DT56a (Femarelle): a natural selective estrogen receptor modulator (SERM)

A selective estrogen receptor modulator (SERM) is defined as a substance with dissimilar effects on different tissues: agonist in some and antagonists in others. The natural compound DT56a (Femarelle) was shown to activate estrogen receptors in human cultured female derived osteoblasts. It was also shown to relieve menopausal symptoms and to increase bone mineral density with no effect on sex steroid hormone levels and on the endometrial thickness. DT56a, similarly to estradiol-17β (E₂), stimulated the specific activity of creatine kinase (CK) in skeletal and vascular tissues of female rats, as a marker of estrogen receptor (ER) activation. However, in the uterus, CK was activated only by E₂ but not by DT56a. In order to prove that DT56a is a SERM, we examined the mutual interaction between DT56a and E₂, at supra physiological doses, in different tissues in both intact and ovariectomized female rats, as well as in human cultured vascular and bone cells. Administration of DT56a or E₂ stimulated CK in all tissues tested, but when given simultaneously, in intact immature female rats, DT56a completely abolished E₂ stimulation of CK in all organs except in the diaphyseal bone where the inhibition was partial. In ovariectomized female rats, DT56a abolished E₂'s stimulation of CK in diaphyseal bone, thymus, uterus and pituitary but caused a partial inhibition in aorta, left ventricle and epiphyseal cartilage. In human bone cells E₂ stimulation of CK, of alkaline phosphatase (AP) activity and of DNA synthesis was completely abolished by DT56a in post-menopausal cells and partially inhibited in pre-menopausal cells. In human vascular cells, inhibition of DNA synthesis by E₂ was completely abolished by DT56a and E₂-induced CK was partially inhibited by DT56a. The results support the finding that DT56a is a SERM; it stimulated different parameters similar to E₂, but when given simultaneously, at supra physiological doses, inhibited these E₂'s effects. Further investigations regarding intra and extra cellular mechanism of action of DT56a are currently performed.     

DT56a stimulates gender-specific human cultured bone cells in vitro

DT56a found to have SERM-like properties is used for the treatment of menopausal symptoms and osteoporosis. In vivo experiments demonstrated that DT56a displayed selective estrogenic activity; it stimulated creatine kinase (CK) specific activity in the skeletal tissues but not on the uterus of ovariectomized rats. DT56a, when applied together with estradiol-17beta (E(2)), completely inhibited the E(2)-stimulated CK, as demonstrated by other SERMs. DT56a stimulated bone formation in a rat model as measured by histological and histomorphometrical parameters. In a clinical study, administration of DT56a (Femarelle) resulted in a considerable elevation of bone mineral density and relief of menopausal symptoms. The aim of the present study was to analyze the effects of DT56a in vitro on human-derived bone cultured osteoblasts (Ob), by measuring its effects, at different concentrations, on DNA synthesis, CK and alkaline phosphatase (ALP) specific activities as well as changes in intracellular [Ca(2+)](i) concentrations. DT56a stimulated CK and DNA synthesis in both pre- and post-menopausal female Ob with maximal effect at 100 ng/ml for both age groups. In addition, DT56a stimulated ALP in Ob from both pre- and post-menopausal women with maximal effect at lower dose of 50 ng/ml, with higher response of pre-menopausal cells. Raloxifene (Ral) inhibited all DT56a-stimulated changes in Ob from both age groups. DT56a, when given together with E(2), completely antagonized E(2)-stimulated effects demonstrating its nature as a phyto-SERM. DT56a also, dose dependency, stimulated the intracellular levels of [Ca(2+)](i) with maximal effect at 10 ng/ml. Male-derived Ob did not respond to DT56a in any parameter. In summary, DT56a stimulated sex-specifically female-derived Ob, indicating its unique nature compared to the compounds currently used for postmenopausal osteoporosis by being bone-forming and not only an anti-resorptive agent.     

