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 (HLMs) and expressed P450s to study the metabolic disposition of norendoxifen. 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. 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-1 mg-1 . The apparent hepatic intrinsic clearances of norendoxifen formation were 0.74 µl mg-1 min. CYP3A5 and CYP2D6 were the major enzymes capable of norendoxifen formation from endoxifen with the rates of 0.26 and 0.86 pmol pmol-1 P450 × min. CYP1A2, 3A2, 2C9, and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6-fold lower. One micromolar ketoconazole (CYP3A inhibitor) showed an inhibitory effect on the rates of norendoxifen formation by 45%, but 1 µm quinidine (CYP2D6 inhibitor) does not show any inhibitory effect. Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP3A5.

Association of CYP2C19*2 and associated haplotypes with lower norendoxifen concentrations in tamoxifen-treated Asian breast cancer patients.

AIM: The aim was to examine the influence of CYP2C19 variants and associated haplotypes on the disposition of tamoxifen and its metabolites, particularly norendoxifen (NorEND), in Asian patients with breast cancer. METHODS: Sixty-six CYP2C19 polymorphisms were identified in healthy Asians (n = 240), of which 14 were found to be tightly linked with CYP2C19*2, CYP2C19*3 and CYP2C19*17. These 17 SNPs were further genotyped in Asian breast cancer patients receiving tamoxifen (n = 201). Steady-state concentrations of tamoxifen and its metabolites were quantified using liquid chromatography­mass spectrometry. Non-parametric tests and regression methods were implemented to evaluate genotypic­phenotypic associations and haplotypic effects of the SNPs. RESULTS: CYP2C19 functional polymorphisms and their linked SNPs were not significantly associated with plasma concentrations of tamoxifen and its main metabolites N-desmethyltamoxifen, (Z)-4-hydroxytamoxifen and (Z)-Endoxifen. However, CYP2C19*2 and its seven linked SNPs were significantly associated with lower NorEND concentrations, MRNorEND/NDDM and MRNorEND/(Z)-END. Specifically, patients carrying the CYP2C19*2 variant allele A had significantly lower NorEND concentrations [median (range), GG vs. GA vs. AA: 1.51 (0.38­3.28) vs. 1.28 (0.30­3.36) vs. 1.15 ng ml−1 (0.26­2.45, P = 0.010)] as well as significantly lower MRNorEND/(Z)-END [GG vs. GA vs. AA: 9.40 (3.27­28.35) vs. 8.15 (2.67­18.9) vs. 6.06 (4.47­14.6), P < 0.0001] and MRNorEND/NDDM [GG vs. GA vs. AA: 2.75 (0.62­6.26) vs. 2.43 (0.96­4.18) vs. 1.75 (1.10­2.49), P < 0.00001]. CYP2C19 H2 haplotype, which included CYP2C19*2, was also significantly associated with lower NorEND concentrations (P = 0.0020), MRNorEND/NDDM (P < 0.0001) and MRNorEND/(Z)-END (P < 0.0001), indicating significantly lower formation rates of NorEND. CONCLUSION: These data highlight the potential relevance of CYP2C19 pharmacogenetics in influencing NorEND concentrations in tamoxifen-treated patients, which may influence treatment outcomes.

A new Suzuki synthesis of triphenylethylenes that inhibit aromatase and bind to estrogen receptors α and ß.

The design and synthesis of dual aromatase inhibitors/selective estrogen receptor modulators (AI/SERMs) is an attractive strategy for the discovery of new breast cancer therapeutic agents. Previous efforts led to the preparation of norendoxifen (4) derivatives with dual aromatase inhibitory activity and estrogen receptor binding activity. In the present study, some of the structural features of the potent AI letrozole were incorporated into the lead compound (norendoxifen) to afford a series of new dual AI/SERM agents based on a symmetrical diphenylmethylene substructure that eliminates the problem of E,Z isomerization encountered with norendoxifen-based AI/SERMs. Compound 12d had good aromatase inhibitory activity (IC50=62.2nM) while also exhibiting good binding activity to both ER-α (EC50=72.1nM) and ER-ß (EC50=70.8nM). In addition, a new synthesis was devised for the preparation of norendoxifen and its analogues through a bis-Suzuki coupling strategy.