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.
Footnotes
This work was supported by the National Key Technology R&D Program in the 11th Five-year Plan of China [2008ZX10002-019]; the National Basic Research Program of China [2009CB522808]; and the National Science and Technology Pillar Program in the 11th Five-year Plan of China [2006BAI11B08, 2008BAI51B02].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.030734.
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ABBREVIATIONS:
- UGT
- UDP-glucuronosyltransferase
- PH-302
- 2-((2-(1H-imidazol-1-yl)-6-methylpyrimidin-4-yl)(3-((benzo-[d][1,3]dioxol-5-ylmethyl)(methyl)amino)propyl)amino)acetamide
- HLM
- human liver microsome(s)
- HIM
- human intestinal microsome(s)
- RLM
- rat liver microsomes
- PLM
- minipig liver microsome(s)
- UDPGA
- UDP-glucuronic acid
- HPLC
- high-performance liquid chromatography
- UFLC
- ultra-fast liquid chromatography
- ESI
- electrospray ionization
- DAD
- diode array detector
- CV
- coefficient of variation.
- Received October 20, 2009.
- Accepted February 16, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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