Effects of 16-month treatment with the cathepsin K inhibitor ONO-5334 on bone markers,mineral density,strength and histomorphometry in ovariectomized cynomolgus monkeys

We examined the effects of ONO-5334, a cathepsin K inhibitor, on bone markers, BMD, strength and histomorphometry in ovariectomized (OVX) cynomolgus monkeys. ONO-5334 (1.2, 6 and 30 mg/kg/day, p.o.), alendronate (0.05 mg/kg/2 weeks, i.v.), or vehicle was administered to OVX monkeys (all groups N = 20) for 16 months. A concurrent Sham group (N = 20) was also treated with vehicle for 16 months. OVX significantly increased bone resorption and formation markers and decreased BMD in lumbar vertebra, femoral neck, proximal tibia and distal radius. Alendronate suppressed these parameters to a level similar to that in the Sham-operated monkeys. ONO-5334 at doses 6 and 30 mg/kg decreased bone resorption markers to a level roughly half of that in the Sham group, while keeping bone formation markers level above that in the Sham monkeys. Changes in DXA BMD confirmed that ONO-5334 at doses 6 and 30 mg/kg increased BMD to a level greater than that in the Sham group in all examined sites. In the proximal tibia, in vivo pQCT analysis showed that ONO-5334 at doses 6 and 30 mg/kg suppressed trabecular BMD loss to the sham level. However, ONO-5334 increased cortical BMD, cortical area and cortical thickness to a level greater than that in the Sham group, suggesting that ONO-5334 improves both cortical BMD and cortical geometry. Histomorphometric analysis revealed that ONO-5334 suppressed bone formation rate (BFR) at osteonal site in the midshaft femur but did not influence OVX-induced increase in BFR at either the periosteal or endocortical surfaces. Unlike alendronate, ONO-5334 increased osteoclasts surface (Oc.S/BS) and serum tartrate-resistant acid phosphatise 5b (TRAP5b) activity, highlighting the difference in the mode of action between these two drugs. Our results suggest that ONO-5334 has therapeutic potential not only in vertebral bones, but also in non-vertebral bones.     

Effects of ONO-5334, a novel orally-active inhibitor of cathepsin K, on bone metabolism

In the present study, we examined the in vitro and in vivo pharmacological effects of ONO-5334, a novel inhibitor of cathepsin K, on bone metabolism. In vitro experiments indicated that ONO-5334 is a potent inhibitor of cathepsin K with Ki value of 0.1 nM. Although this compound inhibited other cysteine proteases, such as cathepsin S, L and B, its inhibitory activity for these enzymes was 8 to 320 fold lower than that for cathepsin K. ONO-5334 also inhibited human osteoclasts bone resorption in vitro at a concentration more than 100 fold lower than that of alendronate, a bisphosphonate. While alendronate disrupted actin ring and induced pyknotic nuclei in osteoclasts, ONO-5334 did not have such effects, suggesting that this compound does not affect osteoclasts viability. In in vivo experiments, oral administration of ONO-5334 dose-dependently reduced plasma calcium level increased by parathyroid hormone related peptide in thyroparathyroidectomized rats. Furthermore, in vivo experiment using normal monkeys demonstrated that ONO-5334 decreases serum and urine C-telopeptide of type I collagen level, a bone resorption marker, soon after oral dosing. These levels were consistently decreased below pre-dose levels by repeated oral dosing with ONO-5334 for 7 days. ONO-5334 on the other hand did not affect bone formation markers, serum osteocalcin and bone specific alkaline phosphatase. These findings indicate that ONO-5334 is a specific inhibitor for cathepsin K and thus may be a novel therapeutic agent for metabolic bone diseases.     

