Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of Daphnetin

Si Cheng Liang, Guang Bo Ge, Hui Xin Liu, Yan Yan Zhang, Li Ming Wang, Jiang Wei Zhang, Lu Yin, Wei Li, Zhong Ze Fang, Jing Jing Wu, Guo Hui Li, Ling Yang

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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.

Original languageEnglish (US)
Pages (from-to)973-980
Number of pages8
JournalDrug Metabolism and Disposition
Volume38
Issue number6
DOIs
StatePublished - Jun 1 2010

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Glucuronosyltransferase
Forensic Anthropology
Microsomes
Phenol
Uridine Diphosphate Glucuronic Acid
Miniature Swine
daphnetin
In Vitro Techniques
Oral Medicine
Liver
Liver Microsomes
Liquid Chromatography
China
Mass Spectrometry
Rheumatoid Arthritis
Protein Isoforms

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmaceutical Science

Cite this

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of Daphnetin. / Liang, Si Cheng; Ge, Guang Bo; Liu, Hui Xin; Zhang, Yan Yan; Wang, Li Ming; Zhang, Jiang Wei; Yin, Lu; Li, Wei; Fang, Zhong Ze; Wu, Jing Jing; Li, Guo Hui; Yang, Ling.

In: Drug Metabolism and Disposition, Vol. 38, No. 6, 01.06.2010, p. 973-980.

Research output: Contribution to journalArticle

Liang, SC, Ge, GB, Liu, HX, Zhang, YY, Wang, LM, Zhang, JW, Yin, L, Li, W, Fang, ZZ, Wu, JJ, Li, GH & Yang, L 2010, 'Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of Daphnetin', Drug Metabolism and Disposition, vol. 38, no. 6, pp. 973-980. https://doi.org/10.1124/dmd.109.030734
Liang, Si Cheng ; Ge, Guang Bo ; Liu, Hui Xin ; Zhang, Yan Yan ; Wang, Li Ming ; Zhang, Jiang Wei ; Yin, Lu ; Li, Wei ; Fang, Zhong Ze ; Wu, Jing Jing ; Li, Guo Hui ; Yang, Ling. / Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of Daphnetin. In: Drug Metabolism and Disposition. 2010 ; Vol. 38, No. 6. pp. 973-980.
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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.",
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AU - Ge, Guang Bo

AU - Liu, Hui Xin

AU - Zhang, Yan Yan

AU - Wang, Li Ming

AU - Zhang, Jiang Wei

AU - Yin, Lu

AU - Li, Wei

AU - Fang, Zhong Ze

AU - Wu, Jing Jing

AU - Li, Guo Hui

AU - Yang, Ling

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