Estrogen receptor binding and bioactivity of individual isomers of active tamoxifen metabolites and their glucuronides

A147

Tamoxifen (TAM), (1-[4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1-(Z)-ene), is a non-steroidal anti-estrogen that has been commonly used for the prevention and treatment of estrogen receptor-positive breast cancer. The active metabolites of TAM including 4-hydroxy-TAM and endoxifen are subjected to conjugation with glucuronic acid and excretion via the biliary system. Although the loss of anti-estrogenic activity of glucuronides of both 4-OH-TAM and endoxifen has been reported, studies examining the mechanism of this loss of activity have not been previously performed. In the present study, the estrogen receptor (ER) binding affinity for glucuronides of active TAM metabolites were determined by in vitro competitive binding assay and their anti-estrogenic properties were further evaluated by 17β-estradiol (E₂)-induced MCF-7 cell proliferation as compared to their unconjugated counterparts. Both trans-4-OH-TAM and trans-endoxifen exhibited significantly higher relative binding affinity (RBA) for ER-alpha (RBA=195 and 158, respectively) than their respective cis isomers (RBA = 2.9 for cis-4-OH-TAM and 4.5 for cis-endoxifen; p<0.01 for both). The RBA of the trans isomers of both 4-OH-TAM and endoxifen was approximately equivalent to that observed for E₂ (RBA=100). All glucuronide conjugates of active TAM metabolites exhibited significantly (p<0.001) reduced ER-alpha binding affinity as compared to their unconjugated counterparts. Consistent with this lowered ER-alpha binding affinity, the glucuronide conjugates of all 4-OH-TAM and endoxifen isomers did not suppress E₂-induced MCF-7 cell proliferation at all concentrations of TAM metabolite tested in this study; only unconjugated TAM metabolites inhibited this effect, with the trans isomers of 4-OH-TAM and endoxifen significantly (p<0.05) more potent than their cis isomers. A virtually identical dose-dependent inhibition of E₂-induced MCF-7 cell proliferation was found for both trans-4-OH-TAM and trans-endoxifen, with maximal inhibition attained at 1 x10^-6 M of TAM metabolite. These data indicate that both 4-OH-TAM and endoxifen exhibit roughly equipotent anti-estrogenic effects on E₂-induced cell proliferation and ER-binding, that the trans isomers of both compounds exhibit significantly more effect on E₂-induced cell proliferation and ER-binding than their cis counterparts, and that conjugation of the same metabolites with glucuronic acid effectively negates this activity. This may have important implications in terms of both whole-body and target-tissue-specific glucuronidation pathways and individual response to TAM therapy and cancer prevention.