RAD1901: a novel, orally bioavailable selective estrogen receptor degrader that demonstrates antitumor activity in breast cancer xenograft models.

Agents that inhibit estrogen production, such as aromatase inhibitors or those that directly block estrogen receptor (ER) activity, such as selective estrogen receptor modulators and selective estrogen receptor degraders, are routinely used in the treatment of ER-positive breast cancers. However, although initial treatment with these agents is often successful, many women eventually relapse with drug-resistant breast cancers. To overcome some of the challenges associated with current endocrine therapies and to combat the development of resistance, there is a need for more durable and more effective ER-targeted therapies. Here we describe and characterize a novel, orally bioavailable small-molecule selective estrogen receptor degrader, RAD1901, and evaluate its therapeutic potential for the treatment of breast cancer. RAD1901 selectively binds to and degrades the ER and is a potent antagonist of ER-positive breast cancer cell proliferation. Importantly, RAD1901 produced a robust and profound inhibition of tumor growth in MCF-7 xenograft models. In an intracranial MCF-7 model, RAD1901-treated animals survived longer than those treated with either control or fulvestrant, suggesting the potential benefit of RAD1901 in the treatment of ER-positive breast cancer that has metastasized to the brain. Finally, RAD1901 preserved ovariectomy-induced bone loss and prevented the uterotropic effects of E2, suggesting that it may act selectively as an agonist in bone but as an antagonist in breast and uterine tissues. RAD1901 is currently under clinical study in postmenopausal women with ER-positive advanced breast cancer.

Effects of abaloparatide, a human parathyroid hormone-related peptide analog, on bone mineral density in postmenopausal women with osteoporosis.

CONTEXT: Abaloparatide is a novel synthetic peptide analog of parathyroid hormone-related protein (PTHrP) that is currently being developed as a potential anabolic agent in the treatment of postmenopausal osteoporosis. OBJECTIVE: This study sought to assess the effects of abaloparatide on bone mineral density (BMD) at the lumbar spine, total hip, and femoral neck in postmenopausal women with osteoporosis. DESIGN: Multi-center, multi-national, double-blind placebo controlled trial in which postmenopausal women were randomly assigned to receive 24 weeks of treatment with daily sc injections of placebo, abaloparatide, 20, 40, or 80 µg, or teriparatide, 20 µg. A 24-week extension was also performed in a subset of subjects. PARTICIPANTS: Postmenopausal women with osteoporosis (n = 222). MAIN OUTCOME MEASURES: BMD by dual-x-ray absorptiometry and biochemical markers of bone turnover. RESULTS: At 24 weeks, lumbar spine BMD increased by 2.9, 5.2, and 6.7% in the abaloparatide, 20-, 40-, and 80-µg groups, respectively, and 5.5% in the teriparatide group. The increases in the 40- and 80-µg abaloparatide groups and the teriparatide group were significantly greater than placebo (1.6%). Femoral neck BMD increased by 2.7, 2.2, and 3.1% in abaloparatide, 20-, 40-, and 80-µg groups, respectively, and 1.1% in the teriparatide group. The increase in femoral neck BMD with abaloparatide, 80 µg was significantly greater than placebo (0.8%). Total hip BMD increased by 1.4, 2.0, and 2.6% in the abaloparatide, 20-, 40-, and 80-µg groups, respectively. The total hip increases in the 40- and 80-µg abaloparatide groups were greater than both placebo (0.4%) and teriparatide (0.5%). CONCLUSIONS: Compared with placebo, 24 weeks of daily sc abaloparatide increases BMD of the lumbar spine, femoral neck, and total hip in a dose-dependent fashion. Moreover, the abaloparatide-induced BMD increases at the total hip are greater than with the marketed dose of teriparatide. These results support the further investigation of abaloparatide as an anabolic therapy in postmenopausal osteoporosis.

Design, Synthesis, and Preclinical Characterization of the Selective Androgen Receptor Modulator (SARM) RAD140.

This report describes the discovery of RAD140, a potent, orally bioavailable, nonsteroidal selective androgen receptor modulator (SARM). The characterization of RAD140 in several preclinical models of anabolic androgen action is also described.

Synthesis of potent, substituted carbazoles as selective androgen receptor modulators (SARMs).

The synthesis and in vitro binding affinity for a novel series of potent androgen receptor modulators is described. One of the more potent compounds (17, RAD35010) was further characterized in vivo where it restored levator ani weight in castrated male rats to near sham level while having no significant effect on prostate weight.

