Evista
The interaction of raloxifene and the active metabolite of the antiestrogen EM-800 (SC 5705) with the human estrogen receptor.

Schafer JI, Liu H, Tonetti DA, Jordan VC.

Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.

A naturally occurring mutation at amino acid 351 (D351Y) in the human estrogen receptor (ER) can change the pharmacology of antiestrogens. Raloxifene is converted from an antiestrogen to an estrogen, whereas the biological properties of the steroidal pure antiestrogen ICI 182,780 are not affected by the D351Y ER (Levenson, A. S., and Jordan, V. C. Cancer Res., 58: 1872-1875, 1998). We propose an assay system that can be used to classify antiestrogens by determining their ability to up-regulate transforming growth factor alpha (TGF-alpha) mRNA in MDA-MB-231 cells stably transfected with either wild-type or D351Y ER. The novel compound EM-800 and its active metabolite, EM-652, have been reported to be p.o. active nonsteroidal pure antiestrogens. Using the D351Y cell line, EM-652 is able to up-regulate TGF-alpha mRNA in a dose-dependent manner and to a similar extent as estradiol, whereas in the wild-type cell line, it acts as an antiestrogen. In addition, the pure antiestrogen ICI 182,780 is capable of inhibiting EM-652-induced TGF-alpha mRNA expression at the D351Y ER. In MCF-7 cells expressing wild-type ER, it has previously been shown that ICI 182,780 decreases ER only at the protein level. EM-652 treatment does not decrease ER protein levels to a similar extent as ICI 182,780 treatment, and, in addition, EM-652 has no effect on ER mRNA levels. In proliferation assays, EM-652 is as effective as raloxifene in inhibiting cell growth. From these studies, we conclude that the reason the pharmacology of EM-652 is similar to that of raloxifene is because they both fit the ER in the same manner, and their biology depends on an interaction of the antiestrogenic side chain with amino acid 351.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10485477&dopt=Abstract raloxifene Evista



Evista
Raloxifene analog LY 117018 effects on central and peripheral beta-endorphin.

Genazzani AR, Bernardi F, Stomati M, Rubino S, Giardina L, Luisi S, Monteleone P, Genazzani AD, Luisi M, Petraglia F.

Department of Reproductive Medicine and Child Development, University of Pisa, Italy.

Raloxifene is a selective estrogen receptor modulator with a benzothiophene structure, that exerts an estrogen-like action on some target tissues and an anti-estrogenic action on the uterus and breasts. A limited number of data are available on the effect of raloxifene on neuroendocrine function. Since beta-endorphin (beta-EP) is considered a marker of neuroendocrine function, the aim of the present study was to evaluate the effects of a 14 day treatment with a raloxifene analog, LY 117018, on beta-EP content in the hypothalamus, hippocampus, anterior and neuro-intermediate pituitary lobe, and in the plasma of fertile and ovariectomized (ovx) rats. The effect of LY 117018 in ovx rats was compared to that of 17 beta-estradiol. beta-EP contents were measured by a specific radioimmunoassay. While ovariectomy determined a significant decrease in beta-EP levels in the anterior and neurointermediate pituitary lobe and plasma (p < 0.01), no changes of beta-EP content in the hypothalamus and hippocampus were found. The administration of 17 beta-estradiol or LY 117018 in ovx rats significantly increased beta-EP concentration in the anterior and neurointermediate pituitary lobe, in the hypothalamus and plasma (p < 0.01), though they did not significantly modify hippocampal beta-EP content. When LY 117018 was administered together with 17 beta-estradiol in ovx animals, a clear anti-estrogenic effect in all organs and in plasma was observed, resulting in significantly lower beta-EP content with respect to the group treated with 17 beta-estradiol alone (p < 0.01). The chronic administration of LY 117018 in fertile rats significantly decreased beta-EP content in the anterior pituitary, hippocampus and plasma (p < 0.01), while it increased beta-EP hypothalamic content and did not change beta-EP content in the neurointermediate lobe. In conclusion, raloxifene analog LY 117018 has an estrogen-like action on neuroendocrine opiatergic pathways when administered alone in ovx rats, while it exerts an anti-estrogen effect in fertile or in ovx rats treated with 17 beta-estradiol.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10533160&dopt=Abstract raloxifene Evista



Evista
Raloxifene and estradiol benzoate both fully restore hippocampal choline acetyltransferase activity in ovariectomized rats.

