Jeremy N. Bradbeer

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type‐selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast‐mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast‐mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration‐dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast‐mediated assay in vitro. Cbz‐Leu‐Leu‐Leu‐H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)‐stimulated resorption in the FRLB assay with an IC‐50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC‐50 of 100 nM. In the adjuvant‐arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz‐Leu‐Leu‐Leu‐H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz‐Leu‐Leu‐Leu‐H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast‐mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

A selective inhibitor of the osteoclastic V-H+-ATPase prevents bone loss in both thyroparathyroidectomized and ovariectomized rats

A potent and selective inhibitor of the osteoclastic V-H(+)-ATPase, (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6, 6-pentamethylpiperidin-4-yl)-2,4-pentadienamide (SB 242784), was evaluated in two animal models of bone resorption. SB 242784 completely prevented retinoid-induced hypercalcemia in thyroparathyroidectomized (TPTX) rats when administered orally at 10 mg/kg. SB 242784 was highly efficacious in the prevention of ovariectomy-induced bone loss in the rat when administered orally for 6 months at 10 mg/kg/d and was partially effective at 5 mg/kg/d. Its activity was demonstrated by measurement of bone mineral density (BMD), biochemical markers of bone resorption, and histomorphometry. SB 242784 was at least as effective in preventing bone loss as an optimal dose of estrogen. There were no adverse effects of compound administration and no effects on kidney function or urinary acidity. Selectivity of the inhibitor was further studied using an in situ cytochemical assay for bafilomycin-sensitive V-H(+)-ATPase using sections of osteoclastoma and numerous other tissues. SB 242784 inhibited the osteoclast enzyme at 1,000-fold lower concentrations than enzymes in any of the other tissues evaluated. SB 242784 demonstrates the utility of selective inhibition of the osteoclast V-H(+)-ATPase as a novel approach to the prevention of bone loss in humans.

Vis‐à‐Vis Cells and the Priming of Bone Formation

Bone formation throughout skeletal growth and remodeling always entails deposition of new bone onto a pre‐existing mineralized surface. In contrast, the initial deposition of bone in development requires the formation, ex novo, of the first mineralized structure in a nonmineralized tissue. We investigated the cellular events associated with this initial bone formation, with specific reference to the respective role of cartilage and bone cells in bones which form via a cartilage model. The cellular architecture of initial osteogenic sites was investigated by light, confocal, and electron microscopy (EM) in the membranous ossification of fetal calvarial bones (not forming via a cartilage model) and in the membranous ossification of the bony collars of endochondral bones. Bone sialoprotein (BSP), which is expressed during early phases of bone deposition and has been proposed to be involved in the control of both mineral formation and bone cell–matrix interactions, was used as a marker of initial bone formation. We found that at all sites, BSP‐producing cells (as identified by intracellular immunoreactivity) are arranged in a characteristic vis‐à‐vis (face to face) pattern prior to the appearance of the first mineralizing BSP‐immunoreactive extracellular matrix. In perichondral osteogenesis, the vis‐à‐vis pattern comprises osteoblasts differentiating from the perichondrium/periosteum and early hypertrophic chondrocytes located at the lateral aspects of the rudiment. By EM, the first mineral and the first BSP‐immunoreactive sites coincide temporally and spatially in the extracellular matrix at the boundary between cartilage and periosteum. We further showed that in an in vitro avian model of chondrocyte differentiation in vitro to osteoblast‐like cells, early hypertrophic chondrocytes replated as adherent cells turned on the expression of high levels of BSP in conjunction with the switch to collagen type I synthesis and matrix mineralization. We propose a model for the priming of bone deposition, i.e., the formation of the first bone structure, in which the architectural layout of cells competent to deposit a mineralizing matrix (the vis‐à‐vis pattern) determines the polarized deposition of bone. For bones forming via a cartilage model, the priming of bone deposition involves and requires cells that differentiate from early hypertrophic chondrocytes.

Orally bioavailable nonpeptide vitronectin receptor antagonists with efficacy in an osteoporosis model

A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.