1A1 was the major isoform that catalyzed this process. Acacetin-7-O-glucuronide formation exhibited clear substrate inhibition kinetics when combined with recombinant UGTs or with liver/intestinal microsomes derived from humans, monkeys, rats, mice, dogs, or pigs. Intrinsic metabolic clearance values of human intestinal microsomes were two-fold greater than those of human liver microsomes. Among the evaluated species, the Km value of dog microsomes (0.86 μM) was greatest in acacetin glucuronidation, while mice exhibited the highest CLint value, 5.05 mL/min/mg. The CLint values of microsomes derived from monkeys and minipigs were 1.99 mL/min/mg and 2.12 mL/min/mg, respectively, exhibiting similar intrinsic metabolic clearance activity to that observed in humans. Conclusion: Monkey may represent a suitable model for experimental studies of acacetin pharmacokinetics owing to a high sequence homology of UGT1A1 and similar UGT1A1 glucuronidation activity to humans. Keywords: Acacetin, UDP-glucuronosyltransferases, Human liver microsomes, Glucuronidation, Species differences
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