Le T. Duong

Cathepsin K Inhibition: A New Mechanism for the Treatment of Osteoporosis

Cathepsin K (CatK), a cysteine protease, is highly expressed by osteoclasts and very efficiently degrades type I collagen, the major component of the organic bone matrix. Robust genetic and pharmacological preclinical studies consistently demonstrate that CatK inhibition increases bone mass, improves bone microarchitecture and strength. Recent advances in the understanding of the molecular and cellular mechanisms involved in bone modeling and remodeling suggest that inhibition of CatK decreases bone resorption, but increases the number of cells of osteoclast lineage. This in turn maintains the signals for bone formation, and perhaps may even increase bone formation on some cortical surfaces. Several CatK inhibitors, including relacatib, balicatib, odanacatib and ONO-5334 had entered clinical development for metabolic bone disorders with increased bone resorption, such as postmenopausal osteoporosis. However, odanacatib (ODN) is the only candidate continuing in development. ODN is a highly selective oral CatK inhibitor dosed once-weekly in humans. In a Phase 2 clinical trial, postmenopausal women treated with ODN had sustained reductions of bone resorption markers, while bone formation markers returned to normal after an initial decline within the first 2xa0years on treatment. In turn areal bone mineral density increased continuously at both spine and hip for up to 5xa0years. ODN has also been demonstrated to improve bone mass in women with postmenopausal osteoporosis previously treated with alendronate and in men with osteoporosis. ODN is currently in a worldwide Phase 3 fracture outcome trial for the treatment of postmenopausal osteoporosis with interim results supporting its anti-fracture efficacy at the spine, hip and non-vertebral sites.

Effects of Odanacatib on bone mineralization density distribution in thoracic spine and femora of ovariectomized adult rhesus monkeys: a quantitative backscattered electron imaging study.

Odanacatib (ODN) has been developed as a selective inhibitor of cathepsin K, the major cysteine protease in osteoclasts. In adult rhesus monkeys, treatment with ODN prevents ovariectomy-induced bone loss in lumbar vertebrae and hip. In this study, we evaluate the effects of ODN on bone mineralization density distribution (BMDD) by quantitative backscattered electron imaging in vertebral spongiosa, distal femoral metaphyseal and cortical shaft from monkeys (aged 16–23xa0years), treated with vehicle (nxa0=xa05) or ODN (6xa0mg/kg, nxa0=xa04 or 30xa0mg/kg, nxa0=xa04, PO daily) for 21xa0months. Dual-energy X-ray absorptiometry was measured in a subset of distal femoral samples. In lumbar vertebrae there was a shift to higher mineralization in samples from ODN-treated groups, compared to vehicle: CaMean (+4xa0%), CaPeak (+3xa0%), CaWidth (−9xa0%), CaLow (−28xa0%) in the 6xa0mg/kg group and CaMean (+5.1xa0%, pxa0<xa00.023), CaPeak (+3.4xa0%, pxa0<xa00.046), CaWidth (−15.7xa0%, pxa0=xa00.06) and CaLow (−38.2xa0%, pxa0<xa00.034) in the 30xa0mg/kg group. In distal femoral metaphyseal cancellous bone, there was a clear tendency toward a dose-dependent increase in matrix mineralization, as in the spine. However, primary and osteonal bone of the distal cortical diaphyses showed no significant change in BMDD, whereas bone mineral density was significantly increased after treatment. In ovariectomized monkeys, this study shows that ODN treatment increased trabecular BMDD, consistent with its previously reported ability to reduce cancellous remodeling. Here, ODN also showed no changes in BMDD in cortical bone sites, consistent with its actions on maintaining endocortical and stimulating periosteal bone formation.

The bone resorption inhibitors odanacatib and alendronate affect post-osteoclastic events differently in ovariectomized rabbits.

