Chemical reactivity and metabolism of norethindrone-4β,5β-epoxide by rat liver microsomes in vitro

Abstract

A method has been developed to separate norethindrone and norethindrone-4β,5β-epoxide by high performance liquid chromatography using isocratic solvent systems with either ODS-reverse phase or conventional silica gel columns. Using these techniques it was found that norethindrone epoxide, prepared chemically, was stable in aqueous buffer (pH 7.4) at 37°C for at least 1 h. Under similar conditions, in the presence of a 10-fold molar excess of cysteine or glutathione, the half life for norethindrone epoxide was 15 and 32 min respectively. In 0.01 M perchloric acid at 37°C the t 1 2 of norethindrone epoxide was 17 min. Norethindrone epoxide was rapidly degraded by rat liver microsomal epoxide hydratase to give a metabolite having properties consistant with it being norethindrone-4,5-dihydrodiol. Epoxide hydratase activities were stimulated about three fold by pretreating rats with phenobarbitone. The pH optimum for this reaction was pH 7.4. Conversion of norethindrone epoxide to norethindrone-dihydrodiol was inhibited by the epoxide hydratase inhibitor 1,2-epoxytrichloropropane. Although norethindrone was extensively metabolised in the presence of NADPH and rat liver microsomes, no conversion to norethindrone-4β,5β-epoxide could be demonstrated, either in the presence or absence of epoxytrichloropropane in the reaction mixture. If norethindrone epoxide was produced under these conditions it was suggested that it either reacted with microsomal proteins at or close to the site or production or was further metabolised. Norethindrone-4β,5β-epoxide did not cause any loss of cytochrome P-450 when incubated with rat liver microsomes in the absence of NADPH. Only in the presence of NADPH did further metabolism of norethindrone epoxide occur leading to the formation of active metabolites capable of breaking down cytochrome P-450. The initial rate of loss of cytochrome P-450 under these conditions was greater with norethindrone than with norethindrone epoxide as the substrate.