"Non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D augment the induction of creatine kinase B by estrogen and selective estrogen receptor modulators (SERMS) in osteoblast-like cells and rat skeletal organs

We have demonstrated previously that daily treatments for 3 days with the so-called "non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D in ROS 17/2.8 osteoblast-like cells, stimulate the specific activity of creatine kinase BB (CK), and that such treatment with these analogs followed by a single treatment with gonadal steroids, upregulates responsiveness and sensitivity to estradiol 17beta (E(2)) for the induction of CK. This study was designed to determine if these same "non-hypercalcemic" vitamin D analogs could upregulate in vivo the response to E(2) and whether substitution of selective estrogen receptor modulators (SERMS) for E(2) would result in the same upregulation. We found that one week or 2 weeks pretreatment of prepubertal rats with vitamin D analogs led to increased induction of CK by E(2) and by the SERMS tamoxifen, tamoxifen methiodide and raloxifene, in epiphysis and diaphysis of the femur but not in the uterus. However, in contrast to their antiestrogenic activity in the uterus, there was no inhibition of E(2) action by the SERMS in skeletal tissues. The induction of mRNA for ckb in ROS 17/2.8 cells by E(2) or SERMS was demonstrated only after vitamin D pretreatment; there was no inhibition of E(2) induction by SERMS. Antagonists of vitamin D dependent calcium transport (transcaltachia) did not inhibit stimulation by vitamin D analogs. These results support the involvement of a nuclear mechanism in the upregulation of induction of CK by E(2), which may be due, in part, to the ability of vitamin D to increase estrogen receptor(s).     

Estrogen directly modulates circadian rhythms of PER2 expression in the uterus

Fluctuations in circulating estrogen and progesterone levels associated with the estrous cycle alter circadian rhythms of physiology and behavior in female rodents. Endogenously applied estrogen shortens the period of the locomotor activity rhythm in rodents. We recently found that estrogen implants affect Period (Per) gene expression in the suprachiasmatic nucleus (SCN; central clock) and uterus of rats in vivo. To explore whether estrogen directly influences the circadian clock in the SCN and/or tissues of the reproductive system, we examined the effects of 17beta-estradiol (E(2)) on PER2::LUCIFERASE (PER2::LUC) expression in tissue explant cultures from ovariectomized PER2::LUC knockin mice. E(2) applied to explanted cultures shortened the period of rhythmic PER2::LUC expression in the uterus but did not change the period of PER2::LUC expression in the SCN. Raloxifene, a selective estrogen receptor modulator and known E(2) antagonist in uterine tissues, attenuated the effect of E(2) on the period of the PER2::LUC rhythm in the uterus. These data indicate that estrogen directly affects the timing of the molecular clock in the uterus via an estrogen receptor-mediated response.     

Differential effects of estrogen and raloxifene on messenger RNA and matrix metalloproteinase 2 activity in the rat uterus

A detailed analysis of the differential effects of estrogen (E) compared to raloxifene (Ral), a selective estrogen receptor modulator (SERM), following estrogen receptor (ER) binding in gynecological tissues was conducted using gene microarrays, Northern blot analysis, and matrix metalloproteinase (MMP) 2 activity studies. We profiled gene expression in the uterus following acute (1 day) and prolonged daily (5 wk) treatment of E and Ral in ovariectomized rats. Estrogen regulated twice as many genes as Ral, largely those associated with catalysis and metabolism, whereas Ral induced genes associated with cell death and negative cell regulation. Follow-up studies confirmed that genes associated with matrix integrity were differentially regulated by Ral and E at various time points in uterine and vaginal tissues. Additional experiments were conducted to determine the levels of MMP2 activity in uterus explants from ovariectomized rats following 2 wk of treatment with E, Ral, or one of two additional SERMs: lasofoxifene, and levormeloxifene. Both E and lasofoxifene stimulated uterine MMP2 activity to a level twofold that of Ral, whereas levormeloxifene elevated MMP2 activity to a level 12-fold that of Ral. These data show that one of the significant differences between E and Ral signaling in the uterus is the regulation of genes and proteins associated with matrix integrity. This may be a potential key difference between the action of SERMs in the uterus of postmenopausal women.     