The pairing of a selective estrogen receptor modulator, bazedoxifene, with conjugated estrogens as a new paradigm for the treatment of menopausal symptoms and osteoporosis prevention.

The menopausal transition is associated with decreased ovarian function and concomitant decline in estrogen production, which may result in physiological effects such as hot flashes, reduced bone mass, and altered lipid profile. It is well established that these unfavorable changes are effectively offset with estrogen therapy (ET) or, in women with a uterus, estrogens in combination with a progestin (hormone therapy). Selective estrogen receptor (ER) modulators (SERMs), which exhibit both ER agonist and antagonist activities depending on the target tissue, have been regarded as offering the potential to provide the benefits of ET and hormone therapy with an improved safety and tolerability profile. To date, no SERM alone has demonstrated an ideal benefit-risk profile for menopausal therapy. The tissue-selective estrogen complex, or the pairing of a SERM with estrogens, may provide an optimal blend of ER agonist and antagonist activities. We evaluated the physiological profile of this novel therapeutic paradigm by using various in vivo models to assess uterine, vasomotor, lipid, and skeletal responses to a tissue-selective estrogen complex partnering bazedoxifene with conjugated estrogens (CE). Bazedoxifene at 3.0 mg/kg effectively antagonized CE-induced uterine stimulation without reversing the positive effects of CE on vasomotor instability. When paired with CE, bazedoxifene at 3.0 mg/kg reduced total cholesterol levels by up to 20% compared with CE alone and significantly increased total bone density relative to control. These preclinical findings showed that the appropriate dose combination of bazedoxifene/CE exhibits positive vasomotor, lipid, and skeletal responses with minimal uterine stimulation.

Quantitative analysis of gene regulation by seven clinically relevant progestins suggests a highly similar mechanism of action through progesterone receptors in T47D breast cancer cells.

Progesterone (P4) is an essential reproductive steroid hormone required for many aspects of female reproductive physiology. Progestins are compounds that demonstrate progesterone-like activity and are used in oral contraception, hormone therapy, and treatment of some reproductive disorders, but differ widely in their chemical structures, potency, and pharmacokinetics. While numerous studies have assessed progestins on specific endpoints, little is known about the activation of global gene expression by progestins. We used Affymetrix GeneChip U133A expression arrays to examine the action of P4 and six clinically relevant synthetic progestins (3-ketodesogestrel, drospirenone, levonorgestrel, medroxyprogesterone acetate, norethindrone acetate, and trimegestone) on the progesterone receptor (PR)-positive T47Dco and the PR-negative T47D-Y breast cancer cell lines. Excluding drospirenone, one or more of the progestins-regulated 329 genes, with 30 genes regulated by at least 2.0-fold by all progestins in the T47Dco cells. The synthetic progestins show a high degree of similarity in their transcriptional responses, and each progestin regulates between 77 and 91% of the genes regulated by P4. Independent quantitative RT-PCR analysis confirmed a similar regulation for S100P, PPL, IL20RA, NET1, ATP1A1, HIG2, and CXCL12 (SDF-1) by all seven progestins. Attempts to find differentially regulated genes by any progestin compared to all other treatments failed, suggesting any differences are quantitative, not qualitative. This analysis demonstrates a high degree of similarity among these progestins on PR-regulated gene expression in T47D cells, suggesting a similar and fairly specific mode of action.

Bazedoxifene acetate: a selective estrogen receptor modulator with improved selectivity.

We assessed the preclinical characteristics of a novel, stringently screened selective estrogen receptor modulator, bazedoxifene acetate, including its ability to bind to and activate estrogen receptors and promote increased bone mineral density and bone strength in rats, and the effects impacting the uterine endometrium, breast cancer cell proliferation, and central nervous system-associated vasomotor responses in an animal model. Bazedoxifene bound to estrogen receptor-alpha with an IC50 of 26 nm, an affinity similar to that of raloxifene. Bazedoxifene did not stimulate proliferation of MCF-7 cells but did inhibit 17beta-estradiol-induced proliferation with an IC50 of 0.19 nm. In an immature rat uterine model, bazedoxifene (0.5 and 5.0 mg/kg) was associated with less increase in uterine wet weight than either ethinyl estradiol (10 microg/kg) or raloxifene (0.5 and 5.0 mg/kg). Histological analysis revealed that coadministration of bazedoxifene also appeared to reduce raloxifene-stimulated endometrial luminal epithelial cell and myometrial cell hypertrophy. In ovariectomized rats, bazedoxifene was associated with significant increases in bone mineral density at 6 wk, compared with control, and better compressive strength of bone samples from the L4 vertebrae, compared with samples from ovariectomized animals. In the morphine-addicted rat model of vasomotor activity, bone-sparing doses of bazedoxifene alone were not associated with 17beta-estradiol inhibition of increased vasomotor activity. Bazedoxifene acetate represents a promising new treatment for osteoporosis, with a potential for less uterine and vasomotor effects than selective estrogen receptor modulators currently used in clinical practice. Controlled clinical trial data will be needed to confirm these effects.

