Estrogens elicit many biomedically important responses in different target tissues, and the respective roles of the two estrogen receptors, ERalpha and ERbeta, in mediating these bioactivities is incompletely understood. In this study, we investigated the activity of an ERalpha-selective agonist ligand, propyl pyrazole triol (PPT), in several rat animal models to define the involvement of ERalpha in these biological responses. In a short-term (4 d) uterotrophic assay, PPT was found to be as efficacious as 17alpha-ethinyl-17beta-estradiol in stimulating uterine weight gain and up-regulating complement 3 gene expression. In a 6-wk chronic model, PPT completely prevented the ovariectomy-induced body weight increase and loss of bone mineral density. It also increased uterine weight and markedly reduced plasma cholesterol levels in these mature animals. PPT was also effective in the brain. It increased progesterone receptor mRNA in the arcuate and ventromedial nuclei of the hypothalamus and prevented experimentally induced hot flushes. Our findings indicate that several physiologically relevant estrogen-induced tissue responses can be effectively evoked via ERalpha alone. By providing an approach that is complementary to that of analyzing the phenotype and response of ER knockout animals, our findings also demonstrate that ER subtype-selective ligands can play a valuable role in enhancing our understanding of how estrogens work through the two ER subtypes.

The discovery of a second estrogen receptor (ER), called ERbeta, in 1996 sparked intense interest within the scientific community to discover its role in mediating estrogen action. However, despite more than 6 yr of research into the function of this receptor, its physiological role in mediating estrogen action remains unclear and controversial. We have developed a series of highly selective agonists for ERbeta and have characterized their activity in several clinically relevant rodent models of human disease. The activity of one such compound, ERB-041, is reported here. We conclude from these studies that ERbeta does not mediate the bone-sparing activity of estrogen on the rat skeleton and that it does not affect ovulation or ovariectomy-induced weight gain. In addition, these compounds are nonuterotrophic and nonmammotrophic. However, ERB-041 has a dramatic beneficial effect in the HLA-B27 transgenic rat model of inflammatory bowel disease and the Lewis rat adjuvant-induced arthritis model. Daily oral doses as low as 1 mg/kg reverse the chronic diarrhea of HLA-B27 transgenic rats and dramatically improve histological disease scores in the colon. The same dosing regimen in the therapeutic adjuvant-induced arthritis model reduces joint scores from 12 (maximal inflammation) to 1 over a period of 10 d. Synovitis and Mankin (articular cartilage) histological scores are also significantly lowered (50-75%). These data suggest that one function of ERbeta may be to modulate the immune response, and that ERbeta-selective ligands may be therapeutically useful agents to treat chronic intestinal and joint inflammation.

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.

The unexpected discovery of a second form of the estrogen receptor (ER), designated ERbeta, surprised and energized the field of estrogen research. In the 9 yr since its identification, the remarkable efforts from academic and industrial scientists of many disciplines have made significant progress in elucidating its biology. A powerful battery of tools, including knockout mice as well as a panel of receptor-selective agonists, has allowed an investigation into the role of ERbeta. To date, in vivo efficacy studies are limited to rodents. Current data indicate that ERbeta plays a minor role in mediating estrogen action in the uterus, on the hypothalamus/pituitary, the skeleton, and other classic estrogen target tissues. However, a clear role for ERbeta has been established in the ovary, cardiovascular system, and brain as well as in several animal models of inflammation including arthritis, endometriosis, inflammatory bowel disease, and sepsis. The next phase of research will focus on elucidating, at a molecular level, how ERbeta exerts these diverse effects and exploring the clinical utility of ERbeta-selective agonists.

We present the structure-based optimization of a series of estrogen receptor-beta (ERbeta) selective ligands. X-ray cocrystal structures of these ligands complexed to both ERalpha and ERbeta are described. We also discuss how molecular modeling was used to take advantage of subtle differences between the two binding cavities in order to optimize selectivity for ERbeta over ERalpha. Quantum chemical calculations are utilized to gain insight into the mechanism of selectivity enhancement. Despite only two relatively conservative residue substitutions in the ligand binding pocket, the most selective compounds have greater than 100-fold selectivity for ERbeta relative to ERalpha when measured using a competitive radioligand binding assay.