ONO-5334, a cathepsin K inhibitor,improves bone strength by preferentially increasing cortical bone mass in ovariectomized rats

This study compared the effects of ONO-5334, a cathepsin K inhibitor, with those of alendronate on bone mass and strength in ovariectomized rats. Ovariectomy resulted in significant elevation in urinary deoxypyridinoline and plasma C-terminal cross-linking telopeptide of type I collagen (CTX) 8 weeks after surgery. Peripheral quantitative computed tomography analysis showed that total, trabecular, and cortical bone mineral content (BMC) decreased in the proximal tibia, which was paralleled with a significant decline in bone strength. Treatment with ONO-5334 (0.12, 0.6, 3 or 15 mg/kg) once daily for 8 weeks dose-dependently restored the decrease in total BMC and bone mineral density (BMD) in the proximal tibia and suppressed urinary deoxypyridinoline and plasma CTX levels. Alendronate (1 mg/kg, once daily) also fully restored these bone mass parameters. Separate analysis of trabecular and cortical bones, however, showed that ONO-5334 only partially restored trabecular BMD and BMC at 15 mg/kg, whereas alendronate fully restored these parameters. On the other hand, ONO-5334 increased both cortical BMD and BMC with an effect more potent than that of alendronate. Bone geometric analysis indicated that ONO-5334 at 15 mg/kg decreased endosteal circumference without affecting periosteal circumference, resulting in marked increase in cortical thickness. Interestingly, the effects of ONO-5334 on bone strength parameters were more prominent than those of alendronate, although the two test compounds had a similar effect on total BMC. Taken together, our results indicate that ONO-5334 has pharmacological characteristics different from those of alendronate and may offer a unique therapy for patients with osteoporosis.     

Effects of novel cathepsin K inhibitor ONO-5334 on bone resorption markers: a study of four sustained release formulations with different pharmacokinetic patterns

The purpose of the study was clarify the effect of the cathepsin K inhibitor ONO-5334 on bone resortion markers using sustained release (SR) formulations with different pharmacokinetic (PK) patterns, and identify the optimal SR formulation. The PK profiles and pharmacodynamic effect on bone resorption markers of 4 SR candidates formulations were evaluated in healthy postmenopausal women within a randomized, 2-part, open-label crossover study. In Part A, subject received a single dose of each formulation orally in the fed state. In Part B, two selected formulations were evaluated in the fasted state. From the results from Part A, C _max was reduced and plasma concentrations of ONO-5334 were sustained with all SR formulations compared with an immediate release tablet. In pharmacodynamics, the level of C-terminal telopeptide of type I collagen (CTX) in serum and urine were inhibited with SR tablets rather than with granules. C _max and area under the concentration–time curve from time 0 to the last measurable time point (AUC_0?t ) of SR tablets were higher than those of granules. From Part B, C _max in the fasted condition was lower than that in the fed condition with two SR tablets. In contrast, C _{24 h} in the fasted condition was slightly higher than that in the fed condition, but AUC_0?t was similar. The inhibitory effect on CTX in serum and urine may depend on the PK pattern of ONO-5334. The SR tablets was well tolerated in postmenopausal women and has the optimal SR profiles on pharmacodynamics effect on bone resortion markers and PK profile. These results suggest that SR tablets of ONO-5334 are an excellent drug candidate for osteoporosis.     

Balicatib, a cathepsin K inhibitor, stimulates periosteal bone formation in monkeys

Summary  

Balicatib, an inhibitor of the osteoclastic enzyme cathepsin K, was tested in ovariectomized monkeys, a model for osteoporosis. As expected, ovariectomy-induced bone mass changes were partially prevented by balicatib treatment. Bone turnover was significantly decreased at most sites, but unlike most bone resorption inhibitors, periosteal bone formation rates were increased.     

