The Cytochrome P450 Enzyme Responsible For The Production of Z-Norendoxifen In Vitro

Background 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 (HLMs) and expressed P450s to study the metabolic disposition of norendoxifen. Methods To validate that norendoxifen was the metabolite of endoxifen, metabolites in HLMs 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 CYP4A11). Formation rates of norendoxifen were evaluated in the absence and presence of P450 isoform specific inhibitors using HLMs. Results The peak of norendoxifen was found in the incubations consisting of endoxifen, HLMs and cofactors. The retention times of norendoxifen, Endoxifen and the internal standard (diphenhydramine) were 7.81, 7.97 and 5.86 min, respectively. The Km (app) and Vmax (app) values of norendoxifen formation from endoxifen in HLM was 47.8 μM and 35.39 pmol/min/mg. The apparent hepatic intrinsic clearances [Clint app] of norendoxifen formation were 0.74 μl/ mg × min. CYP3A5 and CYP2D6 were the major enzymes capable of norendoxifen formation from endoxifen with rates of 0.26 and 0.86 pmol/pmol P450×min. CYP1A2, 3A2, 2C9 and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6-fold lower.1μM ketoconazole (CYP3A inhibitor) showed an inhibitory effect on rates of norendoxifen formation by 45%, but 1μM quinidine (CYP2D6 inhibitor) does not show an inhibitory effect. Conclusion Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP3A5. This article is protected by copyright. All rights reserved.