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

• DOI: 10.1124/jpet.111.184721
• Publication Date: 2011-10-05T03:23:09Z
• Authors: Stacie M. Bratton; Carrie M. Mosher; Farid Khallouki; Moshe Finel; Michael H. Court; Jeffery H. Moran; Anna Radominska-P...
• Keywords: 570; Glucuronides - metabolism; Tissue Banks; 03 medical and health sciences; Glucuronides; 0302 clinical medicine; Tandem Mass Spectrometry; Microsomes; 615; Recombinant Proteins - metabolism; Warfarin - isolation & purification; Humans; Glucuronosyltransferase - metabolism; Glucuronosyltransferase; Chromatography, High Pressure Liquid; Ultraviolet; Liver - enzymology; Chromatography; Stereoisomerism; Warfarin - metabolism; Recombinant Proteins; 3. Good health; Kinetics; Liver - metabolism; Spectrophotometry; High Pressure Liquid; Microsomes, Liver; Spectrophotometry, Ultraviolet; Warfarin; Warfarin - analogs & derivatives

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