Cytochrome P450 3A4 regiospecific oxygenation of raloxifene: Evidence for the formation of the di‐quinone methide

Abstract

While the bioactivation of xenobiotics to toxic intermediates through cytochrome P450 (CYP)‐catalyzed oxygenation reactions is a well recognized process, the production of reactive electrophilic intermediates by P450s through dehydrogenation reactions is not well characterized. Raloxifene is a second generation selective estrogen receptor modulator used for the treatment and prevention of osteoporosis in postmenopausal women. Previous studies have shown CYP3A4‐mediated metabolism of raloxifene produces two hydroxylated metabolites and several glutathione (GSH) adducts. The hydroxylated metabolites and one of the GSH adducts could theoretically be derived from the same dehydrogenated intermediate. It is postulated that raloxifene is dehydrogenated to a reactive di‐quinone methide. To evaluate the formation of the di‐quinone methide, raloxifene was incubated with CYP3A4 in the presence of H218O or 18O2. LC/MS analysis showed that 3′‐hydroxyraloxifene incorporated oxygen only from 18O2, while the source of the oxygen for the 7‐hydroxyraloxifene was inconclusive. These results demonstrate CYP3A4 selectively oxygenates raloxifene from 18O2 at the 3′‐position, but not at the 7‐position. Furthermore, the lack of CYP3A4‐mediated oxygenation at the 7‐position suggests that the di‐quinone methide is hydrated by active‐site H2O, but labeled H218O could not serve as the source of hydration. Supported by NIH grant # GM074249 form the National Institute of General Medical Sciences.