Minodronic acid (ONO-5920/YM529) prevents decrease in bone mineral density and bone strength, and improves bone microarchitecture in ovariectomized cynomolgus monkeys

This study examined the effect of the highly potent nitrogen-containing bisphosphonate, minodronic acid (ONO-5920/YM529), on bone mineral density (BMD), bone turnover, bone microarchitecture and bone strength in ovariectomized (OVX) cynomolgus monkeys. Skeletally mature female cynomolgus monkeys, aged 9-17 years, were ovariectomized or sham-operated. Minodronic acid was administered orally once a day in doses of 0, 0.015, and 0.15 mg/kg from the day after surgery for 17 months. Bone resorption markers (urinary N-terminal cross-linking telopeptide of type I collagen and deoxypyridinoline), bone formation markers (serum osteocalcin and bone alkaline phosphatase) and lumbar vertebral BMD were measured at baseline and at 4, 8, 12 and 16 months after surgery. Treatment with minodronic acid dose-dependently inhibited OVX-induced increase in bone turnover markers and decrease in lumbar vertebral BMD, and minodronic acid at 0.15 mg/kg completely prevented these changes. At 17 months after surgery, minodronic acid also suppressed bone resorption (Oc.S/BS and N.Oc/BS) and bone formation (OS/BS, MS/BS, MAR, BFR/BS, and BFR/BV) in the lumbar vertebral bodies and tibia. In the mechanical tests, ultimate load on lumbar vertebral bodies and femoral neck of the OVX-control animals were significantly reduced compared to the sham animals. Minodronic acid prevented these reductions in bone strength at 0.15 mg/kg. There was significant correlation between BMD and bone strength, suggesting that the increase in bone strength was associated with the increase in BMD produced by minodronic acid. In micro-CT analysis of the lumbar vertebral bodies, minodronic acid improved trabecular architecture, converting rod structures into plate structures, and preventing the increase in trabecular disconnectivity at 0.15 mg/kg. In conclusion, similar to patients with postmenopausal osteoporosis, reduction in bone strength of lumbar vertebral bodies and femoral neck was clearly demonstrated in OVX cynomolgus monkeys. Minodronic acid prevented these reductions at a once-daily oral administration. Also, minodronic acid prevented OVX-induced changes in bone turnover, bone mass and bone microarchitecture. Long-term minodronic acid treatment was well tolerated and no adverse effects could be detected. These results suggest that minodronic acid may be a clinically useful drug for osteoporosis.     

Effects of a prostaglandin EP4 agonist, ONO-4819, and risedronate on trabecular microstructure and bone strength in mature ovariectomized rats

The effects of a prostaglandin EP4 agonist, ONO-4819, and risedronate, a representative anti-resorptive drug, on trabecular microarchitecture and biomechanical properties were investigated in mature estrogen-deficient rats; and effects which affected microstructural components that contributed to the improvement of bone strength were also determined. Thirty-three-week-old OVX rats were treated with various doses of ONO-4819, risedronate, or their combination for 11 weeks. Bone mineral density (BMD), trabecular microstructure, and biomechanical strength were determined at the proximal tibia by peripheral quantitative CT, micro CT, and finite element analysis, respectively. Bone histomorphometry was performed at the same site. The results of trabecular structure analysis indicated that whereas risedronate functioned mainly in maintaining trabecular connectivity, ONO-4819 converted the fragile rod-like trabeculae caused by estrogen deficiency to a plate-like structure. In addition, ONO-4819 is one of the few drugs that are capable of increasing trabecular thickness. When the 2 drugs were combined, the beneficial effects of each drug on the trabecular microarchitecture were maintained, resulting in their additive effects on bone strength. The results of bone histomorphometry suggest that ONO-4819 caused an increase in the rate of bone formation by stimulating the differentiation/recruitment of osteoblasts as well as their mineralizing function. ONO-4819 did not stimulate bone resorption, but rather exerted an anti-resorptive function within a certain dose range. ONO-4819 and risedronate increased BMD and improved trabecular structure and biomechanical strength in an additive and independent manner. Thus, EP4 agonist ONO-4819 in combination with risedronate may be an effective treatment for osteoporosis.     

Balicatib, a cathepsin K inhibitor, stimulates periosteal bone formation in monkeys

Summary  

Balicatib, an inhibitor of the osteoclastic enzyme cathepsin K, was tested in ovariectomized monkeys, a model for osteoporosis. As expected, ovariectomy-induced bone mass changes were partially prevented by balicatib treatment. Bone turnover was significantly decreased at most sites, but unlike most bone resorption inhibitors, periosteal bone formation rates were increased.     

