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—100µ g/kg/2×/week), the anabolic parathyroid hormone (PTH (1–34)—15µ g/kg/5×/week), or the experimental anti-resorptive odanacatib (ODN 7.5 µM/day), which suppresses bone resorption without suppressing bone formation. Treatments were administered for 10 months commencing 6 months 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.

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.

Two independent biological replicates of estrogen depletion were employed with differing drug treatment conditions. Data Set I consisted of 9-month-old New Zealand white female rabbits treated as follows: sham-operated (n = 11), ovariectomized (OVX; n = 12), OVX+200 mu g kg(-1) alendronate (ALN), 3 x a week for 27 weeks (n = 12) and OVX+10 mg kg(-1) Cathepsin-K inhibitor (CatKI) daily for 27 weeks. Data Set II consisted of 6-month-old New Zealand white female rabbits that were sham-operated (n = 12), OVX (n = 12) or OVX+0.05mg kg(-1) 17 beta-estradiol (ERT) 3 x a week for 13 weeks (n = 12). Samples from the cortical femur were polished and demineralized to make them suitable for atomic force microscopy (AFM) imaging. Type I collagen fibrils present in bundles or sheets, running parallel to each other, were combined into a class termed Parallel. Fibrils present outside of such structures, typically in images with an angular range of non-parallel fibrils, were combined into a class termed Oblique. The percentage of fibrils coded as Parallel for Sham animals in Data Sets I and II was 52% and 53%, respectively. The percentage of fibrils coded as Parallel for OVX animals in Data Sets I and II was 35% in both cases. ALN and ERT drug treatments reduced the change from 18 to 12%, whereas CatKI treatment reduced the change to 5%.

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ~6 and 15 weeks post-fracture in two separate studies using the unilateral transverse mid-ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre-treated for 3-4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice-weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ~6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment-related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN-treated rabbits at week-6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K-(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre- and post-osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post-surgery returned callus BMD to Veh. Peak load of ODN- or ALN-treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5 alpha-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens. (C) 2014 Elsevier Ltd. All rights reserved.

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.

ABSTRACT Treatment with the cathepsin K (CatK) inhibitor odanacatib (ODN) protects against bone loss and maintains normal biomechanical properties in the spine and hip of ovariectomized (OVX) preclinical models. Here, we characterized the effects of ODN on the dynamics of cortical modeling and remodeling, and dimension and strength of the central femur in adult OVX-rhesus monkeys. Animals were treated with vehicle or ODN (6 or 30 mg/kg, once per day [q.d., p.o.]) in prevention mode for 21 months. Calcein and tetracycline double-labeling were given at 12 and 21 months, and the femoral cross-sections were subjected to dynamic histomorphometric and cement line analyses. ODN treatment significantly increased periosteal and endocortical bone formation (BFR/BS), accompanied with an increase in endocortical mineralizing surface (102%, p < 0.01) with the 6 mg/kg dose. ODN at both doses reduced remodeling hemiosteon numbers by 51% and 66% (p < 0.05), respectively, and ODN 30 mg/kg numerically reduced activation frequency without affecting wall thickness. On the same endocortical surface, ODN increased all modeling-based parameters, while reducing intracortical remodeling, consistent with the observed no treatment effects on cortical porosity. ODN 30 mg/kg markedly increased cortical thickness (CtTh, p < 0.001) and reduced marrow area (p < 0.01). Lastly, ODN treatment increased femoral structural strength (p < 0.001). Peak load was positively correlated with the increases in bone mineral content (BMC) (r2 = 0.9057, p < 0.0001) and CtTh (r2 = 0.6866, p < 0.0001). Taken together, by reducing cortical remodeling-based and stimulating modeling-based bone formation, ODN significantly improved cortical dimension and strength in OVX monkeys. This novel mechanism of CatK inhibition in stimulating cortical formation suggests that ODN represents a novel therapeutic approach for the treatment of osteoporosis. © 2014 American Society for Bone and Mineral Research.

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.

BACKGROUND:Selective and reversible inhibitors of human Cathepsin K (CatK), including odanacatib (ODN), have been developed as potential therapeutics for the treatment of osteoporosis. Inhibitors of human CatK show significantly less potency for the rodent enzymes compared with that for the human or rabbit enzymes; thus the Schenk model in growing rabbit was developed as a screening assay for the in vivo activity of CatK inhibitors in blocking bone resorption.METHODS:In this study, the efficacy of the selective inhibitors L-833905, L-006235, L-873724, and L-1037536 (ODN) of human CatK in the rapidly growing rabbit 'Schenk' model (age seven weeks) was compared to vehicle, using the bisphosphonate, alendronate (ALN), as a positive control, to assess inhibition of bone resorption. An enzyme inhibition assay (EIA) and an in vitro bone resorption assay using rabbit osteoclasts on bovine cortical bone slices were performed to evaluate the potency of these CatK inhibitors. Bone mineral density of the distal femur (DFBMD) was measured after ten days of treatment using ex vivo DXA densitometry.RESULTS:Results of the EIA using rabbit CatK and the rabbit bone resorption assay showed that three of the four compounds (L-006235, L-873724, and ODN) had similar potencies in the reduction of collagen degradation. L-833905 appeared to be a weaker inhibitor of CatK. Taking into account the respective in vitro potencies and pharmacokinetic profiles via oral administration, the efficacy of these four CatK inhibitors was demonstrated in a dose-related manner in the growing rabbit. Significant increases in DFBMD in animals dosed with the CatK inhibitors compared to vehicle were seen.CONCLUSIONS:Efficacy of the CatK inhibitors in the Schenk rabbit correlated well with that in the in vitro rabbit bone resorption assay and in the ovariectomized rabbit model as previously published. Hence, these studies validated the rabbit Schenk assay as a rapid and reliable in vivo model for prioritizing human CatK inhibitors as potential therapeutic agents.