Diabetes modulates differentially creatine kinase-specific activity responsiveness to estradiol-17beta and to raloxifene in rat organs

Diabetes mellitus increases the risk for CVD in women. While there is considerable evidence suggesting beneficial effects of estrogen on decreasing lipid peroxidation, atherosclerotic processes, and cardiovascular diseases, diabetes negates most estrogen protective effects as well as the skeletal protective effects of estrogens, which are not discernable in diabetic women. In the present study, we examined the in vivo effects of estradiol-17beta (E2), on creatine kinase (CK)-specific activity, in estrogen-responsive organs from healthy and diabetic rats. Healthy or diabetic (streptozotocin-induced) female rats were injected with either E2 (10-50 microg/rat) or raloxifene (Ral; 500-1,000 microg/rat). Twenty-four hours following the injection, animals were sacrificed; their organs removed and assayed for CK-specific activity. CK-specific activity in different organs [Left ventricle of heart (Lv), uterus (Ut), aorta (Ao), para uterine adipose tissue (Ad), epiphyseal cartilage (Ep), and diaphyseal bone (Di)] from healthy animals, was stimulated with increased doses of E2, with maximum at 20 microg/rat. Age-matched diabetic female rats exhibited a remarkable decreased response to E2 in all organs except Ut. In contrast, the response to Ral was not altered in diabetic rats. Similar results were observed in organs from ovariectomized female rats (Ovx), healthy or diabetic. These results support our previous in vitro findings, demonstrating that hyperglycemia decreases CK response to E2 but not to Ral in cultured human vascular and bone cells. In summary, diabetes mellitus decreases CK response to E2 but not that of Ral in skeletal and vascular tissues. The decreased response to E2 detected in organs derived from diabetic rats might be due to changes in nuclear and/or membrane estrogen receptors and/or other genomic and non-genomic pathways, as was shown in in vitro cellular models.     

DT56a (Femarelle), contrary to estradiol-17β, is effective in human derived female osteoblasts in hyperglycemic condition

We have reported previously, that female-derived cultured osteoblasts (hObs) responded to DT56a (Femarelle) measured by the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness and (3)[H] thymidine incorporation into DNA (DNA synthesis). Since the skeletal protective effects of estrogens are not discernable in hyperglycemic diabetic women, we sought to analyze the effect of estrogenic compounds on CK and DNA synthesis in hObs when grown in high glucose concentration (HG). Cells were grown either in normal glucose (NG) (4.5g/L; 22mM) or HG (9.0g/L; 44mM) for 7 days. HG increased constitutive CK but, the response of CK activity and DNA synthesis to estradiol-17β (E(2)) treatment was reduced. In contrary, DT56a was found to be active (as measured by CK activity and DNA synthesis) in both NG and HG. HG decreases the hormonal responsiveness and might block important effects of estrogenic compounds, most likely contributing to their decreased skeletal preserving properties in hyperglycemic women. In hObs from post-menopausal women grown in HG, ERs mRNA expressions were unchanged. On the other hand, in hObs from pre-menopausal women HG increased ERs mRNA expressions. Since DT56a unlike E(2) is active in HG environment as well as in normal glucose, it may be an effective bone restoring agent in diabetic post-menopausal women.     

Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifen on creatine kinase B activity in vitro and in vivo

We have compared the cell and tissue selective estrogenic and antiestrogenic activities of tamoxifen, raloxifene, ICI 164,384 and a permanently ionized derivative of tamoxifen — tamoxifen methiodide (TMI). This non-steroidal antiestrogen has limited ability to cross the blood brain barrier and is therefore less likely to cause the central nervous system disturbances caused by tamoxifen. We have used the stimulation of the specific activity of the “estrogen induced protein”, creatine kinase BB, as a response marker in bone, cartilage, uterine and adipose cells and in rat skeletal tissues, uterus and mesometrial adipose tissue. In vitro, TMI, tamoxifen and raloxifene mimicked the agonistic action of 17β-estradiol in ROS 17/2.8 rat osteogenic osteosarcoma, female calvaria, and SaOS₂ human osteoblast cells. In Ishikawa endometrial cancer cells, tamoxifen showed reduced agonistic effects and raloxifene showed no stimulation. However, as antagonists, tamoxifen and raloxifene were equally effective in Ishikawa or SaOS₂ cells. In immature rats, all four of the antiestrogens inhibited estrogen action in diaphysis, epiphysis, uterus and mesometrial adipose tissue; when administered alone, tamoxifen stimulated creatine kinase (CK) specific activity in all these tissues. Raloxifene and TMI, however, stimulated only the skeletal tissues and had no stimulatory effect in the uterus or mesometrial fat, and the pure antiestrogen ICI 164,384 showed no stimulatory effect in any of the tissues. The simultaneous injection of estrogen, plus an antiestrogen which acted as an agonist, resulted in lower CK activity than after injection of either agent alone. These differential effects, in vivo and in vitro, may point the way to a wider therapeutic choice of an appropriate antiestrogen which, although antagonizing E₂ action in mammary cancer, can still protect against osteoporosis and cardiovascular disease and not stimulate the uterus with its attendant undesirable changes, or interfere with the beneficial action of E₂ in the brain.     

