title: Multiple UDP-glucuronosyltransferases in human liver microsomes glucuronidate both R- and S-7-hydroxywarfarin into two metabolites.
abstract: The widely used anticoagulant Coumadin (R/S-warfarin) undergoes oxidation by cytochromes P450 into hydroxywarfarins that subsequently become conjugated for excretion in urine. Hydroxywarfarins may modulate warfarin metabolism transcriptionally or through direct inhibition of cytochromes P450 and thus, UGT action toward hydroxywarfarin elimination may impact levels of the parent drugs and patient responses. Nevertheless, relatively little is known about conjugation by UDP-glucuronosyltransferases in warfarin metabolism. Herein, we identified probable conjugation sites, kinetic mechanisms and hepatic UGT isoforms involved in microsomal glucuronidation of R- and S-7-hydroxywarfarin. Both compounds underwent glucuronidation at C4 and C7 hydroxyl groups based on elution properties and spectral characteristics. Their formation demonstrated regio- and enantioselectivity by UGTs and resulted in either Michaelis-Menten or substrate inhibition kinetics. Glucuronidation at the C7 hydroxyl group occurred more readily than at the C4 group, and the reaction was overall more efficient for R-7-hydroxywarfarin due to higher affinity and rates of turnover. The use of these mechanisms and parameters to model in vivo clearance demonstrated that contributions of substrate inhibition would lead to underestimation of metabolic clearance than that predicted by Michaelis-Menten kinetics. Lastly, these processes were driven by multiple UGTs indicating redundancy in glucuronidation pathways and ultimately metabolic clearance of R- and S-7-hydroxywarfarin.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: C Preston
authors.initials: CP
authors.lastname: Pugh
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA; Department of Chemistry, Hendrix College, Conway, AR, USA.
authors.forename: Dakota L
authors.initials: DL
authors.lastname: Pouncey
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Hartman
authors.affiliation: Department of Chemistry, Hendrix College, Conway, AR, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Nshimiyimana
authors.affiliation: Department of Chemistry, Hendrix College, Conway, AR, USA.
authors.forename: Linda P
authors.initials: LP
authors.lastname: Desrochers
authors.affiliation: Department of Chemistry, Hendrix College, Conway, AR, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Goodwin
authors.affiliation: Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Boysen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA. Electronic address: MillerGroverP@uams.edu.
authors.forename: Grover P
authors.initials: GP
authors.lastname: Miller
mesh_terms: D000925:Anticoagulants / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D014859:Warfarin / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: D000925:Anticoagulants; D014859:Warfarin; D014453:Glucuronosyltransferase
keywords: 7-Hydroxywarfarin; Glucuronidation; In vitro; Metabolism; UGT (UDP-glucuronosyltransferase); Warfarin