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Dual-energy x-ray absorptiometry of raloxifene effects on the lumbar vertebrae and femora of ovariectomized rats.

Sato M, McClintock C, Kim J, Turner CH, Bryant HU, Magee D, Slemenda CW.

Department of Endocrine Research, Lilly Research Laboratories, Indianapolis, Indiana.

A new potential therapeutic agent for postmenopausal osteoporosis, raloxifene, previously known as keoxifene, was evaluated by x-ray densitometry and more traditional techniques in quantitating the short-term (4-5 weeks) effects of ovariectomy on bones from 6-month-old rats. A Hologic QDR 1000/W and, to a limited extent, a Lunar DPXL, was used to quantitate ovariectomy, estrogen replacement, and raloxifene effects on vertebrae, femora, and tibiae. Both instruments performed well with precisions of 1.6% (Hologic) and 0.9% (Lunar) for anesthetized rats, which improved to 0.4% (Hologic) and 0.5% (Lunar) when the same rats were frozen. The lumbar vertebrae L1-4 showed a 12% decrease in bone mineral density 4 weeks after ovariectomy, compared with a 9% decrease for femora. Tibiae were also examined, but edge-detection problems prevented reproducible analysis of this site in vivo. The decrease in bone mineral density postovariectomy, especially for femora, was found to include both an increase in the projected area and a slight but not significant decrease in the bone mineral content of L1-4 and femora. These changes in density parameters of femora were supported by a decrease in dry weight and volume and a marginal increase in the second moment of inertia I for the identical femora examined ex vivo. Examination of individual lumbar vertebrae L1-5 suggested that the bone mineral density of L3 changes most dramatically in response to ovariectomy, but present techniques lack the spatial resolution and precision to quantitate bone changes reliably in individual vertebrae. 17 beta-Estradiol administered at 100 micrograms/kg/day subcutaneously inhibited ovariectomy effects on L1-4 bone mineral density, femoral moment of inertia, dry weight, and volume and to a lesser extent, femoral bone mineral density. A nonsteroidal compound, raloxifene HCl, at 1 mg/kg/day per os, had bone effects and effects on body weight that were largely indistinguishable from those of 17 beta-estradiol; however, raloxifene did not produce the uterotrophic effects observed with estrogen. The half-maximal efficacious dose of raloxifene on L1-4 bone mineral density was between 0.1 and 1.0 mg/kg/day per os. These data show that dual-energy x-ray absorptiometry compares favorably with traditional methods in quantitating bone changes caused by ovariectomy in small rodents, that L1-4 is a more sensitive region than whole femora in evaluating the effect of estrogen deficiency on bone loss, and the raloxifene may have promise as a treatment for conditions characterized by excessive bone loss after ovariectomy.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8053401&dopt=Abstract raloxifene Evista



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The effects of raloxifene on tibia histomorphometry in ovariectomized rats.

Evans G, Bryant HU, Magee D, Sato M, Turner RT.

Department of Orthopedics, Mayo Clinic, Rochester, Minnesota 55905.

Tissue-specific estrogen agonists may be useful in protecting against osteoporosis and the increased risk of coronary heart disease in postmenopausal women with minimal undesired effects on reproductive tissues. The actions of the mixed estrogen agonist/antagonist raloxifene on selected estrogen target tissues were determined in ovariectomized (OVX) rats immediately postovariectomy. Five groups of 75-day-old Sprague-Dawley rats were studied; baseline controls, sham-operated controls, OVX controls, OVX animals treated with estrogen (0.1 mg 17 alpha-ethynyl estradiol/kg.day), and OVX animals treated with raloxifene (3 mg/kg.day). Fluorochrome labels were given on days 1, 28, and 34. The baseline controls were killed on day 2, and the remaining groups on day 35. Ovariectomy increased tibial longitudinal growth rate as well as measurements related to radial growth and cancellous bone turnover. Ovariectomy decreased cancellous bone area and uterine weight, and increased serum cholesterol, bone elongation, and radial bone growth. Estrogen treatment prevented these changes in OVX rats. Raloxifene prevented cancellous osteopenia as well as the changes in radial bone growth, bone resorption, and blood cholesterol, but was less effective in reducing cancellous bone formation and did not prevent uterine atrophy. These findings suggest that raloxifene is a target-specific, mixed estrogen agonist/antagonist. At the concentration studied, raloxifene had potent estrogenic activity on bone resorption and serum cholesterol, a lesser effect on bone formation, and minimal activity on uterine wet weight.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8156931&dopt=Abstract raloxifene Evista



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Endocrine and antiprostatic effects of raloxifene (LY156758) in the male rat.

