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Evaluation of the major metabolites of raloxifene as modulators of tissue selectivity.

Dodge JA, Lugar CW, Cho S, Short LL, Sato M, Yang NN, Spangle LA, Martin MJ, Phillips DL, Glasebrook AL, Osborne JJ, Frolik CA, Bryant HU.

Endocrine Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, U.S.A.

Raloxifene (LY139481 HCl) is a selective estrogen receptor modulator (SERM) which blocks the effects of estrogen on some tissues, such as the breast and uterus, while mimicking estrogen in other tissues, such as bone. To study the origins of this unique pharmacology, we have prepared the major metabolites of raloxifene as chemical probes for examining the estrogen receptor function in vitro and in vivo. In human breast cancer cell (MCF-7) related assays, these glucuronide conjugates show little affinity for the estrogen receptor and are more than two orders of magnitude less potent at inhibiting cell proliferation than raloxifene. In non-traditional estrogen target tissue, such as bone, these metabolites are less effective than the parent at inhibiting cytokine-stimulated bone resorbing activity in rat osteoclasts or producing transforming growth factor beta-3 (TGF-beta3). In animal models, tissue distribution studies with radiolabelled metabolite indicate that conversion to raloxifene occurs readily in a variety of tissues including the liver, lung, spleen, kidney, bone and uterus. Differential conversion of metabolite in target organs, such as bone and the uterus, is not observed indicating that the origin of raloxifene's pharmacology does not result from tissue-selective deconjugation of metabolite to parent.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9328215&dopt=Abstract raloxifene Evista



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Characterization of the "estrogenicity" of tamoxifen and raloxifene in HepG2 cells: regulation of gene expression from an ERE controlled reporter vector versus regulation of the endogenous SHBG and PS2 genes.

Barkhem T, Andersson-Ross C, Hoglund M, Nilsson S.

Karo Bio AB, Novum, Huddinge, Sweden.

The estrogenic character of tamoxifen and raloxifene was studied on three different genes, an ERE-reporter construct and two endogenous genes, sex hormone binding globulin (SHBG) and pS2, in two variants of the human liver carcinoma cell line HepG2. On the ERE-reporter construct and the pS2 gene both tamoxifen and raloxifene acted as pure estrogen antagonists, whereas on the SHBG gene they functioned as partial estrogens/antiestrogens at concentrations below 1 microM and as full "agonists" at concentrations higher than 1 microM. The fold stimulatory effect of tamoxifen and raloxifene on SHBG protein expression was similar in the estrogen receptor (ER) expressing HepG2 cells (HepER3) and the parental non-ER expressing HepG2 cells at concentrations above 1 microM. In contrast, the 17beta-estradiol analogue moxestrol stimulated SHBG expression only in the HepER3 cells. Both tamoxifen and raloxifene had an additive effect to estrogen receptor-dependent SHBG gene expression in the HepER3 cells in the presence of saturating concentrations of moxestrol. However, a significant difference was observed in that a much higher concentration of moxestrol was required to see an additive effect of raloxifene compared to tamoxifen. The cytokine IL1-beta completely blocked the tamoxifen-dependent induction of SHBG gene expression in HepER3 cells, but only partly blocked the effect of moxestrol mediated by the ER. In conclusion, our results suggest that the mechanism for the liver-selective "estrogenic" character of tamoxifen and raloxifene is mediated by a non-ER dependent pathway.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9366498&dopt=Abstract raloxifene Evista



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Molecular determinants of tissue selectivity in estrogen receptor modulators.

Grese TA, Sluka JP, Bryant HU, Cullinan GJ, Glasebrook AL, Jones CD, Matsumoto K, Palkowitz AD, Sato M, Termine JD, Winter MA, Yang NN, Dodge JA.

Endocrine Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA. grese lilly.com

Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: (A) the presence of phenolic hydroxyls, (B) different substituents on the basic amine, (C) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and (D) the imposition of a carbonyl "hinge" between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of in vitro and in vivo assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9391160&dopt=Abstract raloxifene Evista



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Heterogeneity of binding sites and bioeffects of raloxifene on the human leukemic cell line FLG 29.1.

Fiorelli G, Martineti V, Gori F, Benvenuti S, Frediani U, Formigli L, Zecchi S, Brandi ML.

Department of Clinical Physiopathology, University of Florence, School of Medicine, Italy.