Isoflavones and bone: animal and human evidence of efficacy

Previous reports of soy extracts and isoflavone-enriched preparations studied in animals and humans have found that these molecules, when given at appropriate doses, have positive effects on the skeleton, including improvements in bone mineral content (BMC) and bone mineral density (BMD). A reduction in fracture risk of human subjects has not yet been shown in a prospective trial. Isoflavones, which exist in significant amounts only in soybeans, exert estrogen-like effects in human bone cells because of their unique organic structures that are similar to that of estradiol. The discovery of the b isoform of the estrogen receptor (ER) suggests that the molecular regulation of bone remodeling by estrogens, or estrogen-like molecules, including isoflavones, is more complex than previously thought. Depending on the type of ER present in a particular tissue, isoflavones may act as weak estrogen agonists or as weak estrogen antagonists. For example, isoflavones act as weak estrogen agonists in osteoblasts, but in reproductive cells, such as in the breast and uterus, they behave as weak estrogen antagonists. Weak agonistic effects of the isoflavones include stimulation of osteoblast proliferation and differentiation and increasing the production of cytokines that may inhibit osteclastic activity. The selective beneficial effects of estrogen-like molecules in bone tissues, compared to the anti-estrogenic effects in cells of reproductive tissues, make isoflavones attractive for the promotion of bone health. Relatively greater values of BMC and BMD of Asian populations with high consumption of soy isoflavones throughout life, compared to those with lower intakes, indirectly support the skeletal benefits of this pattern of intake of these estrogen-like molecules.     

Belamcanda chinensis and the thereof purified tectorigenin have selective estrogen receptor modulator activities.

Belamcanda chinensis (BC) belongs to the family of iridaceae and the isoflavone tectorigenin has been isolated from the rhizome of this plant. Whether this isoflavone has estrogenic, possibly selective estrogen receptor modulator activities and if so, whether they are mediated via the estrogen receptor alpha or beta is unknown at present. Therefore, we performed binding studies with recombinant human ERalpha and ERbeta to show that tectorigenin binds to both receptor subtypes. In ERalpha-expressing MCF7 and ERbeta-expressing MDA-MB231 reporter gene transfected cells tectorigenin causes transactivation. When given intravenously to ovariectomized (ovx) rats, it inhibits pulsatile pituitary LH secretion. In postmenopausal women estrogen-unopposed LH pulses correlate with hot flushes. Therefore, suppression of pulsatile LH secretion may be beneficial in women suffering from hot flushes. Upon chronic application to ovx rats a BC extract containing 5% Belamcanda at a daily dose of 33 mg or 130 mg of the extract had no effect on uterine weight or on estrogen-regulated uterine gene expression while estrogenic effects in the bone, on bone mineral density of the metaphysis of the tibia could be established. Hence, tectorigenin may have antiosteoporotic effects also in postmenopausal women. Immunohistochemical staining of proliferating cell nuclear antigen--a proliferation marker--in the mammary gland did not indicate a mammotrophic effect of the tectorigenin-containing BC extract at both tested doses. In summary, tectorigenin or the B. chinensis extract containing tectorigenin had a strong hypothalamotropic and osteotropic effect but no effect in the uterus or the mammary gland. Therefore, tectorigenin may be in the future a clinically useful selective estrogen receptor modulator.     

Effects of raloxifene, a selective estrogen receptor modulator, on thymus, T cell reactivity, and inflammation in mice