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once-weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen-deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6?mg/kg, or ODN 30?mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle-treated OVX monkeys. Treatment with ODN also led to dose-dependent increases in serum 1-CTP and maintained estrogen deficiencyelevated Trap-5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L1 to L4) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose-dependently increased L1 to L4 BMD by 7% in the 6?mg/kg group (p?<?0.05 versus OVX-vehicle) and 15% in the 30?mg/kg group (p?<?0.05 versus OVX-vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R?=?0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN-treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long-term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates. (c) 2012 American Society for Bone and Mineral Research

The objectives here were to evaluate the effects of odanacatib (ODN) at doses exceeding the clinical exposure on biomechanical properties of lumbar vertebrae (LV), hip and central femur (CF), and compare ODN to alendronate (ALN) on bone remodeling/modeling in ovariectomized (OVX) monkeys. Ten days post-surgery, animals were treated with vehicle (VEH), ODN-L (2 mg/kg/day, p.o.), ODN-H (8/4 mg/kg/day), or ALN (30 μg/kg/week, s.c.) for 20 months. An intact group was also included. ODN-L provided systemic exposures of 1.8-fold of clinical exposure. ODN-H started at 20-fold for 5.5 months, and then reduced to 7.8-fold of clinical exposure, compared to ALN at approximated clinical exposure. From cross sectional analyses, LV density and peak load in ODN at both doses or ALN were not different from VEH or Intact. However, cortical thickness of femoral neck (FN) and CF in ODN were higher (21–34%, p < 0.05) than VEH, due to smaller endocortical (Ec) perimeter of FN (10–11%; p < 0.05) and CF (9–12%; ODN-L, p < 0.05), and larger CF periosteal (Ps) perimeter (2–12%; ODN-H, p < 0.001) versus VEH. ODN groups also showed slightly higher cortical porosity and Ps non-lamellar bone in CF. ODN-H treatment resulted in higher CF peak load (p < 0.05) versus VEH. For all bone sites analyzed, a positive, linear relationship (r2 = 0.46–0.69, p < 0.0001) of peak load to density or structural parameters was demonstrated. No treatment-related differences in the derived intrinsic strength properties were evidenced as compared between groups. ALN reduced all remodeling surfaces without affecting Ps modeling. Trabecular and intracortical remodeling were reduced in ODN groups, similar to ALN. Ec mineralizing surface in ODN-H trended to be lower than VEH by month 20, but Ec bone formation indices in ODN groups generally were not different from VEH. Ps modeling in ODN groups was significantly higher than other treatment groups. This study overall demonstrated the bone safety profile of ODN and its unique mechanism on cortical bone supporting the clinical application for osteoporosis treatment.

Two cathepsin K inhibitors (CatKIs) were compared with alendronate (ALN) for their effects on bone resorption and formation in ovariectomized (OVX) rabbits. The OVX model was validated by demonstrating significant loss (9.8% to 12.8%) in lumbar vertebral bone mineral density (LV BMD) in rabbits at 13-weeks after surgery, which was prevented by estrogen or ALN. A potent CatKI, L-006235 (L-235), dosed at 10mg/kg per day for 27 weeks, significantly decreased LV BMD loss (p<.01) versus OVX-vehicle control. ALN reduced spine cancellous mineralizing surface by 70%, whereas L-235 had no effect. Similarly, endocortical bone-formation rate and the number of double-labeled Haversian canals in the femoral diaphysis were not affected by L-235. To confirm the sparing effects of CatKI on bone formation, odanacatib (ODN) was dosed in food to achieve steady-state exposures of 4 or 9mM/day in OVX rabbits for 27 weeks. ODN at both doses prevented LV BMD loss (p<.05 and p<.001, respectively) versus OVX-vehicle control to levels comparable with sham or ALN. ODN also dose-dependently increased BMD at the proximal femur, femoral neck, and trochanter. Similar to L-235, ODN did not reduce bone formation at any bone sites studied. The positive and highly correlative relationship of peak load to bone mineral content in the central femur and spine suggested that ODN treatment preserved normal biomechanical properties of relevant skeletal sites. Although CatKIs had similar efficacy to ALN in preventing bone loss in adult OVX rabbits, this novel class of antiresorptives differs from ALN by sparing bone formation, potentially via uncoupling bone formation from resorption. (C) 2011 American Society for Bone and Mineral Research.