Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay

Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in α-MEM medium, supplemented with 10% of selected fetal calf serum, 1 μM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFκB ligand (RANKL, 50 ng/ml), and transforming growth factor-β1 (TGF-β1, 5 ng/ml). Thus, in addition to widely recognized osteoclast-generating factors M-CSF and RANKL, other medium supplements and lengthy culture times were necessary. This assay reliably detected inhibition of osteoclast formation (multinucleated cells positive for tartrate-resistant acid phosphatase) and activity (resorbed area and collagen fragments released from bone slices) in dose response curves with several classes of bone resorption inhibitors. Therefore, this assay can be applied for monitoring bone-resorbing activity of novel drugs and as an clinical test for determining the capacity of blood cells to generate bone-resorbing osteoclasts. Isolation of large quantities of active human osteoclast mRNA and protein is also made possible by this assay

ATF936, a novel oral calcilytic, increases bone mineral density in rats and transiently releases parathyroid hormone in humans.

Parathyroid hormone (PTH), when injected daily as either the intact hormone PTH(1-84) or the active fragment PTH(1-34) (teriparatide), is an efficacious bone anabolic treatment option for osteoporosis patients. Injections lead to rapid and transient spikes in hormone exposure levels, a profile which is a prerequisite to effectively form bone. Oral antagonists of the calcium-sensing receptor (calcilytics) stimulate PTH secretion and represent thus an alternative approach to elevate hormone levels transiently. We report here on ATF936, a novel calcilytic, which triggered rapid, transient spikes in endogenous PTH levels when given orally in single doses of 10 and 30mg/kg to growing rats, and of 1mg/kg to dogs. Eight weeks daily oral application of 30mg/kg of ATF936 to aged female rats induced in the proximal tibia metaphysis increases in bone mineral density, cancellous bone volume and cortical and trabecular thickness as evaluated by computed tomography. In healthy humans, single oral doses of ATF936 produced peak PTH levels in plasma after a median time of 1h and levels returned to normal at 24-h post-dose. The average maximum PTH concentration increase from baseline was 1.9, 3.6, and 6.0-fold at doses of 40, 70, and 140mg. ATF936 was well tolerated. The sharp, transient increase in PTH levels produced by the oral calcilytic ATF936 was comparable to the PTH profile observed after subcutaneous administration of teriparatide. In conclusion, ATF936 might hold potential as an oral bone-forming osteoporosis therapy

Penta-Substituted Benzimidazoles as Potent Antagonists of the Calcium Sensing Receptor (CaSR-Antagonists)

A series of novel benzimidazole derivatives has been designed via a scaffold morphing approach based on known calcilytics chemotypes. Subsequent lead optimisation led to the discovery of penta-substituted benzimidazoles that exhibit attractive in vitro and in vivo calcium sensing receptor (CaSR) inhibitory profiles. In addition, synthesis and structure activity relationship data are provided

1-Alkyl-4-phenyl-6-alkoxy-1H-quinazolin-2-ones: A Novel Series of Potent Calcium-Sensing Receptor Antagonists

Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when administered by daily s.c. injections exposure of short duration results, a pre-requisite for an anabolic response. Instead of applying exogenous PTH, mobilization of endogenous stores of the hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface. Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH release to the blood stream. The starting point for the Novartis calcilytics project was the proprietary structure 2a found by screening of the internal compound collection for inhibition of CaSR signaling function using a fluorimetric assay for intracellular calcium (FLIPR). The compound had an IC50 of 1.5 M and was an interesting starting point for lead optimization being a close analogue of Proquazone, an analgesic developed by Sandoz in the 1980s. Optimization of the series resulted in an increase in in vitro potency by a factor of >100. Key to potency improvement of 2a was the replacement of the 6-methoxy substituent by an propargyloxy moiety (7h). The part of the structure most tolerant to changes was the N(1) substituent. Compounds 10 with N-benzyl groups and especially the thio-analog 11k were among the most potent derivatives. The binding mode of our compounds was predicted based on molecular modeling and confirmed by testing with mutated receptors. While the compounds readily induced PTH release after i.v. application special formulation was needed for oral activity. The required profile was achieved by using micro-emulsions. Excellent PK/PD correlation was found in rats and dogs. High levels of PTH were reached in plasma within minutes which revert to baseline in about 1-2 hours in both species