Raloxifene is a selective estrogen receptor modulator approved for prevention of osteoporosis in postmenopausal women. It is selective by virtue of having estrogen agonistic effects in bone, vessels, and blood lipids, while it is antagonistic with mammary and uterine tissue. The aim of the study was to examine whether the raloxifene analogue LY117018 (LY) has estrogenic effects on the thymus, T cell responsiveness, and inflammation. Oophorectomized normal mice were treated with subcutaneous injections of equipotent antiosteoporotic doses of LY (3 mg/kg) and 17beta-estradiol (E2) (0.1 mg/kg) or vehicle as controls. Effects on thymus were studied by analyses of thymus weight, cellularity, and CD4 and CD8 phenotype expression and histology, while inflammation was determined as T-cell-mediated delayed-type hypersensitivity (DTH) and granulocyte-mediated footpad swelling. LY lacked the suppressive properties of E2 on DTH and granulocyte-mediated inflammation. Furthermore, LY induced only minor thymus atrophy compared with E2 and did not, in contrast to E2, alter the thymic CD4/CD8 phenotypes. These results clearly demonstrate that raloxifene principally lacks the modulatory effects of estrogen on T cell responsiveness and inflammation. Our data are discussed in the context of recent findings in estrogen receptor biology and also with respect to estrogen-mediated alteration of autoimmune rheumatic diseases.     

A novel carborane analog, BE360, with a carbon-containing polyhedral boron-cluster is a new selective estrogen receptor modulator for bone

Carboranes are a class of carbon-containing polyhedral boron-cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors. Estrogen deficiency results in marked bone loss due to increased osteoclastic bone resorption in females, but estrogen replacement therapy is not generally used for postmenopausal osteoporosis due to the risk of uterine cancer. We synthesized a novel carborane compound BE360 to clarify its anti-osteoporosis activity. BE360 showed a high binding affinity to estrogen receptors (ER), ERα and ERβ. In ovariectomized (OVX) mice, femoral bone volume was markedly reduced and BE360 dose-dependently restored bone loss in OVX mice. However, BE360 did not exhibit any estrogenic activity in the uterus. BE360 also restored bone loss in orchidectomized mice without androgenic action in the sex organs. Therefore, BE360 is a novel selective estrogen receptor modulator (SERM) that may offer a new therapy option for osteoporosis.     

Regulation of proliferation of rat cartilage and bone by sex steroid hormones

We have demonstrated previously that 17β-estradiol (E₂) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E₂ on bone is sex specific. E₂ is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E₂ stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E₂. The basal activity and response of CK changes with the varying endogenous levels of E₂ in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E₂ in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.     

Estradiol and raloxifene decrease the formation of multinucleate cells in human bone marrow cultures

Estrogen (E2) deficiency is responsible for increased bone turnover in the postmenopausal period, and it can be prevented by estrogen replacement therapy. The way estrogen acts on bone cells is not fully understood. Human bone marrow cell cultures may be a reliable model for studying the action of steroids on osteoclastogenesis in vitro. We examine the effects of estradiol and Raloxifene, a selective estrogen receptor modulator, on human primary bone marrow cells cultured for 15 days. 17beta-estradiol and Raloxifene significantly decreased the number of tartrate-resistant acid phosphatase multinucleate cells from osteoclast precursors on day 15. Estrogen receptor alpha (ER-alpha) mRNA was present in bone marrow mononuclear cells cultured for 5 days, but there was no estrogen receptor beta (ER-beta) mRNA, suggesting that this effect was mediated by ER-alpha. 15-day cultures no longer contained ER-alpha mRNA, suggesting that estrogen acts on early events of osteoclast differentiation. Finally, 10-8 M 17beta-estradiol has no effect on the release of IL-6 and IL-6-sr into the medium of marrow mononuclear cells cultured for 5 or 15 days. Osteoclast apoptosis was not affected by estradiol or Raloxifene after 15 days of culture under our conditions. In conclusion, we have shown that both estradiol and Raloxifene inhibit osteoclast differentiation in human bone marrow mononuclear cultures. The biological effect that can mimic in vivo differentiation could be mediated through ER-alpha.     

Estrogenic activity of B-fluorinated o-carborane-1,2-bisphenol synthesized via S(N)Ar reaction

We previously identified o-carborane bisphenol BE360 (4) as a selective estrogen receptor modulator (SERM), which ameliorated bone loss without inducing estrogenic action in uterus of OVX and ORX mice. Here, we synthesized a fluorinated derivative, B-fluorinated o-carborane bisphenol BE310 (5) by means of S(N)Ar reaction. Compound 5 was a partial ER agonist, like 4, with little change of ERα and ERβ selectivity as compared with 4. However, its agonistic activity was 40 times weaker than that of 4. Thus, 5 is a novel SERM candidate with potential for reduced estrogenic side effects, and in vivo evaluation as an anti-osteoporosis agent seems warranted.