Coumadin (R-, S-Warfarin) is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide inter-patient 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 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 towards these separated enantiomers have been assessed using high performance liquid chromatography (HPLC-UV/Vis) analysis and product confirmations have been made using HPLC-MS/MS. 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. Specifically, 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.
- Received June 3, 2011.
- Revision received September 26, 2011.
- Accepted September 27, 2011.
- The American Society for Pharmacology and Experimental Therapeutics