Wu X, Glinn MA, Ostrowski NL, Su Y, Ni B, Cole HW, Bryant HU, Paul SM.

Neuroscience and Endocrine Divisions, Lilly Research Laboratories, Eli Lilly and Company, DC 0530, Lilly Corporate Center, Indianapolis, IN, USA.

Selective estrogen receptor modulators (SERMs) demonstrate tissue-specific estrogen receptor (ER) agonist or antagonist properties. Raloxifene, a prototypical SERM, has ER agonist properties in bone and on cholesterol metabolism but full antagonist properties in the uterus and breast. To characterize the ER agonist/antagonist profile of raloxifene in the brain, we have examined its effect on the activity of a known estrogen-responsive gene product, choline acetyltransferase (ChAT), in the hippocampus and other brain regions of 6-month-old ovariectomized (OVX) Sprague-Dawley rats. Three weeks post-ovariectomy, animals received estradiol benzoate (EB, 0.03 mg or 0.3 mg kg(-1) day(-1) for 3 or 10 days); raloxifene HCl (3.0 mg kg(-1) day(-1) for 3 or 10 days), tamoxifen (3.0 mg kg(-1) day(-1) for 10 days) or vehicle (20% CDX). As previously reported, ChAT activity decreased by approximately 20%-50% in the hippocampus of OVX compared with SHAM-operated control rats with no change in ChAT activity observed in the hypothalamus. Raloxifene or EB reversed the OVX-induced decrease in ChAT activity in the hippocampus but did not change ChAT activity in the hypothalamus. Animals that received combined EB (0.03 mg/kg) plus raloxifene (1 mg/kg) or tamoxifen alone (3.0 or 10 mg/kg) also showed increased hippocampal ChAT activity. Raloxifene failed to increase uterine weight and blocked the estrogen-induced increase in uterine weight, while another SERM, tamoxifen, increased uterine weight. These data demonstrate that raloxifene has estrogen-like properties on hippocampal ChAT activity in vivo, and suggest that benzothiophene SERMs may exert estrogen-like beneficial effects on cholinergic neurotransmission in brain without producing peripheral stimulation of breast or uterine tissue.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10564741&dopt=Abstract raloxifene Evista



Evista
Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

Williams B, Brooks AN, Aldridge TC, Pennie WD, Stephenson R, McArdle CA.

Division of Medicine, University of Bristol, Bristol, UK.

GnRH acts via phospholipase C (PLC) activating G-protein coupled receptors to stimulate secretion of gonadotrophins from gonadotrophs. These cells are also regulated by gonadal steroids, which act centrally to influence GnRH secretion, and peripherally to modulate GnRH action. We have shown that oestradiol can stimulate proliferation and modulate GnRH-stimulated [(3)H]inositol phosphate ([(3)H]IP(x)) accumulation (used as a measure of PLC activity) in a gonadotroph-derived cell line (alphaT3-1). Here we show that when alphaT3-1 cells were incubated in medium with 2% foetal calf serum (FCS), [(3)H]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory effect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and raloxifene on the proportion of cells in the S-phase of the cell cycle (as measured by flow cytometry of propidium iodide-labelled cells) and on oestrogen receptor activity (as measured by trans-activation of the oestrogen-response elements in the vitellogenin promoter) were quantified. In addition, RT-PCR revealed expression of alpha and beta (but not beta2) subtypes of oestrogen receptors. Thus, oestrogen is an essential mitogen for alphaT3-1 cells, its mitogenic effect is oestrogen receptor mediated and is associated with a marked alteration of cell cycle distribution, and the full extent of these effects are best revealed in the presence of raloxifene. Using this strategy, we found that cells cultured for 4 days with 10 nM raloxifene expressed GnRH receptors (K(d) for (125)I-buserelin 4.33 nM) and that their activation by GnRH caused a concentration-dependent increase in [(3)H]IP(x) (in cells labelled with [(3)H]inositol) and inositol 1,4,5 trisphophate (in unlabelled cells). Addition of 10 nM oestradiol (to overcome receptor blockade by raloxifene) reduced GnRH receptor number by 31% but increased maximal effects on [(3)H]IP(x) and Ins(1,4,5)P(3) approximately 4-fold. The effects of oestradiol on GnRH receptor number and signalling were not, however, mimicked by culture for 2 days in medium with 10% FCS and the S-phase blocker, thymidine (15 mM). This treatment increased the proportion of cells in the S-phase 2- to 3-fold but did not alter GnRH receptor number or signalling. Other treatments which altered cell cycle transition (hydroxyurea, colcemid, methotrexate) also failed to alter GnRH receptor number or signalling and no correlation was seen between GnRH receptor number or GnRH-stimulated [(3)H]IP(x) accumulation and the proportion of cells in the S-phase or G2/M-phases of the cell cycle. Thus, oestradiol has pronounced effects on GnRH signalling, proliferation and cell cycle distribution in alphaT3-1 cells, but these trophic effects do not underlie the modulation of GnRH signalling.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10607935&dopt=Abstract raloxifene Evista



Evista
Uterine effects of 3-year raloxifene therapy in postmenopausal women younger than age 60.

