Iwaniec, UT

Abstract Background: Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We and others have previously demonstrated that endoxifen is the most active tamoxifen metabolite responsible for eliciting the anti-cancer effects of this drug and that endoxifen concentrations are an important factor with regard to tamoxifen efficacy. These studies have led to the development of endoxifen as a novel anti-breast cancer drug for which phase I clinical trials are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast gene expression profiles were compared to that of estrogen, tamoxifen, raloxifene and lasofoxifene by microarray and RT-PCR analyses in both estrogen receptor alpha (ERα) and ERβ expressing cell lines. The in vivo effects of an anti-cancer dose of endoxifen (50mg/kg/day) on the skeleton were first analyzed in 3-month-old ovariectomized C57BL/6 mice using Dual-energy X-ray absorptiometry, peripheral Quantitative Computed Tomography, micro-Computed Tomography and histomorphometry. In a second set of studies, a pre-clinical rat model was used to determine the effects of endoxifen (10mg/kg/day) on the skeleton in both a pre- and post-menopausal setting. Results: Endoxifen treatment of ERα and ERβ expressing mouse osteoblast cells led to dramatically different gene expression profiles when compared to that of estrogen and other anti-estrogens. In ovariectomized mice, daily administration of endoxifen led to significant increases in bone mineral density and content throughout the skeleton relative to vehicle control treated animals. The numbers and activity of both osteoblasts and osteoclasts were also found to be significantly higher in endoxifen treated mice. In the pre-clinical model system, endoxifen treatment of 4 month-old ovariectomized Sprague-Dawley rats significantly protected against bone loss following estrogen depletion primarily due to suppression of osteoclast mediated bone resorption. Importantly, in sham operated rats (thus retaining ovarian function), endoxifen treatment enhanced bone volume and trabecular thickness and did not suppress osteoclast activity. Conclusions: These data are the first to examine the effects of the novel breast cancer therapy, endoxifen, on bone and reveal that the molecular mechanisms of action of this compound are substantially different than that of other SERMs. Endoxifen was shown to protect against bone loss following estrogen depletion in both mice and rats and interestingly, enhanced bone mass in ovary intact rats, an observation that is in stark contrast to the known effects of tamoxifen which induces bone loss in the “pre-menopausal” setting. These studies suggest that endoxifen may have superior bone-beneficial effects compared to tamoxifen, and if efficacy is confirmed in later phase trials, endoxifen may represent a better drug of choice for a sub-set of breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-01.

Abstract Background Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We have recently demonstrated that endoxifen is the most active tamoxifen metabolite with regard to inhibiting the growth of ERα+ breast cancer cells and these studies have led to the development of endoxifen as a novel anti-breast cancer drug for which first-in-human studies are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast (OB) and osteoclast (OC) maturation and gene expression were monitored by cell differentiation assays and real-time PCR. Dual-energy X-ray absorptiometry (DXA), peripheral Quantitative Computed Tomography (pQCT) and micro-Computed Tomography (μCT) were used to determine changes in bone density, mass and architecture following 45 days of oral endoxifen administration (50mg/kg/day) to 3-month-old ovariectomized (OVX) C57BL/6 mice relative to vehicle control treated animals. Alterations in the numbers and activity of OBs and OCs were determined by histomorphometry and serum levels of P1NP and CTX-1 respectively. Results: Endoxifen treatment of mouse derived bone marrow stromal cells and human OBs led to significant increases in the expression of critical bone marker genes such as Runx2, osterix, osteocalcin, osteoprotegerin and alkaline phosphatase in a dose dependent manner. Daily administration of endoxifen to OVX mice led to significant increases in total body bone mineral density (BMD) (6%) and content (BMC) (9%), which was accompanied by a 50% decrease in fat tissue mass as determined by DXA. pQCT analysis of the tibial metaphysis revealed dramatic increases in BMD (35%) and BMC (20%), as well as trabecular density (52%), cortical content (62%), cortical area (60%) and cortical thickness (78%). μCT analysis of the femoral metaphysis revealed increases in bone volume/total volume (200%), trabecular number (38%) and trabecular thickness (18%), as well as decreased trabecular spacing (29%). Interestingly, there was nearly a 50% increase in the numbers of OCs derived from endoxifen treated mice which was associated with elevated expression of OC marker genes such as NFATcl, RANK, c-fms and cathepsin-K compared to control treated animals. Approximately 4 times as many OBs and OCs were observed on the bone surfaces of endoxifen treated mice which correlated with nearly 2-fold increases in serum levels of the bone formation (P1NP) and resorption (CTX-1) markers. Conclusions: These data are the first to demonstrate that endoxifen has anabolic effects on the mouse skeleton which are similar to that of estrogen. Additionally, these data reveal that endoxifen's mechanism of action in bone is different than that reported for tamoxifen and other selective estrogen receptor modulators in mice as it increases, rather than decreases, bone formation and remodeling. Therefore, the use of endoxifen for the treatment of endocrine responsive breast cancer may avoid the detrimental skeletal effects of many conventional endocrine therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-09.