Minodronic acid ameliorates vertebral bone strength by increasing bone mineral density in 9-month treatment of ovariectomized cynomolgus monkeys

The effect of treatment for 9 months with minodronic acid, a nitrogen-containing bisphosphonate, on vertebral mechanical strength was examined in ovariectomized (OVX) cynomolgus monkeys. Forty skeletally mature female monkeys were randomized into four OVX groups and one sham group (n = 8) based on lumbar bone mineral density (BMD). OVX animals were treated orally with 15 and 150 μg/kg QD of minodronic acid or 500 μg/kg QD alendronate as a reference drug. Measurements of bone turnover markers and lumbar BMD were conducted at 0, 4 and 8 months. Measurements of bone mechanical strength and minodronic acid concentration in vertebral bodies were also performed. OVX resulted in a decrease in lumbar BMD and an increase in bone turnover markers at 4 and 8 months, compared to the sham group, and the ultimate load on the lumbar vertebra was decreased in OVX animals. Minodronic acid and alendronate prevented the OVX-induced increase in bone turnover markers and decrease in lumbar BMD. Minodronic acid at 150 μg/kg increased the ultimate load on lumbar vertebra compared to untreated OVX animals. Regression analysis revealed that the ultimate load was correlated with lumbar BMD and bone mineral content (BMC), and most strongly with the increase in lumbar BMD and BMC over 8 months. In a separate analysis within the sham-OVX controls and minodronic acid and alendronate treatment groups, the ultimate loads were also correlated with BMD and BMC. The load-BMD (BMC) correlation in the minodronic acid group showed a trend for a shift to a higher load from the basal relationship in the sham-OVX controls. These results indicate that treatment with minodronic acid for 9 months increases vertebral mechanical strength in OVX monkeys, mainly by increasing BMD and BMC.     

Decreased bone remodeling and porosity are associated with improved bone strength in ovariectomized cynomolgus monkeys treated with denosumab, a fully human RANKL antibody

This study examined the effects of denosumab, an anti-RANKL antibody that inhibits bone resorption, on bone histomorphometry in adult ovariectomized cynomolgus monkeys (OVX cynos). A month after surgery, OVX cynos were treated with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50mg/kg/month) for 16months (n=14-20/group). Sham controls were treated with vehicle (Sham-Veh; n=17). Areal and volumetric BMD, urine NTx, and serum osteocalcin were measured at baseline and months 3, 6, 12, and 16. Double fluorochrome labels were injected prior to iliac and rib biopsies at month 6 and month 12, and prior to sacrifice at month 16. Histomorphometry was performed on these biopsies, the tibial diaphysis, the L2 vertebra, and the proximal femur. Strength of humeral cortical beams, femur diaphysis, femur neck, and trabecular cores of L5-L6 vertebrae was determined by destructive biomechanical testing. There was no evidence of woven bone, osteomalacia, or other bone histopathologic changes with OVX or with denosumab. OVX-Veh animals exhibited significantly greater bone remodeling at all skeletal sites relative to Sham-Veh controls. Both doses of denosumab markedly inhibited bone remodeling at all sites, including significant reductions in trabecular eroded surfaces (48-86% lower than OVX-Veh controls), cortical porosity (28-72% lower), and dynamic parameters of bone formation (81-100% lower). Decreased fluorochrome labeling with denosumab was related to reductions in cortical porosity and trabecular eroded surfaces, and regression analyses suggested that these reductions contributed to denosumab-related increments in BMD and bone strength. Denosumab-treated animals with the lowest levels of fluorescent labeling exhibited the greatest structural bone strength values at each site. Intracortical remodeling had no relationship with material properties including ultimate strength, elastic modulus or toughness (r(2)=0.00-0.01). These data suggest that remodeling inhibition with denosumab improved structural strength without altering material properties under these experimental conditions. Greater structural strength in the denosumab-treated animals can be primarily explained by the combined effects of increased trabecular and cortical bone mass, and reductions in trabecular eroded surfaces and cortical porosity.     

