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  • Multiple UDP-glucuronosyltransferases in human liver microsomes glucuronidate both R- and S-7-hydroxywarfarin into two metabolites.

Multiple UDP-glucuronosyltransferases in human liver microsomes glucuronidate both R- and S-7-hydroxywarfarin into two metabolites.

Archives of biochemistry and biophysics (2014-12-03)
C Preston Pugh, Dakota L Pouncey, Jessica H Hartman, Robert Nshimiyimana, Linda P Desrochers, Thomas E Goodwin, Gunnar Boysen, Grover P Miller
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
4,7-Dihydroxycoumarin, 97%
Mefenamic acid, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Acetic acid-12C2, 99.9 atom % 12C
Sigma-Aldrich
Magnesium chloride solution, 0.1 M
USP
Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard
Supelco
Ethanol solution, certified reference material, 2000 μg/mL in methanol
Supelco
Acetic acid, analytical standard
Supelco
Warfarin, PESTANAL®, analytical standard
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~0.025 M in H2O
Sigma-Aldrich
Benzylideneacetone, ≥98%, FG
Sigma-Aldrich
(1S,2S)-(−)-1,2-Diphenylethylenediamine, 97%
Sigma-Aldrich
Acetic acid, ≥99.5%, FCC, FG
Sigma-Aldrich
7-Ethoxycoumarin
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Supelco
Tetrahydrofuran, Selectophore, ≥99.5%
Supelco
Tetrahydrofuran, analytical standard
Sigma-Aldrich
Magnesium chloride solution, PCR Reagent, 25 mM MgCI2 solution for PCR
Sigma-Aldrich
Mefenamic acid
Supelco
Warfarin, analytical standard
Supelco
Mefenamic acid, analytical standard
Supelco
Tetrahydrofuran, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, 2 M in H2O
Sigma-Aldrich
Acetic acid, natural, ≥99.5%, FG
Sigma-Aldrich
Acetic acid solution, suitable for HPLC
Sigma-Aldrich
Methanol, BioReagent, ≥99.93%
Sigma-Aldrich
Methanol, NMR reference standard
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Acetic acid, for luminescence, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Acetic acid, glacial, ACS reagent, ≥99.7%
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%