Neubauer BL, Best KL, Clemens JA, Gates CA, Goode RL, Jones CD, Laughlin ME, Shaar CJ, Toomey RE, Hoover DM.

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285.

The benzothiophene anti-estrogen, raloxifene [LY156758; (6-hydroxy-2-(4-hydroxyphenyl) benzo(b)thien-3-yl)(4-(2-1-piperidinyl)ethoxy)phenyl methanone hydrochloride] has selective estrogen pharmacological antagonist activity in female rats. The present studies were done in the male rat to assess activity of raloxifene related to inhibition of prostatic growth and effects on the hypothalamic-pituitary-gonadal axis. Raloxifene did not compete for binding of the androgen, [3H]-methyltrienolone (R1881) in cytosolic extracts of ventral prostate. Similarly, the compound did not inhibit prostatic 5 alpha-reductase or testicular 17 alpha-hydroxy/C17,20-lyase activities. Raloxifene had no effect on the ventral prostatic uptake of [3H]-R1881 in vivo. Administration of estradiol to castrated male rats stimulated fourfold increases of in vitro ventral prostatic binding of [3H]-R1881. Raloxifene was devoid of agonist activity in castrated animals, because the compound had no stimulatory effect on prostatic androgen receptor binding activity. When raloxifene was coadministered with estradiol, the compound markedly antagonized the estrogen-induced increase of prostatic [3H]-R1881 binding, confirming its antiestrogenic properties in male rats. Serum prolactin was also elevated significantly (P < 0.05) with a single injection of raloxifene (20.0 mg/kg). In these same animals, serum FSH was significantly (P < 0.05) decreased by one dose (10.0 mg/kg) of the compound. Luteinizing hormone levels in castrated male rats were unaffected by raloxifene administration. Raloxifene treatment of castrated males significantly (P < 0.05) antagonized the stimulatory response of the ventral prostate (VP) to exogenous androgens in a dose-dependent manner. Raloxifene treatment of intact male rats for 14 and 28 days produced significant (P < 0.05) dose-dependent regression of the VP and seminal vesicles (SV). The VP regressive responses to raloxifene were associated with a decline in serum testosterone levels. Histological analysis of the VPs in raloxifene-treated rats was consistent with an androgen-deprived state. These findings support the contention that raloxifene is a pure estrogen antagonist and a physiological antagonist of androgen action in male rats. These pharmacological properties provide support for further structure-activity and mechanistic investigations with benzothiophenes in the medical management of prostatic neoplasia.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8234067&dopt=Abstract raloxifene Evista



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Raloxifene (LY139481 HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats.

Black LJ, Sato M, Rowley ER, Magee DE, Bekele A, Williams DC, Cullinan GJ, Bendele R, Kauffman RF, Bensch WR, et al.

Department of Skeletal Disease Research, Eli Lilly and Co., Indianapolis, Indiana 46285.

There is a medical need for an agent with the positive effects of estrogen on bone and the cardiovascular system, but without the negative effects on reproductive tissue. Raloxifene (LY139481 HCI) is a benzothiophene derivative that binds to the estrogen receptor and inhibits the effects of estrogen on the uterus. In an ovariectomized (OVX) rat model we investigated the effects of raloxifene on bone loss (induced by estrogen deficiency), serum lipids, and uterine tissue. After oral administration of raloxifene for 5 wk (0.1-10 mg/kg per d) to OVX rats, bone mineral density in the distal femur and proximal tibia was significantly greater than that observed in OVX controls (ED50 of 0.03-0.3 mg/kg). Serum cholesterol was lower in the raloxifene-treated animals, which had a minimal effective dose of 0.1 mg/kg and an approximate oral ED50 of 0.2 mg/kg. The effects of raloxifene on bone and serum cholesterol were comparable to those of a 0.1-mg/kg per d oral dose of ethynyl estradiol. Raloxifene diverged dramatically from estrogen in its lack of significant estrogenic effects on uterine tissue. Ethynyl estradiol produced a marked elevation in a number of uterine histologic parameters (e.g., epithelial cell height, stromal eosinophilia). These data suggest that raloxifene has promise as an agent with beneficial bone and cardiovascular effects in the absence of significant uterine effects.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8282823&dopt=Abstract raloxifene Evista