An oral cathepsin K inhibitor ONO-5334 inhibits N-terminal and C-terminal collagen crosslinks in serum and urine at similar plasma concentrations in postmenopausal women

Relationships between the plasma concentration of a cathepsin K inhibitor (ONO-5334) and inhibition of bone resorption markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in serum and urinary NTX/creatinine and CTX/creatinine were examined in 10 postmenopausal women. The subjects received slow-release tablets of 100 mg ONO-5534 under fasted or fed conditions in a study with a crossover design. Inhibition of serum NTX and CTX levels and plasma concentrations of ONO-5334 were monitored at 0, 24, 48 and 168 h after dosing. Changes in urinary NTX/creatinine and CTX/creatinine levels in second morning urine were evaluated on 0, 1, 2 and 7 days after dosing. Data were analyzed using sigmoid maximal drug effect (E_max) models. The maximal inhibition, estimated E_max values, were − 31.8% for serum NTX, − 53.1% for serum CTX, − 67.2% for urinary NTX/creatinine, and − 95.2% for urinary CTX/creatinine. The estimated half maximal effective plasma concentrations (EC₅₀) of ONO-5334 and confidence intervals were 1.79 (1.01 to 3.16) ng/mL for serum NTX, 2.07 (1.63 to 2.62) ng/mL for serum CTX, 1.85 (1.30 to 2.61) ng/mL for urinary NTX/creatinine, and 1.98 (0.94 to 3.76) ng/mL for urinary CTX/creatinine. EC₅₀ values for the four crosslinks did not significantly differ, as indicated by the overlapping 95% confidence intervals. The highest signal-to-noise ratio was achieved with serum CTX, and was 2-fold higher than that on serum NTX. Inhibition for serum NTX and CTX, and urinary NTX/creatinine and CTX/creatinine by ONO-5334 were all correlated with correlation coefficients ranging from 0.55 to 0.80. In conclusion, data of ONO-5334 slow-releasing tablets in postmenopausal women were well fitted in E_max model. In all measured telopeptides, the maximal inhibition was obtained at urinary CTX/creatinine level, but serum CTX had the highest signal-to-noise ratio. Inhibition for all measured telopeptides by ONO-5334 were all correlated. The estimated half maximal effective plasma concentrations were not significantly different between all measured telopeptides.     

Intermittent intravenous administration of the bisphosphonate ibandronate prevents bone loss and maintains bone strength and quality in ovariectomized cynomolgus monkeys

Using a clinically relevant regimen, this study investigated the effects of treatment with ibandronate, a highly potent nitrogen-containing bisphosphonate, on bone loss, biochemical markers of bone turnover, densitometry, histomorphometry, biomechanical properties, and bone concentration in aged ovariectomized monkeys. Sixty-six female cynomolgus monkeys, aged 9 years and older, were ovariectomized (OVX) or sham operated. Intravenous (iv) bolus injections of ibandronate at 10, 30, or 150 microg/kg or placebo were administered at 30-day intervals (corresponding to intervals of 3 months in humans), starting at OVX, for 16 months. OVX significantly decreased bone mass at the lumbar spine, proximal femur, femoral neck, and radius and increased bone turnover in a time-dependent manner, as assessed by dual energy X-ray absorptiometry, peripheral quantitative computed tomography, or histomorphometry. Ibandronate iv bolus injections administered at 30 microg/kg every 30 days prevented osteopenia induced by estrogen depletion. OVX-induced increases in bone turnover (as determined by activation frequency, bone formation rate, and biochemical markers of bone turnover, including urinary N-telopeptide and deoxypyridinoline excretion and serum values for osteocalcin and bone-specific alkaline phosphatase) were suppressed on treatment, and bone mass, architecture, and strength were preserved at clinically relevant sites. Treatment with high-dose (150 microg/kg/dose) iv bolus injections of ibandronate further increased bone mass and improved bone strength at both the spine and femoral neck, without adversely affecting bone quality. In contrast, treatment with a 10 microg/kg/dose only partially prevented the OVX-induced effects. These data support the potential for the long-term administration of ibandronate by intermittent iv bolus injections in humans to prevent osteoporosis and improve bone quality at clinically relevant sites.     

