title: Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of daphnetin.
abstract: Daphnetin has been developed as an oral medicine for treatment of coagulation disorders and rheumatoid arthritis in China, but its in vitro metabolism remains unknown. In the present study, the UDP-glucuronosyltransferase (UGT) conjugation pathways of daphnetin were characterized. Two metabolites, 7-O-monoglucuronide daphnetin (M-1) and 8-O-monoglucuronide daphnetin (M-2), were identified by liquid chromatography/mass spectrometry and NMR when daphnetin was incubated, respectively, with liver microsomes from human (HLM), rat (RLM), and minipig (PLM) and human intestinal microsomes (HIM) in the presence of UDP-glucuronic acid. Screening assays with 12 human recombinant UGTs demonstrated that the formations of M-1 and M-2 were almost exclusively catalyzed by UGT1A9 and UGT1A6, whereas M-1 was formed to a minor extent by UGT1A3, 1A4, 1A7, 1A8, and 1A10 at a high substrate concentration. Kinetics studies, chemical inhibition, and correlation analysis were used to demonstrate that human UGT1A9 and UGT1A6 were major isoforms involved in the daphnetin glucuronidations in HLM and HIM. By in vitro-in vivo extrapolation of the kinetic data measured in HLM, the hepatic clearance and the corresponding hepatic extraction ratio were estimated to be 19.3 ml/min/kg b.wt. and 0.93, respectively, suggesting that human clearance of daphnetin via the glucuronidation is extensive. Chemical inhibition of daphnetin glucuronidation in HLM, RLM, and PLM showed large species differences although the metabolites were formed similarly among the species. In conclusion, the UGT conjugation pathways of daphnetin were fully elucidated and its C-8 phenol group was more selectively catalyzed by UGTs than by the C-7 phenol.
authors.affiliation: Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.
authors.forename: Si-Cheng
authors.initials: SC
authors.lastname: Liang
authors.forename: Guang-Bo
authors.initials: GB
authors.lastname: Ge
authors.forename: Hui-Xin
authors.initials: HX
authors.lastname: Liu
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Zhang
authors.forename: Li-Ming
authors.initials: LM
authors.lastname: Wang
authors.forename: Jiang-Wei
authors.initials: JW
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Yin
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Zhong-Ze
authors.initials: ZZ
authors.lastname: Fang
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Wu
authors.forename: Guo-Hui
authors.initials: GH
authors.lastname: Li
authors.forename: Ling
authors.initials: L
authors.lastname: Yang
mesh_terms: D001741:Black or African American; D002681:China; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D013379:Substrate Specificity; D014468:Umbelliferones / Q000378:metabolism / Q000493:pharmacokinetics*; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism; D044465:White People
chemical_list: D014468:Umbelliferones; D014535:Uridine Diphosphate Glucuronic Acid; D014453:Glucuronosyltransferase; C039952:daphnetin