Sustained Modeling‐Based Bone Formation During Adulthood in Cynomolgus Monkeys May Contribute to Continuous BMD Gains With Denosumab

Denosumab (DMAb) administration to postmenopausal women with osteoporosis is associated with continued bone mineral density (BMD) increases and low fracture incidence through 8 years, despite persistently reduced bone turnover markers and limited fluorochrome labeling in iliac crest bone biopsies. BMD increases were hypothesized to result from additional accrual of bone matrix via modeling‐based bone formation—a hypothesis that was tested by examining fluorochrome labeling patterns in sections from ovariectomized (OVX) cynomolgus monkeys (cynos) treated with DMAb for 16 months. Mature OVX or Sham cynos were treated monthly with vehicle for 16 months, whereas other OVX cynos received monthly 25 or 50 mg/kg DMAb. DMAb groups exhibited very low serum bone resorption and formation biomarkers and near‐absent fluorochrome labeling in proximal femur cancellous bone. Despite these reductions, femoral neck dual‐energy X‐ray absorptiometry (DXA) BMD continued to rise in DMAb‐treated cynos, from a 4.6% increase at month 6 to 9.8% above baseline at month 16. Further examination of cortical bone in the proximal femur demonstrated consistent and prominent labeling on the superior endocortex and the inferior periosteal surface, typically containing multiple superimposed labels from month 6 to 16 over smooth cement lines, consistent with continuous modeling‐based bone formation. These findings were evident in all groups. Quantitative analysis at another modeling site, the ninth rib, demonstrated that DMAb did not alter the surface extent of modeling‐based labels, or the cortical area bound by them, relative to OVX controls, while significantly reducing remodeling‐based bone formation and eroded surface. This conservation of modeling‐based formation occurred concomitantly with increased femoral neck strength and, when coupled with a reduction in remodeling‐based bone loss, is likely to contribute to increases in bone mass with DMAb treatment. Thus, this study provides preclinical evidence for a potential mechanism that could contribute to the clinical observations of continued BMD increases and low fracture rates with long‐term DMAb administration. © 2015 American Society for Bone and Mineral Research.     

Romosozumab Improves Bone Mass and Strength While Maintaining Bone Quality in Ovariectomized Cynomolgus Monkeys

Romosozumab (Romo), a humanized sclerostin antibody, is a bone‐forming agent under development for treatment of osteoporosis. To examine the effects of Romo on bone quality, mature cynomolgus monkeys (cynos) were treated 4 months post‐ ovariectomy (OVX) with vehicle, 3 mg/kg, or 30 mg/kg Romo for 12 months, or with 30 mg/kg Romo for 6 months followed by vehicle for 6 months (30/0). Serum bone formation markers were increased by Romo during the first 6 months, corresponding to increased cancellous, endocortical, and periosteal bone formation in rib and iliac biopsies at months 3 and 6. Dual‐energy X‐ray absorptiometry (DXA) bone mineral density (BMD) was increased by 14% to 26% at the lumbar spine and proximal femur at month 12, corresponding to significant increases in bone strength at 3 and 30 mg/kg in lumbar vertebral bodies and cancellous cores, and at 30 mg/kg in the femur diaphysis and neck. Bone mass remained positively correlated with strength at these sites, with no changes in calculated material properties at cortical sites. These bone‐quality measures were also maintained in the 30/0 group, despite a gradual loss of accrued bone mass. Normal bone mineralization was confirmed by histomorphometry and ash analyses. At the radial diaphysis, a transient, reversible 2% reduction in cortical BMD was observed with Romo at month 6, despite relative improvements in bone mineral content (BMC). High‐resolution pQCT confirmed this decline in cortical BMD at the radial diaphysis and metaphysis in a second set of OVX cynos administered 3 mg/kg Romo for 6 months. Radial diaphyseal strength was maintained and metaphyseal strength improved with Romo as estimated by finite element modeling. Decreased radial cortical BMD was a consequence of increased intracortical remodeling, with no increase in cortical porosity. Romo resulted in marked improvements in bone mass, architecture, and bone strength, while maintaining bone quality in OVX cynos, supporting its bone efficacy and safety profile. © 2016 American Society for Bone and Mineral Research.     

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.     