Molecular and pharmacological properties of a potent and selective novel nonsteroidal progesterone receptor agonist tanaproget.

Progesterone receptor (PR) agonists have several important applications in women's health, such as in oral contraception and post-menopausal hormone therapy. Currently, all PR agonists used clinically are steroids. Because of their interactions with other steroid receptors, steroid-metabolizing enzymes, or other steroid-signaling pathways, these drugs can pose significant side effects in some women. Efforts to discover novel nonsteroidal PR agonists with improved biological properties led to the discovery of tanaproget (TNPR). TNPR binds to the PR from various species with a higher relative affinity than reference steroidal progestins. In T47D cells, TNPR induces alkaline phosphatase activity with an EC(50) value of 0.1 nm, comparable with potent steroidal progestins such as medroxyprogesterone acetate (MPA) and trimegestone (TMG), albeit with a reduced efficacy ( approximately 60%). In a mammalian two-hybrid assay to measure PR agonist-induced interaction between steroid receptor co-activator-1 and PR, TNPR showed similar potency (EC(50) value of 0.02 nm) and efficacy to MPA and TMG. Importantly, in key animal models such as the rat ovulation inhibition assay, TNPR demonstrates full efficacy and an enhanced progestational potency (30-fold) when compared with MPA and TMG. Furthermore, TNPR has relatively weak interactions with other steroid receptors and binding proteins and little effect on cytochrome P450 metabolic pathways. Finally, the three-dimensional crystal structure of the PR ligand binding domain with TNPR has been delineated to demonstrate how this nonsteroidal ligand achieves its high binding affinity. Therefore, TNPR is a structurally novel and very selective PR agonist with an improved preclinical pharmacological profile.

A selective estrogen receptor-beta agonist causes lesion regression in an experimentally induced model of endometriosis.

BACKGROUND: Endometriosis is a common gynaecological problem of uncertain aetiology. It affects primarily young, reproductive-aged women and can result in chronic pelvic pain and infertility. Current approved therapies have significant side-effects and hysterectomy is employed as a final solution. ERB-041 is a selective estrogen receptor-beta (ERbeta) agonist that has anti-inflammatory activity in preclinical models of arthritis and inflammatory bowel disease, but is inactive in many preclinical models of classic estrogen activity. Because endometriosis is now thought to be, at least in part, an inflammatory disease, we evaluated ERB-041's activity in an experimentally induced model of endometriosis. METHODS: Athymic nude mice (ovariectomized or intact) were implanted with tissue fragments of normal human endometrium. After establishment of lesions for 11-14 days, mice were treated with ERB-041 for 15-17 days. Upon euthanasia, the number of lesions, their size and location were noted. Five lesions were recovered for RNA analysis. RESULTS: Across six studies, ERB-041 caused complete lesion regression in 40-75% of the mice studied. The compound appeared to be equally effective in gonad-intact as in ovariectomized mice, and analysed recovered lesions expressed ERalpha but not ERbeta mRNA. CONCLUSIONS: ERB-041 and possibly other ERbeta selective agonists may be a useful new approach to treating endometriosis.

Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) alpha or ERbeta in human osteosarcoma cells: distinct and common target genes for these receptors.

Estrogens exert many important effects in bone, a tissue that contains both estrogen receptors alpha and beta (ERalpha and ERbeta). To compare the actions of these receptors, we generated U2OS human osteosarcoma cells stably expressing ERalpha or ERbeta, at levels comparable with those in osteoblasts, and we characterized their response to 17beta-estradiol (E2) over time using Affymetrix GeneChip microarrays to determine the expression of approximately 12,000 genes, followed by quantitative PCR verification of the regulation of selected genes. Of the approximately 100 regulated genes we identified, some were stimulated by E2 equally through ERalpha and ERbeta, whereas others were selectively stimulated via ERalpha or ERbeta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48-h time course studied. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ERbeta, in keeping with the selective E2 enhancement of the motility of ERbeta-containing cells. On genes regulated equally by E2 via ERalpha or ERbeta, the phytoestrogen genistein preferentially stimulated gene expression via ERbeta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation.

