ObjectiveThe desired clinical profile of SERMs was for the treatment of menopausal symptoms and maintenance of bone mass, with no stimulatory effect on the uterus or mammary gland. To date, no SERM has achieved this goal. Although all SERMs function via estrogen receptors, differences in their chemical structures result in distinct pharmacologic profiles. These studies explored the preclinical uterine activation profiles of 3 SERMs: BZA, RLX, and LFX.DesignPreclinical studies in immature and mature ovariectomized (OVX) rats.Materials and methodsImmature and mature OVX rats were treated daily for 3 days or 6 weeks, respectively, with BZA, RLX, and LFX compared to saline control and ± ethinyl estradiol (EE). Uterine and endometrial endpoints were evaluated, including wet weight, endometrial epithelial cell height, and other histologic parameters.ResultsThe data reveal distinct differences between BZA, RLX, and LFX. In the highly sensitive immature rat model, the effect of BZA on uterine wet weight was less than that of RLX or LFX. Similarly, BZA more effectively antagonizes the stimulatory effect of EE on the uterus in comparison with RLX and LFX (rank order, BZA < RLX < LFX). BZA was the only SERM that completely reversed EE-induced uterine stimulation, as the maximal effect of RLX and LFX converged to their independent stimulatory level (rank order, BZA < RLX < LFX). These increases in wet weight occurred at very low doses, and leveled off with increased dose. The same rank order (BZA < RLX < LFX) of uterine stimulation was observed in the mature OVX rat model. Histologic evaluation demonstrated that BZA did not stimulate a significant increase in luminal epithelial cell height vs. control, whereas RLX and LFX stimulated 2- and 5-fold increases, respectively. The rates of myometrial hypertrophy and epithelial apoptosis correlated with rates of endometrial hypertrophy. When BZA was co-administered with RLX, BZA antagonized the stimulatory effect of RLX on these uterine endpoints.ConclusionsBZA demonstrated the lowest degree of uterine stimulation of the SERMs evaluated and results show how these molecules distinguish themselves on the presented endpoints. These data support the endometrial safety of BZA observed in phase III trials for the prevention and treatment of postmenopausal osteoporosis. ObjectiveThe desired clinical profile of SERMs was for the treatment of menopausal symptoms and maintenance of bone mass, with no stimulatory effect on the uterus or mammary gland. To date, no SERM has achieved this goal. Although all SERMs function via estrogen receptors, differences in their chemical structures result in distinct pharmacologic profiles. These studies explored the preclinical uterine activation profiles of 3 SERMs: BZA, RLX, and LFX. The desired clinical profile of SERMs was for the treatment of menopausal symptoms and maintenance of bone mass, with no stimulatory effect on the uterus or mammary gland. To date, no SERM has achieved this goal. Although all SERMs function via estrogen receptors, differences in their chemical structures result in distinct pharmacologic profiles. These studies explored the preclinical uterine activation profiles of 3 SERMs: BZA, RLX, and LFX. DesignPreclinical studies in immature and mature ovariectomized (OVX) rats. Preclinical studies in immature and mature ovariectomized (OVX) rats. Materials and methodsImmature and mature OVX rats were treated daily for 3 days or 6 weeks, respectively, with BZA, RLX, and LFX compared to saline control and ± ethinyl estradiol (EE). Uterine and endometrial endpoints were evaluated, including wet weight, endometrial epithelial cell height, and other histologic parameters. Immature and mature OVX rats were treated daily for 3 days or 6 weeks, respectively, with BZA, RLX, and LFX compared to saline control and ± ethinyl estradiol (EE). Uterine and endometrial endpoints were evaluated, including wet weight, endometrial epithelial cell height, and other histologic parameters. ResultsThe data reveal distinct differences between BZA, RLX, and LFX. In the highly sensitive immature rat model, the effect of BZA on uterine wet weight was less than that of RLX or LFX. Similarly, BZA more effectively antagonizes the stimulatory effect of EE on the uterus in comparison with RLX and LFX (rank order, BZA < RLX < LFX). BZA was the only SERM that completely reversed EE-induced uterine stimulation, as the maximal effect of RLX and LFX converged to their independent stimulatory level (rank order, BZA < RLX < LFX). These increases in wet weight occurred at very low doses, and leveled off with increased dose. The same rank order (BZA < RLX < LFX) of uterine stimulation was observed in the mature OVX