Ellen R. Rowley

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.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. A series of raloxifene analogs which contain modifications to the 2-arylbenzothiophene core have been prepared and evaluated for the ability to bind to the estrogen receptor and inhibit MCF-7 breast cancer cell proliferation in vitro. Their ability to function as tissue-selective estrogen agonists in vivo has been assayed in a short-term, ovariectomized (OVX) rat model with end points of serum cholesterol lowering, uterine weight gain, and uterine eosinophil peroxidase activity. These studies have demonstrated that (1) the 6-hydroxy and, to a lesser extent, the 4'-hydroxy substituents of raloxifene are important for receptor binding and in vitro activity, (2) small, highly electronegative 4'-substituents such as hydroxy, fluoro, and chloro are preferred both in vitro and in vivo, (3) increased steric bulk at the 4'-position leads to increased uterine stimulation in vivo, and (4) additional substitution of the 2-aryl moiety is tolerated while additional substitution at the 4-, 5-, or 7-position of the benzothiophene results in reduced biological activity. In addition, compounds in which the 2-aryl group is replaced by alkyl, cycloalkyl, and naphthyl substituents maintain a profile of in vitro and in vivo biological activity qualitatively similar to that of raloxifene. Several novel structural variants including 2-cyclohexyl, 2-naphthyl, and 6-carbomethoxy analogs also demonstrated efficacy in preventing bone loss in a chronic OVX rat model of postmenopausal osteopenia, at doses of 0.1-10 mg/kg.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator (SERM) which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. In vivo structure-activity relationships and molecular modeling studies have indicated that the orientation of the basic amine-containing side chain of 1, relative to the stilbene plane, is an important discriminating factor for the maintenance of tissue selectivity. We have constructed a series of analogues of 1 in which this side chain is held in an orientation which is orthogonal to the stilbene plane, similar to the low-energy conformation predicted for raloxifene. Herein, we report on the synthesis of these compounds and on their activity in a series of in vitro and in vivo biological assays reflective of the SERM profile. In particular, we describe their ability to (1) bind the estrogen receptor, (2) antagonize estrogen-stimulated proliferation of MCF-7 cells in vitro, (3) stimulate TGF-beta3 gene expression in cell culture, (4) inhibit the uterine effects of ethynyl estradiol in immature rats, and (5) potently reduce serum cholesterol and protect against osteopenia in ovariectomized (OVX) rats without estrogen-like stimulation of uterine tissue. These data demonstrate that one of these compounds, LY357489,4, is among the most potent SERMs described to date with in vivo efficacy on bone and cholesterol metabolism in OVX rats at doses as low as 0.01 mg/kg/d.

Skeletal effects are described for near-lifetime treatment of young, female rats with recombinant human PTH (1-34) (PTH). Rats (5-8 wk of age) were administered 0, 5, 30, or 75 microg/kg x d sc PTH for up to 2 yr, as part of an oncogenicity evaluation, which is required by regulatory agencies for potential chronic therapies. Proliferative lesions were observed in the skeleton as described in Vahle et al. (1 ); in this paper, we describe the quantitative bone data for this study. In the appendicular skeleton, PTH stimulated trabecular and endocortical mineral apposition to the near exclusion of marrow spaces at 5 microg/kg, with some periosteal apposition at 30 microg/kg, followed by considerable periosteal apposition and altered geometry at 75 microg/kg. Increased bone mass was observed for all treatment groups that substantially exceeded normal levels attained by vehicle controls and exceeded skeletal efficacy reported previously for similar doses in shorter-term studies. Dose-dependent increases in osteocalcin levels and a linear increase in wet weight of femora were observed for the entire treatment duration, suggesting nearly continuous PTH stimulation of osteoblasts and skeletal growth throughout life. Histology showed many osteocytes and prominent osteoblasts, but a conspicuous absence of osteoclasts. Morphometry showed a lack of distinction between trabecular and cortical bone. Biomechanics of vehicle controls showed that optimal mechanical integrity for the normal skeleton is observed at about 11 months of age. PTH greatly strengthened and stiffened vertebra and femora; however, the midshaft showed reduced toughness and increased brittleness with treatment, which was not the case for vertebra. Related studies of 6 and 9 months duration showed that the optimal duration for PTH skeletal efficacy was about 6 months in rats, based on toughness, strength, ultimate displacement, and architecture, especially for cortical bone. Therefore, treatment duration is an under appreciated aspect of PTH pharmacology; and PTH skeletal effects are a complex function of dose and duration. Comparative analyses showed that short-term treatment (6 months or less) is more advantageous than near-lifetime treatment, because PTH stimulates skeletal growth throughout life, resulting in abnormal architecture and untoward biomechanical properties in rats.