Odanacatib (ODN) is a bone resorption inhibitor which differs from standard antiresorptives by its ability to reduce bone resorption without decreasing bone formation. What is the reason for this difference? In contrast with other antiresorptives, such as alendronate (ALN), ODN targets only the very last step of the resorption process. We hypothesize that ODN may therefore modify the remodeling events immediately following osteoclastic resorption. These events belong to the reversal phase and include recruitment of osteoblasts, which is critical for connecting bone resorption to formation. We performed a histomorphometric study of trabecular remodeling in vertebrae of estrogen-deficient rabbits treated or not with ODN or ALN, a model where ODN, but not ALN, was previously shown to preserve bone formation. In line with our hypothesis, we found that ODN treatment compared to ALN results in a shorter reversal phase, faster initiation of osteoid deposition on the eroded surfaces, and higher osteoblast recruitment. The latter is reflected by higher densities of mature bone forming osteoblasts and an increased subpopulation of cuboidal osteoblasts. Furthermore, we found an increase in the interface between osteoclasts and surrounding osteoblast-lineage cells. This increase is expected to favor the osteoclast–osteoblast interactions required for bone formation. Regarding bone resorption itself, we show that ODN, but not ALN, treatment results in shallower resorption lacunae, a geometry favoring bone stiffness. We conclude that, compared to standard antiresorptives, ODN shows distinctive effects on resorption geometry and on reversal phase activities which positively affect osteoblast recruitment and may therefore favor bone formation.

Effects of Parathyroid Hormone, Alendronate and Odanacatib on the mineralisation process in intracortical and endocortical Haversian bone of ovariectomized rabbits

Although cortical bone strength depends on optimal bone composition, the influences of standard therapeutic agents for osteoporosis on bone mineral accrual in cortical bone are not understood. This study compared effects on cortical bone composition of two current therapeutic approaches for osteoporosis: the anti-resorptive bisphosphonate alendronate (ALN), and anabolic intermittent parathyroid hormone (PTH). The experimental anti-resorptive cathepsin K inhibitor, odanacatib (ODN) which inhibits resorption without inhibiting bone formation, was also tested. To determine effects of these agents on Haversian remodeling and mineral accrual, we compared ALN (100μg/kg/2xweek), PTH(1-34) (15μg/kg, 5x/week) and ODN (7.5μM/day) administered for 10 months commencing 6 months after ovariectomy (OVX) in skeletally mature rabbits by histomorphometry. We used synchrotron-based Fourier-transform infrared microspectroscopy (sFTIRM), coupled to fluorochrome labelling, to measure maturation of the cortical matrix in situ at both endocortical and intracortical sites of bone formation. PTH and ODN, but not ALN, treatment increased bone toughness, and PTH treatment stimulated bone formation, not only on endocortical and periosteal bone, but also in intracortical pores. In Sham and OVX rabbits, normal matrix maturation was observed at both endocortical and intracortical sites including: mineral accrual (increasing mineral:matrix), carbonate substitution (carbonate:mineral) and collagen molecular compaction (amide I:II) in situ in endocortical and intracortical bone. ALN treatment reduced bone formation on these surfaces. In ALN-treated bone, while intracortical bone matured normally, endocortical bone did not show a significant increase in mineral:matrix. ODN treatment resulted in slower mineral accrual and limited carbonate substitution. While PTH-treatment did not modify matrix maturation in endocortical bone, the initial stages of mineral accrual were slower in intracortical bone. In conclusion, these three classes of therapy have differing effects on both bone formation, and the process of bone matrix maturation. ALN suppresses bone formation, and the normal process of matrix maturation in endocortical bone. ODN does not suppress bone formation, but limits mineral accrual. PTH stimulates bone formation, and the matrix formed matures normally in endocortical bone. The ability of PTH treatment to stimulate bone formation in intracortical bone may provide a novel additional mechanism by which PTH increases bone strength.

