Abstract
Our understanding of human phase II metabolic pathways which facilitate detoxification and excretion of warfarin (Coumadin) is limited. The goal of this study was to test the hypothesis that there are specific human hepatic and extrahepatic UDP-glucuronosyltransferase (UGT) isozymes, which are responsible for conjugating warfarin and hydroxylated metabolites of warfarin. Glucuronidation activity of human liver microsomes (HLMs) and eight human recombinant UGTs toward (R)- and (S)-warfarin, racemic warfarin, and major cytochrome P450 metabolites of warfarin (4′-, 6-, 7-, 8-, and 10-hydroxywarfarin) has been assessed. HLMs, UGT1A1, 1A8, 1A9, and 1A10 showed glucuronidation activity toward 4′-, 6-, 7-, and/or 8-hydroxywarfarin with Km values ranging from 59 to 480 μM and Vmax values ranging from 0.03 to 0.78 μM/min/mg protein. Tandem mass spectrometry studies and structure comparisons suggested glucuronidation was occurring at the C4′-, C6-, C7-, and C8-positions. Of the hepatic UGT isozymes tested, UGT1A9 exclusively metabolized 8-hydroxywarfarin, whereas UGT1A1 metabolized 6-, 7-, and 8-hydroxywarfarin. Studies with extrahepatic UGT isoforms showed that UGT1A8 metabolized 7- and 8-hydroxywarfarin and that UGT1A10 glucuronidated 4′-, 6-, 7-, and 8-hydroxywarfarin. UGT1A4, 1A6, 1A7, and 2B7 did not have activity with any substrate, and none of the UGT isozymes evaluated catalyzed reactions with (R)- and (S)-warfarin, racemic warfarin, or 10-hydroxywarfarin. This is the first study identifying and characterizing specific human UGT isozymes, which glucuronidate major cytochrome P450 metabolites of warfarin with similar metabolic rates known to be associated with warfarin metabolism. Continued characterization of these pathways may enhance our ability to reduce life-threatening and costly complications associated with warfarin therapy.
Footnotes
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This study was supported by National Institutes of Health Grants DK60109, DK56262, and GM075893 (to A.R.-P.), by Bioterrorism Cooperative Agreement U90/CCU616974-07 (to J.H.M.), by an Association of Public Health Laboratories-Fellowship (to N.C.M. and J.H.M.), by the Academy of Finland (Project 210933), and by the Sigrid Juselius Foundation (to M.F.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.129858.
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ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; GlcUA, glucuronic acid; HLM, human liver microsome; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MRM, multiple reaction monitoring; P450, cytochrome P450.
- Received August 8, 2007.
- Accepted October 4, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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