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Estrogen and raloxifene stimulate transforming growth factor-beta 3 gene expression in rat bone: a potential mechanism for estrogen- or raloxifene-mediated bone maintenance.

Yang NN, Bryant HU, Hardikar S, Sato M, Galvin RJ, Glasebrook AL, Termine JD.

Endocrine Research, Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46285, USA.

Estrogen or raloxifene (LY156758) prevent estrogen deficiency-induced bone loss in animals and humans. We demonstrated in the rat that a 22% reduction in bone mineral density generated by ovariectomy was associated with a 2-fold reduction of transforming growth factor-beta 3 (TGF beta 3) messenger RNA expression in the femur. Administration of 17 beta-estradiol or raloxifene to ovariectomized rats restored both bone mineral density and TGF beta 3 messenger RNA expression in the femur to levels measured in intact animals. In transient transfection assays, the promoter sequence from -38 to + 110 of the human TGF beta 3 gene, which contains no palindromic estrogen response element, was sufficient to mediate 17 beta-estradiol or raloxifene induced-reporter gene expression in presence of the estrogen receptor. Raloxifene activated TGF beta 3 promoter as a full agonist at nanomolar concentrations. In the same cellular system, raloxifene inhibited the estrogen response element-containing vitellogenin promoter expression as a pure estrogen antagonist. In two well characterized osteoclast differentiation models, TGF beta 3 significantly inhibited the differentiation and bone-resorptive activities of murine and avian osteoclasts. These findings suggest that regulation of TGF beta 3 gene expression by raloxifene or estrogen in bone may be an important target to mediate bone maintenance.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8612550&dopt=Abstract raloxifene Evista



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Raloxifene, tamoxifen, nafoxidine, or estrogen effects on reproductive and nonreproductive tissues in ovariectomized rats.

Sato M, Rippy MK, Bryant HU.

Department of Endocrine Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.

For the first time, four well-characterized compounds from four distinct chemical classes were directly compared for efficacy and potency in hone, uteri, lipids, and adipose tissues in an ovariectomized model with 6 month old rats. Five weeks of oral dosing confirmed that ethynyl estradiol, tamoxifen, and raloxifene are potent inhibitors of the loss in volumetric bone mineral density (BMD, mg/cc) induced by ovariectomy, as measured by computed tomography. In the metaphysis of distal femora from ovariectomized rats, analysis showed a significant 12-20% decrease (P< 0.01) in the BMD. Linear regression analysis was used to calculate half-maximal efficacious doses for ethynyl estradiol ED(50) =0.04mg /kg, which was threefold more potent than tamoxifen, which in turn was threefold more potent than raloxifene, which was more efficacious than nafoxidine. In the uterus, raloxifene had minimal effects on the endometrium and smaller effects on uterine eosinophil peroxidase activity than nafoxidine, tamoxifen, or estrogen, respectively. Estrogen was the most potent in reducing cholesterol levels in ovariectomized rats, whereas tamoxifen and nafoxidine were more effective than raloxifene in blocking gain in body weight. Distinct compounds had advantages in the management of bone, uterine, serum cholesterol, and adipose tissues after ovariectomy. The distinct pattern of pharmacological effects may be best understood in terms of their respective chemical structure, specifically estrogens, benzothiophenes (raloxifene), dihydronapthylenes (nafoxidine), and triphenylethylenes (tamoxifen). These data point to advantages of separate compounds in the management of bone, uterine, serum cholesterol, and adipose tissues after estrogen deficiency, and show that the benzothiophene raloxifene has potentially important advantages over estrogen, tamoxifen, or nafoxidine in the uterus.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8666168&dopt=Abstract raloxifene Evista