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Research ArticleMetabolism, Transport, and Pharmacogenomics

Analysis of R- and S-Hydroxywarfarin Glucuronidation Catalyzed by Human Liver Microsomes and Recombinant UDP-Glucuronosyltransferases

Stacie M. Bratton, Carrie M. Mosher, Farid Khallouki, Moshe Finel, Michael H. Court, Jeffery H. Moran and Anna Radominska-Pandya
Journal of Pharmacology and Experimental Therapeutics January 2012, 340 (1) 46-55; DOI: https://doi.org/10.1124/jpet.111.184721
Stacie M. Bratton
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Carrie M. Mosher
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Farid Khallouki
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Moshe Finel
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Michael H. Court
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Jeffery H. Moran
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Anna Radominska-Pandya
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Abstract

Coumadin (R-, S-warfarin) is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide interpatient variability and is typically administered as a racemic (Rac) mixture, which complicates the biotransformation pathways. The goal of the current work was to identify the human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of the separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin and the possible interactions between these enantiomers. The kinetic and inhibition constants for human recombinant 1A family UGTs toward these separated enantiomers have been assessed using high-performance liquid chromatography (HPLC)-UV-visible analysis, and product confirmations have been made using HPLC-mass spectrometry/mass spectrometry. We found that separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin demonstrate significantly different glucuronidation kinetics and can be mutually inhibitory. In some cases significant substrate inhibition was observed, as shown by Km, Vmax, and Ki, comparisons. In particular, UGT1A1 and extrahepatic UGT1A10 have significantly higher capacities than other isoforms for S-7-hydroxywarfarin and R-7-hydroxywarfarin glucuronidation, respectively. Activity data generated using a set of well characterized human liver microsomes supported the recombinant enzyme data, suggesting an important (although not exclusive) role for UGT1A1 in glucuronidation of the main warfarin metabolites, including Rac-6- and 7-hydroxywarfarin and their R- and S-enantiomers in the liver. This is the first demonstration that the R- and S-enantiomers of hydroxywarfarins are glucuronidated, with significantly different enzymatic affinity and capacity, and supports the importance of UGT1A1 as the major hepatic isoform involved.

Footnotes

  • This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grants GM075893, GM061834] (to A.R.-P. and M.H.C., respectively); a Bioterrorism Cooperative Agreement [Grant U90/CCU616974-07] (to J.H.M.); and the Sigrid Juselius Foundation (to M.F.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    http://dx.doi.org/10.1124/jpet.111.184721.

  • ABBREVIATIONS:

    Rac
    racemic
    UDP-GlcUA
    UDP-glucuronic acid
    UGT
    UDP-glucuronosyltransferase
    P450
    cytochrome P450
    HPLC
    high-performance liquid chromatography
    HLM
    human liver microsomes
    MS
    mass spectrometry
    LC-MS/MS
    liquid chromatography-tandem mass spectrometry
    Kis
    substrate inhibition kinetics
    MRM
    multiple reaction monitoring.

  • Received June 3, 2011.
  • Accepted September 27, 2011.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 340 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 340, Issue 1
1 Jan 2012
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Research ArticleMetabolism, Transport, and Pharmacogenomics

R-, S-Hydroxywarfarin Glucuronidation

Stacie M. Bratton, Carrie M. Mosher, Farid Khallouki, Moshe Finel, Michael H. Court, Jeffery H. Moran and Anna Radominska-Pandya
Journal of Pharmacology and Experimental Therapeutics January 1, 2012, 340 (1) 46-55; DOI: https://doi.org/10.1124/jpet.111.184721

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Research ArticleMetabolism, Transport, and Pharmacogenomics

R-, S-Hydroxywarfarin Glucuronidation

Stacie M. Bratton, Carrie M. Mosher, Farid Khallouki, Moshe Finel, Michael H. Court, Jeffery H. Moran and Anna Radominska-Pandya
Journal of Pharmacology and Experimental Therapeutics January 1, 2012, 340 (1) 46-55; DOI: https://doi.org/10.1124/jpet.111.184721
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