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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Glucuronidation of Monohydroxylated Warfarin Metabolites by Human Liver Microsomes and Human Recombinant UDP-Glucuronosyltransferases

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (A.Z., S.B., A.G.-Z., G.P.M., A.R.-P.); Arkansas Public Health Laboratory, Arkansas Department of Health, Little Rock, Arkansas (C.F.L., N.C.M., V.-H.L., J.H.M.); and Drug Discovery and Development Technology Center, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (M.F.)

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 K_m values ranging from 59 to 480 µM and V_max 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.

Address correspondence to: Jeffery H. Moran, Arkansas Public Health Laboratory, Arkansas Department of Health, 201 South Monroe Street, Little Rock, AR 72205. E-mail: Jeffery.Moran{at}arkansas.gov