The formation of estrogen-like tamoxifen metabolites and their influence on enzyme activity and gene expression of ADME genes

Tamoxifen, a widely used breast cancer therapy, is metabolized into compounds with both anti-estrogenic and estrogen-like effects at the estrogen receptor. However, the formation and biological impact of its estrogen-like metabolites remain poorly understood. To address this, we investigated two estrogen-like metabolites—tamoxifen bisphenol and metabolite E—by examining their metabolic pathways, effects on cytochrome P450 (CYP) activity, and influence on the expression of ADME (Absorption, Distribution, Metabolism, and Excretion) genes.

The formation of these metabolites was analyzed using human liver microsomes and Supersomes™, while their cellular metabolism and effects on CYP enzymes were studied in upcyte® hepatocytes. CYP activity was assessed using HPLC-MS/MS, and changes in ADME gene expression were measured via RT-PCR. Metabolite E was generated from tamoxifen by CYP2C19, CYP3A, and CYP1A2, and from desmethyltamoxifen by CYP2D6, CYP1A2, and CYP3A. Tamoxifen bisphenol, in turn, was primarily formed from (E)- and (Z)-metabolite E through the activity of CYP2B6 and CYP2C19, respectively.

In phase II metabolism, UGT2B7, UGT1A8, and UGT1A3 exhibited the highest glucuronidation activity for tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites—including (Z)-4-hydroxytamoxifen, (Z)-endoxifen, and (Z)-norendoxifen—were found to inhibit CYP2C enzyme activity. In contrast, tamoxifen bisphenol consistently induced CYP enzymes, similar to the known inducers rifampicin and phenobarbital.

At the transcript level, tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol, and (E)-metabolite E significantly upregulated *CYP3A4* expression, with increases of up to 5.6-fold. These findings indicate that estrogen-like tamoxifen metabolites are formed through CYP-dependent pathways and are further processed via glucuronidation. The induction of CYP activity by tamoxifen bisphenol, alongside the inhibition of CYP2C enzymes by anti-estrogenic metabolites, suggests potential drug-drug interactions.