An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation.

Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis.

Antagonists of the calcium receptor. 2. Amino alcohol-based parathyroid hormone secretagogues.

When administered as a single agent to rats, the previously reported calcium receptor antagonist 3 elicited a sustained elevation of plasma PTH resulting in no increase in overall bone mineral density. The lack of a bone building effect for analogue 3 was attributed to the large volume of distribution (V(dss)(rat) = 11 L/kg), producing a protracted plasma PTH profile. Incorporation of a carboxylic acid functionality into the amino alcohol template led to the identification of 12 with a lower volume of distribution (V(dss)(12) = 1.18 L/kg) and a shorter half-life. The zwitterionic nature of antagonist 12 necessitated the utility of an ester prodrug approach to increase overall permeability. Antagonist 12 elicited a rapid and transient increase in circulating levels of PTH following oral dosing of the ester prodrug 11 in the dog. The magnitude and duration of the increases in plasma levels of PTH would be expected to stimulate new bone formation.

Leucocyte cathepsin K affects atherosclerotic lesion composition and bone mineral density in low-density lipoprotein receptor deficient mice.

AIMS: Cathepsin K (CatK), an established drug target for osteoporosis, has been reported to be upregulated in atherosclerotic lesions. Due to its proteolytic activity, CatK may influence the atherosclerotic lesion composition and stability. In this study, we investigated the potential role of leucocyte CatK in atherosclerotic plaque remodelling. METHODS AND RESULTS: To assess the biological role of leucocyte CatK, we used the technique of bone marrow transplantation to selectively disrupt CatK in the haematopoietic system. Total bone marrow progenitor cells from CatK(+/+), CatK(+/-), and CatK(-/-) mice were transplanted into X-ray irradiated low-density lipoprotein receptor knockout (LDLr(-/-)) mice. The selective silencing of leucocyte CatK resulted in phenotypic changes in bone formation with an increased total bone mineral density in the CatK(-/-) chimeras and an effect of gene dosage. The absence of leucocyte CatK resulted in dramatically decreased collagen and increased macrophage content of the atherosclerotic lesions while lesion size was not affected. The atherosclerotic lesions also demonstrated less elastic lamina fragmentation and a significant increase in the apoptotic and necrotic area in plaques of mice transplanted with CatK(-/-) bone marrow. CONCLUSION: Leucocyte CatK is an important determinant of atherosclerotic plaque composition, vulnerability, and bone remodelling, rendering CatK an attractive target for pharmaceutical modulation in atherosclerosis and osteoporosis.

Intermittent treatment with parathyroid hormone (PTH) as well as a non-peptide small molecule agonist of the PTH1 receptor inhibits adipocyte differentiation in human bone marrow stromal cells.

Whereas continuous PTH infusion increases bone resorption and bone loss, intermittent PTH treatment stimulates bone formation, in part, via reactivation of quiescent bone surfaces and reducing osteoblast apoptosis. We investigated the possibility that intermittent and continuous PTH treatment also differentially regulates osteogenic and adipocytic lineage commitment of bone marrow stromal progenitor/mesenchymal stem cells (MSC). The MSC were cultured under mildly adipogenic conditions in medium supplemented with dexamethasone, insulin, isobutyl-methylxanthine and troglitazone (DIIT), and treated with 50 nM human PTH(1-34) for either 1 h/day or continuously (PTH replenished every 48 h). After 6 days, cells treated with PTH for 1 h/day retained their normal fibroblastic appearance whereas those treated continuously adopted a polygonal, irregular morphology. After 12-18 days numerous lipid vacuole and oil red O-positive adipocytes had developed in cultures treated with DIIT alone, or with DIIT and continuous PTH. In contrast, adipocyte number was reduced and alkaline phosphatase staining increased in the cultures treated with DIIT and 1 h/day PTH, indicating suppression of adipogenesis and possible promotion of early osteoblastic differentiation. Furthermore, intermittent but not continuous PTH treatment suppressed markers of differentiated adipocytes such as mRNA expression of lipoprotein lipase and PPARgamma as well as glycerol 3-phosphate dehydrogenase activity. All of these effects of intermittent PTH were also produced by a 1 h/day treatment with AH3960 (30 microM), a small molecule, non-peptide agonist of the PTH1 receptor. AH3960, like PTH, activates both the cAMP and calcium signaling pathways. Treatment with the adenylyl cyclase activator forskolin for 1 h/day, mimicked the anti-adipogenic effect of intermittent PTH, whereas pretreatment with the protein kinase-A inhibitor H89 prior to intermittent PTH resulted in almost complete conversion to adipocytes. In contrast, the MAP kinase inhibitor PD 98059 failed to prevent the anti-adipocytic effect of intermittent PTH, suggesting that the inhibitory effect of PTH on adipocyte differentiation is predominantly cAMP-dependent. These results demonstrate a differential effect of PTH1 receptor agonists on the adipocytic commitment and differentiation of adult human bone marrow mesenchymal stem cells. This response may represent an additional mechanism that contributes to the overall bone anabolic action of intermittent PTH.