Differing Effects of Parathyroid Hormone, Alendronate, and Odanacatib on Bone Formation and on the Mineralization Process in Intracortical and Endocortical Bone of Ovariectomized Rabbits

Bone is formed by deposition of a collagen-containing matrix (osteoid) that hardens over time as mineral crystals accrue and are modified; this continues until bone remodeling renews that site. Pharmacological agents for osteoporosis differ in their effects on bone remodeling, and we hypothesized that they may differently modify bone mineral accrual. We, therefore, assessed newly formed bone in mature ovariectomized rabbits treated with the anti-resorptive bisphosphonate alendronate (ALN—100µxa0g/kg/2×/week), the anabolic parathyroid hormone (PTH (1–34)—15µxa0g/kg/5×/week), orxa0the experimental anti-resorptive odanacatib (ODN 7.5xa0µM/day), which suppresses bone resorption without suppressing bone formation. Treatments were administered for 10xa0months commencing 6xa0months after ovariectomy (OVX). Strength testing, histomorphometry, and synchrotron Fourier-transform infrared microspectroscopy were used to measure bone strength, bone formation, and mineral accrual, respectively, in newly formed endocortical and intracortical bone. In Sham and OVX endocortical and intracortical bone, three modifications occurred as the bone matrix aged: mineral accrual (increase in mineral:matrix ratio), carbonate substitution (increase in carbonate:mineral ratio), and collagen molecular compaction (decrease in amide I:II ratio). ALN suppressed bone formation but mineral accrued normally at those sites where bone formation occurred. PTH stimulated bone formation on endocortical, periosteal, and intracortical bone surfaces, but mineral accrual and carbonate substitution were suppressed, particularly in intracortical bone. ODN treatment did not suppress bone formation, but newly deposited endocortical bone matured more slowly with ODN, and ODN-treated intracortical bone had less carbonate substitution than controls. In conclusion, these agents differ in their effects on the bone matrix. While ALN suppresses bone formation, it does not modify bone mineral accrual in endocortical or intracortical bone. While ODN does not suppress bone formation, it slows matrix maturation. PTH stimulates modelling-based bone formation not only on endocortical and trabecular surfaces, but may also do so in intracortical bone; at this site, new bone deposited contains less mineral than normal.

The Impact of Estrogen Depletion and Drug Treatment on Type I Collagen Microstructure

alendronate (ALN) and cathepsin K inhibitors (CatKIs) on bone formation and resorption. 3 One animal group included sham-operated animals, OVX animals, and OVX animals that were treated with either ALN or CatKi. The second animal group included sham-operated animals, OVX animals, and OVX animals given estrogen replacement therapy (ERT). These experiments provide two biologically independent tests of the changes in Type I collagen structure occurring under OVX estrogen depletion conditions. In addition, the impact of ALN, a bis(phosphonate) drug currently used in the clinic, CatKi, an example of an inhibitor that has just completed phase III clinical trials, and ERT on the protein structure was also explored. For all samples, AFM images of cortical femur bone were obtained. A quantitative analysis of the AFM images of the micron level Type I collagen fibril organization indicates a significant decrease in fibrils appearing in ordered structures such as bundles and sheets (18%), and a concomitant increase in fibrils appearing in disordered structures, upon OVX treatment of 6- and 9-month old rabbits. ALN and ERT drug treatment reduce the decrease of fibrils observed ordered structures (12%) and CatKi provide an even greater degree of prevention resulting in only a 5% decrease. These changes in the microscale level structure of the protein are mirrored by change in both structural bone properties, as indicated by average stiffness, and materials bone properties, as indicated by average strain energy. These studies point to the importance of gaining a further understanding of how changes in Type I collagen protein structure are related to estrogen-related disease for purposes of optimizing diagnosis, treatment, and the understanding of the impact on bone quality and function.

Synthetic Signal Peptides of Parathyroid Hormone: Probes for Components of the Secretory Apparatus

Parathyroid hormone (PTH), like the vast majority of secreted proteins and peptide hormones, is initially biosynthesized as a larger precursor, preproPTH (1,2). These precursor proteins have a cleavable N-terminal extension termed the signal sequence or leader sequence (3,4). This sequence is only transiently associated with the secreted protein and is rapidly removed by an enzyme, signal peptidase (located on the lumenal face of the endoplasmic reticular membrane (ER)), once the initiation of secretion has been effected (3). It is generally assumed that secreted proteins all use the same secretory machinery to achieve passage through the ER, although their subsequent passage through the cellular secretory pathway may be different.