Dmd063552 977..983

Esculetin (6,7-dihydroxycoumarin, ET) and 4-methylesculetin (6,7dihydroxy-4-methylcoumarin, 4-ME) are typical coumarin derivatives that are attracting considerable attention because of their wide spectrum of biologic activities, but their metabolism remains unknown. This study aimed to elucidate the in vitro UDPglucuronosyltransferase (UGT) metabolism characteristics of ET and 4-ME. 7-O-monoglucuronide esculetin (ET-G) and 7-Omonoglucuronide 4-methylesculetin (4-ME-G) were identified by liquid chromatography-mass spectrometry (LC-MS) and H-nuclear magnetic resonance (HNMR) when ET or 4-ME was incubated with human liver (HLM) in the presence of UDPglucuronic acid. Screening assays with 12 human expressed UGTs demonstrated that the formations of ET-G and 4-ME-G were almost exclusively catalyzed by UGT1A6 and UGT1A9. Phenylbutazone and carvacrol (UGT1A6 and UGT1A9 chemical inhibitors, respectively) at different concentrations (50, 100, and 200 mM) significantly inhibited the formation of glucuronidates of ET and 4-ME in HLM, UGT1A6, and UGT1A9 when the concentrations of ET and 4-ME ranged from 10 to 300 mM (P < 0.05). Clearance rates of ET in HLM, HIM, UGT1A6, and UGT1A9 were 0.54, 0.16, 0.69, and 0.14 ml/min/mg, respectively. Corresponding clearance rates values of 4-ME were 0.59, 0.03, 0.14, and 0.04 ml/min/mg, respectively. In conclusion, 7-O-monoglucuronidation by UGT1A6 and UGT1A9 was the predominant UGT metabolic pathway for both ET and 4-ME in vitro. The liver is probably the major contributor to the glucuronidation metabolism of ET and 4-ME. ET showed more rapid metabolism than 4-ME in