The Cytochrome P450 Enzyme Responsible for the Production of (Z)‐Norendoxifen in vitro

Norendoxifen, an active metabolite of tamoxifen, is a potent aromatase inhibitor. Little information is available regarding production of norendoxifen in vitro . Here, we conducted a series of kinetic and inhibition studies in human liver microsomes ( HLM s) and expressed P450s to study the metabolic disposition of norendoxifen. To validate that norendoxifen was the metabolite of endoxifen, metabolites in HLM s incubates of endoxifen were measured using a HPLC / MS / MS method. To further probe the specific isoforms involved in the metabolic route, endoxifen was incubated with recombinant P450s ( CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5 and CYP 4A11). Formation rates of norendoxifen were evaluated in the absence and presence of P450 isoform specific inhibitors using HLM s. The peak of norendoxifen was found in the incubations consisting of endoxifen, HLM s, and cofactors. The retention times of norendoxifen, endoxifen, and the internal standard (diphenhydramine) were 7.81, 7.97, and 5.86 min, respectively. The K m (app) and V max (app) values of norendoxifen formation from endoxifen in HLM was 47.8 μ m and 35.39 pmol min −1 mg −1 . The apparent hepatic intrinsic clearances of norendoxifen formation were 0.74 μl mg −1 min. CYP 3A5 and CYP 2D6 were the major enzymes capable of norendoxifen formation from endoxifen with the rates of 0.26 and 0.86 pmol pmol −1 P450 × min. CYP 1A2, 3A2, 2C9, and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6‐fold lower. One micromolar ketoconazole ( CYP 3A inhibitor) showed an inhibitory effect on the rates of norendoxifen formation by 45%, but 1 μ m quinidine ( CYP 2D6 inhibitor) does not show any inhibitory effect. Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP 3A5.