Cohen FJ, Watts S, Shah A, Akers R, Plouffe L Jr.

Lilly Research Laboratories, Eli Lilly and Co., Lilly Corporate Center, Indianapolis, Indiana 46285, USA.

OBJECTIVE: To assess the uterine effects of 3 years of therapy with raloxifene in healthy, postmenopausal women under age 60. METHODS: Integrated data from two identically designed, randomized, double-masked, placebo-controlled clinical trials were analyzed. Nine hundred sixty-nine healthy women with uteri (ages 45 through 60, 2 to 8 years postmenopausal) were assigned randomly to raloxifene 30, 60, or 150 mg per day, or an identical placebo for 3 years. Endometrial thickness was evaluated with transvaginal ultrasonography every 6 months for 2 years and again after 3 years. Further uterine evaluation, including endometrial sampling if necessary, was initiated for vaginal bleeding or findings of endometrial thickness greater than 5 mm. RESULTS: Endometrial thickness was unchanged by raloxifene and not significantly different from placebo at any time. One hundred seventy-two women had at least one episode of endometrial thickness greater than 5 mm or vaginal bleeding distributed equally among all groups. A total of 102 (10.5%) women underwent endometrial sampling at least once: 15 (1.5%) for vaginal bleeding, 78 (8.0%) for endometrial thickness greater than 5 mm, and nine (0.9%) for other reasons. There were no significant treatment differences in the proportion of women sampled, in the clinical findings, or in the histologic diagnoses. CONCLUSION: Raloxifene given to healthy postmenopausal women at doses from 30 to 150 mg per day does not stimulate uterine growth and does not cause vaginal bleeding, spotting, or discharge through 3 years of therapy. Thus, any bleeding during therapy should be deemed unexpected and prompt a clinical evaluation.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10636511&dopt=Abstract raloxifene Evista



Evista
Raloxifene reduces atherosclerosis: studies of optimized raloxifene doses in ovariectomized, cholesterol-fed rabbits.

Bjarnason NH, Haarbo J, Byrjalsen I, Kauffman RF, Knadler MP, Christiansen C.

Centre for Clinical and Basic Research, Ballerup Byvej, Ballerup, Denmark.

OBJECTIVE: We have previously shown that raloxifene, a selective oestrogen receptor modulator, 35 mg/day inhibits atherosclerosis in ovariectomized, cholesterol-fed rabbits. This effect was only partial as compared to 17beta-oestradiol 4 mg/day; however, plasma raloxifene concentrations were low relative to those obtained in raloxifene-treated women. We therefore investigate the effects of raloxifene at higher doses. DESIGN: The study on atherosclerosis in ovariectomized, cholesterol-fed rabbits (n = 80) compared raloxifene 70 mg/day and 210 mg/day to 17beta-oestradiol 4 mg/day and placebo. RESULTS: After 48 weeks of therapy, the aortic cholesterol content in the 70 mg/day and 210 mg/day raloxifene treatment groups were 471 +/- 56 nmol/mg protein and 456 +/- 56 nmol/mg protein, respectively. This was significantly less than in the placebo group (654 +/- 69 nmol/mg protein; P < 0.05). In the oestrogen-treated group, the aortic cholesterol content was 357 +/- 62 nmol/mg protein (P < 0.01 as compared to placebo). Differences in serum lipids between the treatment groups could only partly explain the effect on aortic cholesterol content, indicating that additional anti-atherogenic mechanisms may contribute to the decrease in aortic atherosclerosis. This anti-atherosclerotic activity of raloxifene was observed at plasma concentrations comparable to those in postmenopausal women during raloxifene treatment. CONCLUSIONS: We conclude that clinically relevant raloxifene treatment inhibits aortic atherosclerosis in ovariectomized, cholesterol-fed rabbits.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10671951&dopt=Abstract raloxifene Evista