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; Anna Radominska-Pandya

doi:10.1124/jpet.111.184721

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Analysis of R- and S-hydroxywarfarin glucuronidation catalyzed by human liver microsomes and recombinant UDP-glucuronosyltransferases

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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

The Journal of pharmacology and experimental therapeutics, Vol.340(1), pp.46-55

01/2012

DOI: https://doi.org/10.1124/jpet.111.184721

Handle:

https://hdl.handle.net/2376/106045

PMCID: PMC3251030

PMID: 21972237

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https://doi.org/10.1124/jpet.111.184721View

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Abstract

Recombinant Proteins - metabolism

Tissue Banks

Stereoisomerism

Humans

Microsomes, Liver - metabolism

Warfarin - metabolism

Chromatography, High Pressure Liquid

Spectrophotometry, Ultraviolet

Warfarin - analogs & derivatives

Warfarin - isolation & purification

Tandem Mass Spectrometry

Glucuronides - metabolism

Glucuronosyltransferase - metabolism

Microsomes, Liver - enzymology

Kinetics

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 K(m), V(max), and K(i), 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.

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Title: Analysis of R- and S-hydroxywarfarin glucuronidation catalyzed by human liver microsomes and recombinant UDP-glucuronosyltransferases
Creators: Stacie M Bratton - Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
Carrie M Mosher
Farid Khallouki
Moshe Finel
Michael H Court
Jeffery H Moran
Anna Radominska-Pandya
Publication Details: The Journal of pharmacology and experimental therapeutics, Vol.340(1), pp.46-55
Academic Unit: Veterinary Clinical Sciences, Department of
Publisher: United States
Grant note: R01 GM075893 / NIGMS NIH HHS U90/CCU616974-07 / PHS HHS GM075893 / NIGMS NIH HHS GM061834 / NIGMS NIH HHS R01 GM061834 / NIGMS NIH HHS
Identifiers: 99900546720201842
Language: English
Resource Type: Journal article