Response to continuous and pulsatile PTH dosing: a mathematical model for parathyroid hormone receptor kinetics.

In this paper, we propose a mathematical model for parathyroid hormone receptor (PTH1R) kinetics, focusing on the receptor's response to PTH dosing to discern bone formation responses from bone resorption. The PTH1R is a major target for new osteoporosis treatments, as pulsatile PTH dosing has been shown to induce net bone formation in both animals and humans, and PTH(1-34) was recently FDA approved for the treatment of post-menopausal osteoporosis. PTH has also been shown to cause net bone loss when given continuously, so that the net action of PTH on bone is dependent on the dosing pattern. We have developed a simplified two-state receptor kinetics model for the PTH1R, based on the concepts of Segel et al., to distinguish the activity of active and inactive receptor and receptor-ligand complexes. The goal is to develop a plausible model of the minimal essential biological relationships necessary for understanding the responses to PTH dosing. A two-state model is able to effectively discriminate between continuous and pulsatile PTH dosing using the active species as surrogates for the downstream anabolic response. For continuous PTH dosing, the model predicts a desensitized system dominated by the inactive receptor and complex, consistent with downstream net bone loss that has been demonstrated experimentally. Using pulsatile PTH dosing, the model system predicts a highly sensitized state dominated by the active receptor and complex, corresponding to net bone formation. These results are consistent with the hypothesis that the kinetics of the receptor plays a critical role in the downstream effects of PTH dosing. Moreover, these results indicate that within a range of biologically relevant PTH doses, the two-state model is able to capture the differential behavior of the system for both continuous and pulsatile PTH dosing. The development of such a model provides a rational basis for developing more biologically extensive models that may support the design of optimal dosing strategies for PTH-based anti-osteoporosis treatments. Moreover, this model provides a unique starting point from which to design experiments investigating PTH receptor biology.

Rapid inhibition of thyroxine-induced bone resorption in the rat by an orally active vitronectin receptor antagonist.

An excess of thyroid hormone results in increased bone turnover and loss of bone mass in humans. Exogenous administration of thyroid hormone to rats has served as a model of human hyperthyroidism in which antiresorptive therapies have been tested. We have further refined this model of thyroxine (T4)-induced turnover in the rat. Daily administration of T4 to aged rats for as short as 1 week resulted in elevated bone resorption determined by significantly higher urinary deoxypyridinoline (Dpd) compared with vehicle controls or animals receiving T4 plus estradiol. Three weeks of daily administration of T4 led to significantly lower bone mineral density compared with untreated controls or animals receiving T4 plus estradiol. In a follow-up study, a depot formulation of T4 caused an increase in Dpd identical to that achieved with a bolus dose. SB-273005 [(4S)-2,3,4,5-tetrahydro-8-[2-[6-(methylamino)-2-pyridinyl] ethoxy]-3-oxo-2-(2,2,2-trifluoroethyl)-1H-2-benzazepine-4- acetic acid] a potent antagonist of the integrins alpha(v)beta(3) and alpha(v)beta(5), has been shown previously to inhibit bone resorption in cultures of human osteoclasts and to protect bone in ovariectomized rats. The effect of SB-273005 by oral administration was evaluated in this thyroxine-induced turnover model. Dose-dependent inhibition of resorption was seen with SB-273005 after 7 days of dosing using Dpd as a measure of bone resorption. In summary, it has been demonstrated that the antiresorptive activity of a vitronectin receptor antagonist can be measured after only 7 days of treatment in this refined rat model of thyroxine-induced bone turnover. These data suggest that SB-273005 may be useful for the treatment of metabolic bone diseases, including those resulting from hyperthyroidism.