rat model. Histologic evaluation demonstrated that BZA did not stimulate a significant increase in luminal epithelial cell height vs. control, whereas RLX and LFX stimulated 2- and 5-fold increases, respectively. The rates of myometrial hypertrophy and epithelial apoptosis correlated with rates of endometrial hypertrophy. When BZA was co-administered with RLX, BZA antagonized the stimulatory effect of RLX on these uterine endpoints. The data reveal distinct differences between BZA, RLX, and LFX. In the highly sensitive immature rat model, the effect of BZA on uterine wet weight was less than that of RLX or LFX. Similarly, BZA more effectively antagonizes the stimulatory effect of EE on the uterus in comparison with RLX and LFX (rank order, BZA < RLX < LFX). BZA was the only SERM that completely reversed EE-induced uterine stimulation, as the maximal effect of RLX and LFX converged to their independent stimulatory level (rank order, BZA < RLX < LFX). These increases in wet weight occurred at very low doses, and leveled off with increased dose. The same rank order (BZA < RLX < LFX) of uterine stimulation was observed in the mature OVX rat model. Histologic evaluation demonstrated that BZA did not stimulate a significant increase in luminal epithelial cell height vs. control, whereas RLX and LFX stimulated 2- and 5-fold increases, respectively. The rates of myometrial hypertrophy and epithelial apoptosis correlated with rates of endometrial hypertrophy. When BZA was co-administered with RLX, BZA antagonized the stimulatory effect of RLX on these uterine endpoints. ConclusionsBZA demonstrated the lowest degree of uterine stimulation of the SERMs evaluated and results show how these molecules distinguish themselves on the presented endpoints. These data support the endometrial safety of BZA observed in phase III trials for the prevention and treatment of postmenopausal osteoporosis. BZA demonstrated the lowest degree of uterine stimulation of the SERMs evaluated and results show how these molecules distinguish themselves on the presented endpoints. These data support the endometrial safety of BZA observed in phase III trials for the prevention and treatment of postmenopausal osteoporosis.

A unique mutation in LRP5 is associated with high bone mass in man. Transgenic mice expressing this LRP5 mutation have a similar phenotype with high bone mass and enhanced strength. These results underscore the importance of LRP5 in skeletal regulation and suggest targets for therapies for bone disease. A mutation (G171V) in the low-density lipoprotein receptor related protein 5 (LRP5) has been associated with high bone mass (HBM) in two independent human kindreds. To validate the role of the mutation, several lines of transgenic mice were created expressing either the human LRP5 G171V substitution or the wildtype LRP5 gene in bone. Volumetric bone mineral density (vBMD) analysis by pQCT showed dramatic increases in both total vBMD (30-55%) and trabecular vBMD (103-250%) of the distal femoral metaphysis and increased cortical size of the femoral diaphysis in mutant G171V transgenics at 5, 9, 17, 26, and 52 weeks of age (p < 0.01 for all). In addition, high-resolution microcomputed tomography (microCT) analysis of the distal femorae and lumbar vertebrae revealed an increase (110-232%) in trabecular bone volume fraction caused by both increased trabecular number (41-74%) and increased trabecular thickness (34-46%; p < 0.01 for all) in the mutant G171V mice. The increased bone mass was associated with significant increases in vertebral compressive strength (80-140%) and the increased cortical size with significant increases in femoral bending strength (50-130%). There were no differences in osteoclast number at 17 weeks of age. However, compared with littermate controls, the mutant G171V transgenic mice showed an increase in actively mineralizing bone surface, enhanced alkaline phosphatase staining in osteoblasts, and a significant reduction in the number of TUNEL-positive osteoblasts and osteocytes. These results suggest that the increased bone mineral density in mutant G171V mice was caused by increased numbers of active osteoblasts, which could in part be because of their increased functional lifespan. While slight bone anabolic activity was observed from overexpression of the wildtype LRP5 gene, it is clear that the G171V mutation, rather than overexpression of the receptor itself, is primarily responsible for the dramatic HBM bone effects. Together, these findings establish the importance of this novel and unexpected role of a lipoprotein receptor in regulating bone mass and afford a new model to explore LRP5 and its recent association with Wnt signaling in bone biology.

