Pharmacological Relevance of Endoxifen in a Laboratory Simulation of Breast Cancer in Postmenopausal Patients

Tamoxifen, a nonsteroidal antiestrogen, is a pioneering therapy for the treatment of breast cancer targeted to the tumor estrogen receptor (ER) (1). The laboratory strategy (2) of targeting tumor ER with long-term antihormone therapy accurately translated to clinical practice with enhanced control of disease recurrence following long-term adjuvant therapy in both pre- and postmenopausal patients. This targeted adjuvant treatment strategy ultimately resulted in a 30% decrease in mortality (3–6). Indeed, long-term (five years) adjuvant tamoxifen therapy contributes substantially to national statistics with the reduction in death rate from breast cancer during the past decade (7). Early findings demonstrated that tamoxifen is metabolically activated to 4-hydroxytamoxifen (Figure 1) that has a hundred-fold increase in affinity for the ER (8,9). However, high affinity of a ligand for the ER is an advantage but not a requirement for antiestrogenic activity (10,11). Indeed, studies comparing the antitumor activity of tamoxifen and 4-hydroxytamoxifen (4OHT) in vivo demonstrated that tamoxifen was the superior agent for clinical development (10). 4-Hydroxytamoxifen is cleared from the body faster than tamoxifen while the parent drug tamoxifen accumulates (12). Nevertheless, the subsequent identification of 4-hydroxy-N-desmethyltamoxifen (13–15) (later called endoxifen) (Figure 1) and the demonstration of a reduction of endoxifen production in women taking Selective Serotonin Reuptake Inhibitors (SSRI) (for reduction of hot flashes), which block the enzymatic activity of CYP2D6 (16), suggested that this important drug interaction reduces tamoxifen’s efficacy. Subsequently, a hypothesis was developed and connected with genotypic aberrations in CYP2D6 alleles (17). The hypothesis depends on the concept that different polymorphisms of CYP2D6 can result in altered enzymatic activity of the cytochrome P450 2D6, which results in different rates of tamoxifen metabolism and thus different levels of tamoxifen metabolites N-desmethyltamoxifen (NDMTAM) and endoxifen. Some clinical studies have shown association between the CYP2D6 genotypes and the clinical outcomes (18–20), and some have not found any association (21–23). Numerous papers using retrospective clinical data presented convincing cases for or against the hypothesis. However, the low incidence of poor metabolizers (PM) (24), and the known poor compliance rate (25) of long-term antihormone therapies make the hypothesis difficult to validate in retrospective patient samples. Figure 1. The chemical structures of all metabolites of tamoxifen in this study and their metabolism pathways and metabolizing cytochrome P450 (CYP) enzymes with relative binding affinities (RBAs) for the estrogen receptor (ER). Tamoxifen is metabolically activated ... In this article we address the role of the CYP2D6 genotype during tamoxifen treatment of breast cancer in a laboratory simulation. We have characterized a panel of four representative ER-positive breast cancer cell lines, MCF-7, T47D, BT474, and ZR-75-1, and carefully calibrated their concentration responsiveness to both estradiol (E2) and estrone (E1). We selected concentrations of total estrogen (E1/E2) corresponding to the previously reported circulating levels in postmenopausal breast cancer patients (26,27). We address two hypotheses: 1) that tamoxifen is a prodrug that needs to be metabolically activated to NDMTAM and 4OHT, and 2) that circulating concentrations of tamoxifen and its two metabolites NDMTAM and 4OHT are sufficient to block estrogen-stimulated breast cancer cell growth and gene expression in a postmenopausal patient simulation. Our chosen concentrations of tamoxifen and metabolites have been determined recently for postmenopausal patients genotyped for CYP2D6 as extensive metabolizers (EM), intermediate metabolizers (IM), and PM (28). The experiment was repeated with all cell lines to address the second hypothesis with and without relevant concentrations of endoxifen for each genotype. Overall, metabolic activation to endoxifen appears to play a supportive role in blocking estrogen-induced cell replication in breast cancer cell lines in a simulated model of postmenopausal breast cancer. Conversion to endoxifen is perhaps consolidating the long-term benefits of antiestrogen therapy but is not essential for the immediate antiestrogenic antitumor actions of tamoxifen in the postmenopausal setting.