Alendronate Reduces Bone Toughness of Ribs without Significantly Increasing Microdamage Accumulation in Dogs Following 3 Years of Daily Treatment

Reduced bone toughness, the energy absorption capacity of the tissue, has been consistently documented in vertebrae of animals treated with a wide range of bisphosphonate doses. Data regarding toughness changes in the rib are conflicting, with one report showing no effect and another showing a significant reduction following treatment of beagle dogs with high doses of bisphosphonates. The goal of this study was to evaluate changes in bone toughness and various other tissue-level properties of the rib following 3 years of bisphosphonate treatment with doses at and above those used to treat osteoporosis. Skeletally mature intact beagle dogs were treated daily for 3 years with vehicle (VEH), alendronate 0.2 mg/kg (ALN0.2), or alendronate 1.0 mg/kg (ALN1.0). The lower ALN dose approximates, on a milligram per kilogram basis, that used for treatment of postmenopausal osteoporosis, with the higher dose being five times higher. Ribs were assessed for biomechanical properties, bone turnover rate, microdamage, density, and geometry. Toughness was significantly lower with ALN1.0 (-33%) but not ALN0.2 (-19%) compared to VEH, while neither ultimate stress nor modulus differed among the groups. Bone density, geometry, and structural biomechanical properties were similar among the three groups. There was no significant difference in overall microdamage accumulation among the groups. Intracortical bone formation rate was significantly lower than VEH in both ALN groups (-69% to -90%). These data show that while rib cortical bone experiences significant reductions in turnover following bisphosphonate treatment, it is only in animals treated with doses above those used to treat osteoporosis that toughness is significantly compromised.     

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.     

Bone Mineral and Organic Properties in Postmenopausal Women Treated With Denosumab for Up to 10 years

In postmenopausal women with osteoporosis, denosumab (DMAb) therapy through 10 years resulted in significantly higher degree of mineralization of bone, with a subsequent increase from years 2–3 to year 5 and no further difference between years 5 and 10. Our aim was to assess the variables reflecting the quality of bone mineral and organic matrix (Fourier transform infrared microspectroscopy), and the microhardness of bone (Vickers microindentation). Cross-sectional assessments were performed in blinded fashion on iliac bone biopsies from osteoporotic women (72 from FREEDOM trial, 49 from FREEDOM Extension trial), separately in cortical and cancellous compartments. After 2–3 years of DMAb, mineral/matrix ratio and microhardness of cortical bone were significantly higher compared with placebo, whereas mineral maturity, mineral crystallinity, mineral carbonation, and collagen maturity were not different in both bone compartments. Through 5 years of DMAb, mineral carbonation was significantly lower and mineral/matrix ratio, mineral maturity, and crystallinity were significantly higher versus 2–3 years and were not different between 5 and 10 years, with the exception of mineral maturity in cancellous bone. These data support a transition of mineral to more mature crystals (within physiological range) and the completeness of secondary mineralization within 5 years of DMAb treatment. Microhardness in cortical and cancellous compartments was significantly lower at 5 years of DMAb versus 2–3 years and was not different from years 5 to 10. The lower microhardness at years 5 and 10 is likely the result of maturation of the organic matrix in a persistently low state of bone remodeling over 5 and 10 years. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Differential maintenance of cortical and cancellous bone strength following discontinuation of bone‐active agents