Effects of estrogen replacement therapy on bone turnover in subchondral bone and epiphyseal metaphyseal cancellous bone of ovariectomized cynomolgus monkeys.

ERT decreases the severity of OA in OVX cynomolgus monkeys. We show that bone formation is greater in subchondral bone compared with epiphyseal/metaphyseal cancellous bone of the proximal tibia in these animals and that ERT decreases bone formation in both sites. ERT may decrease the risk of OA by decreasing bone formation in the SC bone. INTRODUCTION: Estrogen replacement therapy (ERT) decreases the risk of osteoporosis and osteoarthritis (OA) in postmenopausal women and has been shown to have direct effects on cells of the bone and cartilage. The effects of ERT have been studied extensively in cancellous bone, but subchondral (SC) bone directly beneath the articular cartilage has not been specifically evaluated. MATERIALS AND METHODS: Adult feral female cynomolgus monkeys were bilaterally ovariectomized (OVX) to simulate menopause; treated with ERT, soy phytoestrogens (SPE), or no hormones (OVX control group) for 3 years; and labeled with calcein before necropsy. At necropsy, the proximal tibias of 20 randomly selected animals from each treatment group were embedded in bioplastic and sectioned. Areas and labels were measured in a carefully defined region of the SC bone and epiphyseal/metaphyseal cancellous (EMC) bone, and derived dynamic and static indices were compared between the SC and EMC bone and among the three treatment groups. Student's t-tests and ANOVA were used to compare the data. RESULTS AND CONCLUSIONS: In both the SC and EMC bone, most of the values for the dynamic indices were highest in the OVX control group, intermediate in the SPE group, and lowest in the ERT group. The mineralizing surface, double-labeled surface, and bone formation rate (surface referent) were significantly higher in the SC bone compared with the EMC bone in the OVX control group. The trabecular bone volume was higher in the SPE-treated group compared with the OVX control group. In conclusion, the bone turnover indices were higher in the SC bone compared with the EMC bone, and ERT decreased these indices in both sites. In addition, SPE was protective against loss of bone volume.     

Effects of combination treatment with alendronate and vitamin K2 on bone mineral density and strength in ovariectomized mice

Bisphosphonates increase bone mineral density (BMD) by suppressing remodeling space and elongating the duration of mineralization. Menatetrenone (vitamin K₂) reduces the incidence of fractures by improving bone quality through enhanced γ-carboxylation of bone glutamic acid residues of osteocalcin in osteoporotic patients. This study investigated the effects of combination treatment with alendronate (ALN) and vitamin K₂ on BMD and bone strength in ovariectomized (OVX) mice. Thirty-three female mice, 16 weeks of age, were assigned to four groups: (1) OVX-control group; (2) oral vitamin K₂ group; (3) subcutaneous ALN group; and (4) ALN + vitamin K₂ group. The treatment was started 4 weeks after OVX and continued for 4 weeks. BMD, geometric parameters measured by peripheral quantitative computed tomography, and mechanical strength at the femoral metaphysis and mid-diaphysis were evaluated after an 8-week treatment period. ALN alone significantly increased total BMD (20%, P < 0.05) and trabecular BMD (25%, P < 0.05), but not the mechanical parameters of the femur, compared with the OVX-control group. Combination treatment with ALN and vitamin K₂ increased not only total BMD (15%, P < 0.05) and trabecular BMD (32%, P < 0.05) but also maximum load (33%, P < 0.05) and breaking energy (25%, P < 0.05) of compression test at the distal metaphysis, and maximum load (20%, P < 0.05) and breaking force (33%, P < 0.05) of three-point bending test at the mid-diaphysis compared with the OVX-control group. These results suggest that ALN, alone or in combination with vitamin K₂, showed significant improvement in BMD, but that the combination treatment was more effective than ALN alone for improving bone strength in OVX mice.     

