Coumadin (R/S-warfarin) is a commonly prescribed anticoagulant for over ∼20 million Americans. Although highly efficacious, positive clinical outcomes during warfarin therapy depend on maintaining the level of warfarin within a narrow therapeutic window. This task is challenging due to large inter-individual variability in patient responses, which have been attributed to diversity in drug metabolism. Consequently, it is important to understand how metabolic capacity is determined among patients. The overall goal of the research described herein is to provide a foundation for the future application of targeted metabolomics in personalizing warfarin therapy. To achieve this goal, four aspects of warfarin metabolism were studied; 1) basic enzyme kinetics of warfarin metabolism, 2) novel inhibition mechanisms which limit metabolic capacity, 3) interactions between oxidative (Cytochrome P450) and conjugative (UDP-Glucuronosyl transferase) activities, and 4) development of analytical methods for quantifying the warfarin metabolic profile. For part I, studies demonstrated that recombinant CYP3A4, CYP3A5 and CYP3A7 metabolized R- and S-warfarin into 10- and 4'-hydroxywarfarin. For R-warfarin, CYP3A4, CYP3A7, and CYP3A5 demonstrated decreasing preference for 10-hydroxylation over 4'-hydroxylation, while there was no regio-selectivity toward S-warfarin. Overall, CYP3A4 was 30 times more efficient than either CYP3A5 or CYP3A7. For part II, we hypothesized that hydroxywarfarins inhibit CYP2C9 metabolism of warfarin. To test this hypothesis, we investigated the ability of all five racemic hydroxywarfarins to block CYP2C9 activity toward S-warfarin using recombinant enzymes and pooled human liver microsomes. These data demonstrated that 10-hydroxywarfarin and 7-hydroxywarfarin are potent inhibitors of CYP2C9. This novel finding demonstrates that the metabolic pathways for R- and S-warfarin interact with each other, potentially explaining a portion of inter-personal variation in metabolic capacity. These data further suggested that hydroxywarfarins may undergo secondary oxidation to generate dihydroxywarfarins. We hypothesized that these molecules would serve as biomarkers of feedback inhibition, potentially proving a valuable clinical indicator of warfarin sensitivity. Experiments demonstrated that dihydroxylation may be possible, but is very inefficient, potentially limiting the usefulness of these molecules as clinical biomarkers. For part III, we tested the hypothesis that glucuronidation of hydroxywarfarins relieves feedback inhibition, thereby increasing total metabolic capacity toward warfarin. Analysis of the experimental metabolic profiles strongly suggests that the metabolic pathways for R and S interact in a manner which is alleviated by coupling. For part IV, a “dual-phase” UPLC-MS/MS method was developed which is capable of separating and quantifying the R and S enantiomers of warfarin and each hydroxywarfarin. This method was made possible by combining two complementary stationary phases (phenyl and chiral) under compatible chromatographic conditions. Application of this method to human plasma samples demonstrated that patients exhibit a wide degree of variation in both the levels and patterns of hydroxywarfarin metabolites. Further studies in urine samples support these observations, and provide important information about the differences between plasma and urine matrices in the analysis of metabolic profiles. Together, the reported advancement in these four areas of research paves the way for improving clinic outcomes for patients receiving warfarin therapy using a targeted metabolomic strategy.
|Advisor:||Miller, Grover P.|
|Commitee:||Boysen, Gunnar, Chambers, Timothy C., Owens, Michael, Raney, Kevin D.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 72/08, Dissertation Abstracts International|
|Keywords:||Biomarkers, Clinical care, Cytochrome p450, Feedback inhibition, Metabolism, Warfarin|
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