The Impact of R10-Hydroxywarfarin on CYP2C9-Mediated S-Warfarin Metabolism

Thesis (Master's)--University of Washington, 2014

Warfarin therapy is highly effective in treating both atrial fibrillation and deep vein thrombosis. Currently, more than 20 million Americans are receiving warfarin therapy. However, increased risk of adverse events are associated with both under and overdosing the drug. Sub–therapeutic plasma levels may be insufficient to prevent the recurrence of a life–threatening clot. Alternatively, if warfarin levels are too high, the risk of dangerous bleeding events is greatly increased. Inter–individual variability in warfarin sensitivity underlies much of the difficulty in managing treatment by influencing dose requirements in a largely unpredictable manner. For example, one clinical study involving 185 subjects reported a 32–fold difference in daily warfarin doses, from 0.5–16 mg/day, needed to achieve a safe, therapeutic degree of anticoagulation. Many major contributors to this variability have been identified, such as age, race, sex, genetics, as well as several clinical factors. It is estimated that roughly 12% of this variability can be attributed to inherited variants of the Cytochrome P450 2C9 (CYP2C9) isoform. This enzyme is critical for the elimination of the more potent S–enantiomer of warfarin. Another 24–30% can be attributable to different variants of the gene <italic>VKORC1 </italic>, which codes for the target enzyme that warfarin inhibits. However, about 40% of the variability in the dose required to achieve a therapeutic level of anticoagulation remains unknown. One additional source of variability in warfarin dose–response could arise from mutual pharmacokinetic interactions between R– and S– warfarin enantiomers. An interaction of this kind would be more significant if it primarily impacted S–warfarin clearance, because this enantiomer is 3–5 fold more potent a vitamin K antagonist than R–warfarin. This has been investigated previously. For example, S–warfarin was found to be a weak inhibitor of R–warfarin metabolism, whereas R–warfarin inhibited the production of S–6– and S–7–hydroxywarfarin in human liver microsomes with K_i ranges of 7.0–8.4 μM and 6.0–6.9 μM respectively. In addition, recently, the racemic 4’–, 6–, 7–, 8–, & 10–hydroxyl metabolites of warfarin have been shown to inhibit CYP2C9 mediated hydroxylation of S–warfarin, both in recombinant enzyme incubations and human liver microsomal incubations. In these experiments, the 10–hydroxywarfarin metabolite was reported to be the most potent inhibitor of CYP2C9–dependent activity towards S–warfarin. Because 10–hydroxywarfarin is a major circulating metabolite of warfarin following multiple dosing, we propose that it may reach high enough plasma concentrations at steady–state to produce a significant impact on the disposition and pharmacological response of S–warfarin. To test this hypothesis, we determined the ability of racemic 10–hydroxywarfarin and its four stereoisomers to inhibit S–warfarin 7–hydroxylation using human liver microsomes. We determined the unbound fraction of the 10–hydroxywarfarin stereoisomers in human plasma in order to establish the relevant unbound concentration that, based on the free–drug hypothesis, determines the potential for inhibition. Pharmacokinetic data were obtained from an ongoing pharmacogenetic warfarin study wherein subjects were randomized to receive warfarin alone or warfarin in the presence of rifampin (one week pretreatment; three weeks total duration). We found that 10–hydroxywarfarin was highly bound to human plasma proteins, with an unbound fraction of 0.98 ± 0.22%. Racemic 10–hydroxywarfarin and its stereoisomers inhibited human liver microsomal S–warfarin 7–hydroxylation with apparent IC₅₀ values ranging from 4.4 to 30 μM. The 9R,10S–hydroxywarfarin metabolite was the most potent inhibitor with an estimated K_i of 3.7 μM. Based on calculated steady–state R–10–hydroxywarfarin stereoisomer concentrations predicted from the single dose clinical study and the estimated K_i values, we concluded that only limited inhibition of CYP2C9–mediated S–warfarin metabolism by R–10–hydroxywarfarin will occur when warfarin is dosed alone or with rifampin (increase in the S–warfarin AUC ratio = 16% and 23%, respectively).