Selective estrogen receptor modulators: discrimination of agonistic versus antagonistic activities by gene expression profiling in breast cancer cells.

Selective estrogen receptor modulators (SERMs) such as tamoxifen are effective in the treatment of many estrogen receptor-positive breast cancers and have also proven to be effective in the prevention of breast cancer in women at high risk for the disease. The comparative abilities of tamoxifen versus raloxifene in breast cancer prevention are currently being compared in the Study of Tamoxifen and Raloxifene trial. To better understand the actions of these compounds in breast cancer, we have examined their effects on the expression of approximately 12,000 genes, using Affymetrix GeneChip microarrays, with quantitative PCR verification in many cases, categorizing their actions as agonist, antagonist, or partial agonist/antagonist. Analysis of gene stimulation and inhibition by the SERMs trans-hydroxytamoxifen (TOT) and raloxifene (Ral) or ICI 182,780 (ICI) and by estradiol (E2) in estrogen receptor-containing MCF-7 human breast cancer cells revealed that (a) TOT was the most E2-like of the three compounds, (b) all three compounds either partially or fully antagonized the action of E2 on most genes, with the order of antagonist activity being ICI > Ral > TOT, (c) TOT and Ral, but not ICI, displayed partial agonist/partial antagonist activity on a number of E2-regulated genes, (d) several stimulatory cell cycle-related genes were down-regulated exclusively by ICI, (e) the estrogen-like activity of Ral nearly always overlapped with that of TOT, indicating that Ral has little unique agonist activity different from that of TOT, and (f) some genes were specifically up-regulated by TOT but not Ral, ICI, or E2. Hence, gene expression profiling can discern fundamental differences among SERMs and provides insight into the distinct biologies of TOT, Ral, and ICI in breast cancer.

Regulation of gene expression by PRA-910, a novel progesterone receptor modulator, in T47D cells.

Progestins play an important role in women's health and are used in oral contraception, hormone therapy, and treatment of reproductive disorders. The effects of progestins upon gene expression in breast epithelium are poorly understood. In an attempt to characterize the molecular mechanism of progestin action, we used a gene expression profiling approach to examine the action of a novel progestin in the T47D cell model, a human breast cancer cell line. PRA-910 is a novel, nonsteroidal progesterone receptor modulator (PRM) with species-specific activities identified in a screen for selective PRMs. To understand the mechanism of action for PRA-910 in T47D cells, we compared its gene regulation to progesterone (P4) and RU486 through Affymetrix U95A GeneChip analysis and TaqMan RT-PCR. PRA-910, P4, and RU486 regulated 50, 108, and 16 genes by threefold or greater versus vehicle, respectively, with 18 genes having similar regulation for P4 and PRA-910. These data confirm and extend previous findings for T47D cells. We also obtained time course, concentration-response, cyclohexamide sensitivity, and PR-specificity data for two progestin-regulated genes, ATP1A1 and CLDN8. Our data demonstrate that PRA-910 has a unique gene regulation profile distinct from both P4 and RU486. Further investigation of the underlying mechanism for these differences is ongoing.

Status and future direction of male contraceptive development.

Control of fertility constitutes a global health issue, as overpopulation and unintended pregnancy have both major personal and societal impact. Although the contraceptive revolution in the 1960s following the development of hormonal-based oral contraceptives for women has had a major impact on societal dynamics in several cultures, little product innovation has occurred since then. One solution to this global health issue lies in the development of new and innovative contraceptives for both women and men, the goal of which is to provide a range of options for people at all stages and walks of life. Currently, three options for male-based contraception exist (i.e. withdrawal, condoms and vasectomy), and these are acknowledged as woefully inadequate. Introduction of new forms of male contraception based on both hormonal and non-hormonal paradigms are wanted and needed; this need is now becoming recognized by both the public and private sectors. New and innovative products will come from our knowledge of the unique physiology and genetics of reproduction, as well as by exploiting existing and future genomics, proteomics and protein network platforms.

Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype.

Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with quantitative PCR verification in many cases, to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. Of the >12,000 genes queried, over 400 showed a robust pattern of regulation, and, notably, the majority (70%) were down-regulated. We observed a general up-regulation of positive proliferation regulators, including survivin, multiple growth factors, genes involved in cell cycle progression, and regulatory factor-receptor loops, and the down-regulation of transcriptional repressors, such as Mad4 and JunB, and of antiproliferative and proapoptotic genes, including B cell translocation gene-1 and -2, cyclin G2, BCL-2 antagonist/killer 1, BCL 2-interacting killer, caspase 9, and TGFbeta family growth inhibitory factors. These together likely contribute to the stimulation of proliferation and the suppression of apoptosis by E2 in these cells. Of interest, E2 appeared to modulate its own activity through the enhanced expression of genes involved in prostaglandin E production and signaling, which could lead to an increase in aromatase expression and E2 production, as well as the decreased expression of several nuclear receptor coactivators that could impact ER activity. Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which this hormone operates to achieve its widespread effects on breast cancer cells.

Hierarchical affinities and a bipartite interaction model for estrogen receptor isoforms and full-length steroid receptor coactivator (SRC/p160) family members.

Nuclear receptor (NR)-mediated transcription is driven by dynamic multiprotein coactivator complexes, the composition of which is thought to determine the biological activity of NRs at specific promoters. The extent to which NRs discriminate between a spectrum of potential binding partners is intuitively a function of the inherent affinities of these individual interactions. Using real time interaction analysis with BIAcore, we evaluated the affinities and kinetics of the interactions of full-length members of the SRC/p160 coactivator family with estrogen receptor alpha (ER alpha) and ER beta bound to a variety of ligands. We substantiate that 17beta-estradiol enhances the affinity of ER-SRC/p160 interactions, whereas 4(OH)-tamoxifen, raloxifene, and ICI-182,780 inhibit these interactions. We show that a well defined, ER isoform-specific hierarchy governs the association of liganded ERs with full length SRC/p160 family members. Moreover, our data indicate that the interaction affinities of the full-length SRC/p160s with ERs are significantly higher then those of the NR interaction domains of the same coactivators, indicating that portions of coactivator molecules distinct from NR interaction domains might participate in receptor-coactivator complex formation. Finally, the interaction kinetics of SRC/p160s with ERs are consistent with a bipartite model, involving initial rapid formation of an unstable intermediate complex, and a subsequent slower reaction leading to its stabilization. We interpret our results as evidence that hierarchical coactivator interaction affinities are an important source of diversity in NR-mediated signaling and that the complexity of receptor-coactivator cross-talk might be best understood in the context of full-length molecules.

Estrogenic effects of 7alpha-methyl-17alpha-ethynylestradiol: a newly discovered tibolone metabolite.

Tibolone is a synthetic steroid that is prescribed to postmenopausal women for relief of climacteric symptoms and prevention of osteoporosis. It has been reported to be metabolized in a tissue-selective manner to three steroids that collectively have weak estrogenic, progestogenic, and androgenic activities. Recently, a new tibolone metabolite, 7alpha-methyl-17alpha-ethynyl-17beta-estradiol (7alpha-Me-EE2), was identified in women. In this report, we describe the pre-clinical estrogenic activities of this metabolite and compare these effects to those obtained with 17alpha-ethynyl-17beta-estradiol (EE2) and 17beta-estradiol (E2). In an in vitro ligand-binding assay, 7alpha-Me-EE2 bound to both human estrogen receptor (ER)-alpha and -beta with IC(50)'s of 1.2 and 3.0 nM, respectively. Using MCF-7 human breast cancer cells that express high levels of ER-alpha, 7alpha-Me-EE2 transactivated an estrogen response element (ERE)-tk-luciferase reporter gene construct with an EC(50) of 0.021 nM. Likewise, 7alpha-Me-EE2 stimulated MCF-7 breast cancer cell proliferation with an EC(50) of 0.002 nM. In immature female rats, subcutaneous (s.c.) administration of 7alpha-Me-EE2 stimulated uterine wet weight gain with an ED(50) of 0.2 microg/kg. Moreover, 7alpha-Me-EE2 induced uterine complement component C3 gene expression, an estrogenic marker of epithelial cell stimulation, with an ED(50) of 0.5 microg/kg. When compared to EE2 and E2, 7alpha-Me-EE2 exhibited equivalent or greater potencies and efficacies in these assays. In summary, these results indicate that 7alpha-Me-EE2 is a very potent estrogen. This steroid appears to be the most potent estrogenic metabolite of tibolone identified to date, and additional studies are, therefore, warranted regarding the role of this metabolite in the biological actions of the drug.