BACKGROUND AND PURPOSETamoxifen is a prodrug that is metabolically activated by 4-hydroxylation to the potent primary metabolite 4-hydroxytamoxifen (4OHT) or via another primary metabolite N-desmethyltamoxifen (NDMTAM) to a biologically active secondary metabolite endoxifen through a cytochrome P450 2D6 variant system (CYP2D6). To elucidate the mechanism of action of tamoxifen and the importance of endoxifen for its effect, we determined the anti-oestrogenic efficacy of tamoxifen and its metabolites, including endoxifen, at concentrations corresponding to serum levels measured in breast cancer patients with various CYP2D6 genotypes (simulating tamoxifen treatment). EXPERIMENTAL APPROACHThe biological effects of tamoxifen and its metabolites on cell growth and oestrogen-responsive gene modulation were evaluated in a panel of oestrogen receptor-positive breast cancer cell lines. Actual clinical levels of tamoxifen metabolites in breast cancer patients were used in vitro along with actual levels of oestrogens observed in premenopausal patients taking tamoxifen. KEY RESULTSTamoxifen and its primary metabolites (4OHT and NDMTAM) only partially inhibited the stimulant effects of oestrogen on cells. The addition of endoxifen at concentrations corresponding to different CYP2D6 genotypes was found to enhance the anti-oestrogenic effect of tamoxifen and its metabolites with an efficacy that correlated with the concentration of endoxifen; at concentrations corresponding to the extensive metabolizer genotype it further inhibited the actions of oestrogen. In contrast, lower concentrations of endoxifen (intermediate and poor metabolizers) had little or no anti-oestrogenic effects. CONCLUSIONS AND IMPLICATIONSEndoxifen may be a clinically relevant metabolite in premenopausal patients as it provides additional anti-oestrogenic actions during tamoxifen treatment.

Tamoxifen has biologically active metabolites: 4-hydroxytamoxifen (4OHT) and endoxifen. The E-isomers are not stable in solution as Z-isomerization occurs. We have synthesized fixed ring (FR) analogues of 4OHT and endoxifen as well as FR E and Z isomers with methoxy and ethoxy side chains. Pharmacologic properties were documented in the MCF-7 cell line, and prolactin synthesis was assessed in GH3 rat pituitary tumor cells. The FR Z-isomers of 4OHT and endoxifen were equivalent to 4OHT and endoxifen. Other test compounds used possessed partial estrogenic activity. The E-isomers of FR 4OHT and endoxifen had no estrogenic activity at therapeutic serum concentrations. None of the newly synthesized compounds were able to down-regulate ER levels. Molecular modeling demonstrated that some compounds would each create a best fit with a novel agonist conformation of the ER. The results demonstrate modulation by the ER complex of cell replication or gene transcription in cancer.

The results indicate that tamoxifen and other metabolites, excluding endoxifen, completely inhibit estrogen-stimulated growth in all cell lines, but additional antiestrogenic action from endoxifen is necessary for complete blockade of estrogen-stimulated genes. Endoxifen is of supportive importance for the therapeutic effect of tamoxifen in a postmenopausal setting.

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