A series of pyrazolo[4,3-d]pyrimidine sulfonamides and pyrazolo[3,4-d]pyrimidine sulfonamides have been synthesized. These compounds increase transcription of a calcitonin-luciferase promoter and production of cellular calcitonin in a calcitonin-secretion/RIA assay with minimized phosphodiesterase type 4 inhibitory activity at 30 microM as compared to structurally related xanthine methylene ketones such as denbufyllene. These two series are notable examples of small molecules that act as CT-inducers, a method to potentially treat bone loss diseases.

A series of xanthine sulfonamides is presented as a class of calcitonin (CT) inducers - a potentially new method for treating diseases associated with postmenopausal bone loss such as osteoporosis. We have found that certain di-n-butylxanthine sulfonamides 4 upregulate CT transcription in a CT-luciferase reporter gene assay (CT-luci) and increase the production and release of CT in a CT secretion/RIA assay (CTS). In addition, these compounds do not have potent PDE4 inhibitory activity as do the related xanthine methylene ketones such as denbufyllene (2). One compound in particular (9) shows a transcription activation ratio (TAR) of 2.1 in CT-luci, a CTS increase of 3.6-fold, and a PDE4 (phosphodiesterase type IV) IC(50) = 4.1 microM. In addition, this compound showed a statistically significant 47% trabecular bone protection in ovariectomized-induced osteopenia (OVX) rats as determined by assay when administered for 4 weeks at 30 mg/kg/day, i. p. by quantitative computed tomography (QCT). When administered p.o., compound 9 shows 50% trabecular bone protection when administered for 3 weeks at 50 mg/kg/day, i.p. This compared with salmon CT which shows 62% trabecular bone protection when administered at 50 IU/kg/day for 4 weeks.

Immunosuppressants have adverse effects on bone mineral metabolism in animal and human studies, with corticosteroids producing low-turnover osteopenia, and cyclosporin-A (CsA) producing high-turnover osteopenia. Rapamycin (RAPA) is a new immunosuppressant reported to be at least 10 times more potent than CsA, and acts via a different pathway to CsA and the other new immunosuppressant FK506. This study investigated the effects of RAPA on bone mineral metabolism in the rat. Forty-two, 10-week-old, male Sprague Dawley rats were divided into three groups, and treated according to the following protocol: group A (control) received RAPA vehicle by daily gavage for 14 days (n = 12); group B (high dose RAPA) received RAPA 2.5 mg/kg/day by daily gavage for 14 days (n = 15); group C (low dose RAPA) received RAPA 1.25 mg/kg/day by daily gavage for 14 days (n = 15). Rats were weighed and bled on days 0, 7, and 14 for measurement of blood ionized calcium, bone Gla protein (BGP), parathyroid hormone (PTH), and 1,25(OH)2D. Tibial bone histomorphometry was determined on day 14 after double-calcein labeling. Weight gain was similar in the two groups treated with RAPA compared with control animals. High-dose RAPA (group B) transiently depressed serum BGP levels on day 7, with elevated blood ionized calcium levels on day 7, and lowered 1,25(OH)2D levels on day 14. Serum PTH levels were unchanged. Low dose RAPA (group C) did not affect calciotropic hormones. Histomorphometric analyses of tibial metaphyses revealed that parameters of bone formation and resorption were not significantly different in the groups treated with RAPA (group B and C) compared with control animals (group A). Trabecular bone volume (BV/TV) in group B (high-dose RAPA) (15.39 ± 1.01%) and C (low-dose RAPA) (15.38 ±0.57%) was not significantly altered compared with group A (control) (16.42 ± 0.86%). Short-term treatment with RAPA, unlike CsA, does not result in excess resorption and loss of bone volume. The depressed serum 1,25(OH)2D levels seen with high-dose RAPA therapy may adversely effect bone mineral metabolism in the long term.