The benzothiophene divarative raloxifene is known to mimic estrogen in human bone remodeling. To investigate the "in vitro" properties of raloxifene on osteoclast precursors, the human leukemic cell line FLG 29.1, which differentiates toward the osteoclastic phenotype, was examined for raloxifene binding and for evidence of its bioeffects. Scatchard and Hill analysis of binding data with the tritiated raloxifene demonstrated the presence of two classes of binding sites in both nuclear and cytosol fractions with Kd values of approximately 1 nM and approximately 5 nM, respectively. In addition, analysis of binding data using tritiated 17 beta E2 as ligand at high concentrations (10-40 nM) and either unlabeled 17 beta E2 or raloxifene as competitors gave similar results demonstrating the presence of type II EBS in these cells. Picomolar concentrations of raloxifene significantly (p < 0.05) inhibited cell proliferation. Moreover, the compound at nanomolar concentrations induced a significant dose- and time-dependent increase of progesterone receptor, and activated apoptotic cell death. These findings clearly demonstrate that raloxifene acts as an estrogen agonist in FLG 29.1 cells, acting through the estrogen receptor and, possibly, via multiple cooperative binding component(s).

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9398606&dopt=Abstract raloxifene Evista



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Lack of effect of raloxifene on coronary artery atherosclerosis of postmenopausal monkeys.

Clarkson TB, Anthony MS, Jerome CP.

Comparative Medicine Clinical Research Center, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27157, USA. tclarkson cpm.bgsm.edu

Raloxifene has been shown to have estrogen agonist effects on bone and cholesterol metabolism while having estrogen antagonist effects on mammary gland and uterus. Reported here are the results of a study to determine whether raloxifene had the estrogen agonist effect of inhibiting coronary artery atherogenesis and to compare its effects with those of traditional conjugated equine estrogens (CEE) treatment. Ovariectomized (surgically postmenopausal) cynomolgus monkeys were fed a moderately atherogenic diet and treated with a placebo, raloxifene (1 mg/kg x day), raloxifene (5 mg/kg x day), or CEE (Premarin) at a dose that mimicked that of 0.625 mg/day in women. The effects of raloxifene on plasma lipid concentrations were generally comparable to those reported in postmenopausal women treated with raloxifene: reductions in low density lipoprotein cholesterol concentrations and no significant effects on high density lipoprotein cholesterol. We found no evidence that raloxifene had an estrogen agonist effect on coronary arteries. Treatment with CEE resulted in about a 70% reduction in coronary artery plaque size relative to that in the placebo group, whereas neither the low nor the high dose of raloxifene had an effect on coronary artery plaque size. The low dose raloxifene group had about 2 times more atherosclerosis and the high dose group had about 3 times more atherosclerosis than the CEE group.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9506715&dopt=Abstract raloxifene Evista



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The key to the antiestrogenic mechanism of raloxifene is amino acid 351 (aspartate) in the estrogen receptor.

Levenson AS, Jordan VC.

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

The crystallization of the ligand-binding domain (LBD) of the estrogen receptor (ER) with 17beta-estradiol and raloxifene [A. M. Brzozowski et al., Nature (Lond.), 389: 753-758, 1997] now provides a molecular basis for the biological activity of complexes as either agonists or antagonists. It is well established that the critical structural feature of antiestrogens is a correctly positioned alkylaminoethoxy side chain. The X-ray crystallography clearly shows that the alkylaminoethoxy side chain of raloxifene causes a specific and inappropriate molecular perturbation of the LBD and that the nitrogen in the side chain must hydrogen bond with aspartate 351 in the LBD of ER. We previously identified and characterized a naturally occurring mutation in the ER from a tamoxifen-stimulated transplantable human breast tumor line. The mutation is at AA351 of LBD, where the aspartate is changed to tyrosine (Asp351Tyr). In this report, we compared and contrasted the pharmacology of raloxifene to block or induce E2-stimulated increase in TGF-alpha mRNA in stable transfectants of ER-negative human breast cancer cells with the cDNAs from wild-type, mutant-amino acid (AA) 400 ER and mutant-AA 351 ER. Our results show that the mutation at AA 351 that replaces aspartate by tyrosine specifically changes the pharmacology of raloxifene from an antiestrogen to an estrogen. By contrast, a mutation at AA 400 does not, and the antiestrogenic properties of raloxifene are retained. These data and the fact that the nitrogen in the side chain must specifically interact with aspartate 351 makes this the key to the antiestrogenic activity of raloxifene.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9581827&dopt=Abstract raloxifene Evista