Denosumab, a fully human RANKL antibody, reduced bone turnover markers and increased trabecular and cortical bone mass, density, and strength in ovariectomized cynomolgus monkeys

Denosumab is a fully human monoclonal antibody that inhibits RANKL, a protein essential for osteoclast formation, function, and survival. Osteoclast inhibition with denosumab decreased bone resorption, increased bone mineral density (BMD), and reduced fracture risk in osteoporotic women. The effects of 16months of continuous osteoclast inhibition on bone strength parameters were examined in adult ovariectomized (OVX) cynomolgus monkeys (cynos). One month after surgery, OVX cynos (n=14-20/group) were treated monthly with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50mg/kg). Sham-operated controls were treated with vehicle (n=17). OVX-Veh exhibited early and persistent increases in the resorption marker CTx, followed by similar increases in the formation marker BSAP, consistent with increased bone remodeling. Denosumab reduced CTx and BSAP throughout the study to levels significantly lower than in OVX-Veh or Sham-Veh, consistent with reduced remodeling. Increased remodeling in OVX-Veh led to absolute declines in areal BMD of 4.3-7.4% at the lumbar spine, total hip, femur neck, and distal radius (all p<0.05 vs baseline). Denosumab significantly increased aBMD at each site to levels exceeding baseline or OVX-Veh controls, and denosumab significantly increased cortical vBMC of the central radius and tibia by 7% and 14% (respectively) relative to OVX-Veh. Destructive biomechanical testing revealed that both doses of denosumab were associated with significantly greater peak load for femur neck (+19-34%), L3-L4 vertebral bodies (+54-55%), and L5-L6 cancellous cores (+69-82%) compared with OVX-Veh. Direct assessment of bone tissue material properties at cortical sites revealed no significant changes with denosumab. For all sites analyzed biomechanically, bone mass (BMC) and strength (load) exhibited strong linear correlations (r(2)=0.59-0.85 for all groups combined). Denosumab did not alter slopes of load-BMC regressions at any site, and denosumab groups exhibited similar or greater load values at given BMC values compared with OVX-Veh or Sham. In summary, denosumab markedly reduced biochemical markers of bone remodeling and increased cortical and trabecular bone mass in adult OVX cynos. Denosumab improved structural bone strength parameters at all sites analyzed, and strength remained highly correlated with bone mass. There was no evidence for reduced material strength properties of cortical bone with denosumab over this time period, which approximates to 4years of remodeling in the slower-remodeling adult human skeleton. These data indicate that denosumab increased bone strength by increasing bone mass and preserving bone quality.     

Effect of SI-591, a new class of cathepsin K inhibitor with peptidomimetic structure,on bone metabolism in vitro and in vivo

SI-591[N-[1-[[[(1S)-3-[[(3S)-hexahydro-2-oxo-1H-azepin-3-yl]amino]-1-(1-methylethyl)-2,3-dioxopropyl]amino]carbonyl]cyclohexyl]-2-furancarboxamide] is an orally bioavailable compound that was synthesized as one of several unique peptidomimetic compounds without a basic group. This compound was found to have the ability to inhibit cathepsin K, a lysosomal cysteine protease. Cathepsin K is known to be expressed in osteoclasts and involved in bone loss processes. In this study, SI-591 was shown to inhibit the activity of various purified cathepsin molecules at nanomolar concentrations but had high selectivity for cathepsin K over other subtypes including B and L. SI-591 also decreased the level of CTX-I, a bone resorption marker, which was released from osteoclasts in vitro in a dose-dependent manner. The mobilization of calcium from the bones to the blood stream is known to increase in rats fed with a low calcium diet; SI-591 inhibited this increase in serum calcium level at an oral dose of 3 mg/kg. Furthermore, SI-591 significantly decreased the level of CTX-I and DPD, bone resorption markers, at oral doses of 10 mg/kg or less in ovariectomized rats, while it did not affect the level of BGP, a bone formation marker. In addition, SI-591 prevented bone mineral density loss in the lumber vertebrae and femurs in ovariectomized rats. These results suggest that SI-591 inhibits bone resorption without affecting osteoblast maturation. Therefore, SI-591, a novel cathepsin K inhibitor, could be a promising agent for the treatment of postmenopausal osteoporosis.     

Effects of genistein on vertebral trabecular bone microstructure,bone mineral density,microcracks, osteocyte density,and bone strength in ovariectomized rats

Until now, the effects of phytoestrogen on bone in both women and ovarian hormone-deficient animal models of osteoporosis have remained uncertain. We have aimed here to investigate the effect of genistein (GEN) on trabecular bone quality in ovariectomized (OVX) rats. Forty 7-month-old female Sprague-Dawley rats were randomly divided into the following four groups: OVX, sham-operated (SHAM), treated with 17beta-estradiol (EST, 10 microg x kg(-1) x day(-1)), and GEN (5 mg x kg(-1) x day(-1)). At 15 weeks postoperation, the compressive test was performed on the L5 vertebral body; additionally, microcomputed tomography (micro-CT) assessment was performed to estimate the bone mineral density (BMD) and microstructure parameters of the L6 vertebral body. After fatigue damage testing, the L6 vertebral body was bulk-stained in 1% basic fuchsin and embedded in methylmethacrylate. The L4 vertebral body was embedded in methylmethacrylate for dynamic histomorphometry analysis without staining. Mounted bone slices were used to measure microcrack parameters, empty osteocyte lacuna density (e.Lc.Dn), and osteocyte density (Ot.N/T.Ar). Maximum loading (ML) and Ot.N/T.Ar were significantly lower in the OVX group than in the other groups. E.Lc.Dn was significantly decreased in GEN and EST groups compared to the OVX group. ML was significantly decreased in the GEN group compared to the SHAM group. Microcrack density, microcrack surface density, and microcrack length were significantly increased in the OVX group compared to the other groups. Mineral apposition rate was significantly decreased in the OVX group compared to the SHAM and GEN groups. Bone formation rate was significantly decreased in the OVX group compared to other groups. There were no significant differences with regard to mineralizing surface among the four groups. Volumetric BMD at organ was significantly lower in OVX, EST, and GEN groups than in the SHAM group. Bone mineral content was significantly lower in the OVX group than in the SHAM group. Bone volume fraction and trabecular number were significantly decreased in OVX, EST, and GEN groups compared to the SHAM group. Structure model index was significantly lower in the SHAM group than in OVX, EST, and GEN groups. Trabecular separation was significantly increased in the OVX group compared to SHAM and EST groups. There were no significant differences with regard to the trabecular thickness (Tb,Th) between SHAM, GEN, and OVX groups. Tb.Th was significantly lower in the EST group than in the SHAM group. Connectivity density (Conn.D) was significantly lower in the OVX group than in SHAM and GEN groups, and Conn. D was significantly lower in the EST group than in GEN. In conclusion, the present study demonstrates that GEN preserved the biomechanical quality of the trabecular bone regardless of the microstructure and BMD.     

Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation,bone mineral density,and bone strength

The development of bone‐rebuilding anabolic agents for treating bone‐related conditions has been a long‐standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin‐neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin‐neutralizing monoclonal antibody (Scl‐AbIV) to gonad‐intact female cynomolgus monkeys. Two once‐monthly subcutaneous injections of Scl‐AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl‐AbIV treatment had clear anabolic effects, with marked dose‐dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl‐AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle‐treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest‐dose group. Taken together, the marked bone‐building effects achieved in this short‐term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis. © 2010 American Society for Bone and Mineral Research