Osteoporotic patients treated with antiresorptive or anabolic agents experience an increase in bone mass and a reduction in incident fractures. However, the effects of these medications on bone quality and strength after a prolonged discontinuation of treatment are not known. We evaluated these effects in an osteoporotic rat model. Six‐month‐old ovariectomized (OVX) rats were treated with placebo, alendronate (ALN, 2 µg/kg), parathyroid hormone [PTH(1–34); 20 µg/kg], or raloxifene (RAL, 2 mg/kg) three times a week for 4 months and withdrawn from the treatments for 8 months. Treatment with ALN, PTH, and RAL increased the vertebral trabecular bone volume (BV/TV) by 47%, 53%, and 31%, with corresponding increases in vertebral compression load by 27%, 51%, and 31%, respectively (p < .001). The resulting bone strength was similar to that of the sham‐OVX control group with ALN and RAL and higher (p < .001) with PTH treatment. After 4 months of withdrawal, bone turnover (BFR/BS) remained suppressed in the ALN group versus the OVX controls (p < .001). The vertebral strength was higher than in the OVX group only in ALN‐treated group (p < .05), whereas only the PTH‐treated animals showed a higher maximum load in tibial bending versus the OVX controls (p < .05). The vertebral BV/TV returned to the OVX group level in both the PTH and RAL groups 4 months after withdrawal but remained 25% higher than the OVX controls up to 8 months after withdrawal of ALN (p < .05). Interestingly, cortical bone mineral density increased only with PTH treatment (p < .05) but was not different among the experimental groups after withdrawal. At 8 months after treatment withdrawal, none of the treatment groups was different from the OVX control group for cortical or cancellous bone strength. In summary, both ALN and PTH maintained bone strength (maximum load) 4 months after discontinuation of treatment despite changes in bone mass and bone turnover; however, PTH maintained cortical bone strength, whereas ALN maintained cancellous bone strength. Additional studies on the long‐term effects on bone strength after discontinuation and with combination of osteoporosis medications are needed to improve our treatment of osteoporosis. © 2011 American Society for Bone and Mineral Research.     

Intermittent Parathyroid Hormone After Prolonged Alendronate Treatment Induces Substantial New Bone Formation and Increases Bone Tissue Heterogeneity in Ovariectomized Rats

Postmenopausal osteoporosis is often treated with bisphosphonates (eg, alendronate, [ALN]), but oversuppression of bone turnover by long‐term bisphosphonate treatment may decrease bone tissue heterogeneity. Thus, alternate treatment strategies after long‐term bisphosphonates are of great clinical interest. The objective of the current study was to determine the effect of intermittent parathyroid hormone (PTH) following 12 weeks of ALN (a bisphosphonate) treatment in 6‐month‐old, ovariectomized (OVX) rats on bone microarchitecture, bone remodeling dynamics, and bone mechanical properties at multiple length scales. By using in vivo μCT and 3D in vivo dynamic bone histomorphometry techniques, we demonstrated the efficacy of PTH following ALN therapy for stimulating new bone formation, and increasing trabecular thickness and bone volume fraction. In healthy bone, resorption and formation are coupled and balanced to sustain bone mass. OVX results in resorption outpacing formation, and subsequent bone loss and reduction in bone tissue modulus and tissue heterogeneity. We showed that ALN treatment effectively reduced bone resorption activity and regained the balance with bone formation, preventing additional bone loss. However, ALN treatment also resulted in significant reductions in the heterogeneity of bone tissue mineral density and tissue modulus. On the other hand, PTH treatment was able to shift the bone remodeling balance in favor of formation, with or without a prior treatment with ALN. Moreover, by altering the tissue mineralization, PTH alleviated the reduction in heterogeneity of tissue material properties induced by prolonged ALN treatment. Furthermore, switching to PTH treatment from ALN improved bone's postyield mechanical properties at both the whole bone and apparent level compared to ALN alone. The current findings suggest that intermittent PTH treatment should be considered as a viable treatment option for patients with prior treatment with bisphosphonates. © 2017 American Society for Bone and Mineral Research.     

Tissue‐Level Mechanisms Responsible for the Increase in Bone Formation and Bone Volume by Sclerostin Antibody

Bone formation can be remodeling‐based (RBF) or modeling‐based (MBF), the former coupled to bone resorption and the latter occurring directly on quiescent surfaces. Unlike osteoanabolic therapies such as parathyroid hormone (PTH) 1‐34 that increase bone remodeling and thus both formation and resorption, sclerostin antibody (Scl‐Ab) increases bone formation while decreasing bone resorption. With this unique profile, we tested our hypothesis that Scl‐Ab primarily elicited MBF by examining bones from Scl‐Ab–treated ovariectomized (OVX) rats and male cynomolgus monkeys (cynos). Histomorphometry was performed to quantify and characterize bone surfaces in OVX rats administered vehicle or Scl‐Ab (25 mg/kg) subcutaneously (sc) twice/week for 5 weeks and in adolescent cynos administered vehicle or Scl‐Ab (30 mg/kg) sc every 2 weeks for 10 weeks. Fluorochrome‐labeled surfaces in L₂ vertebra and femur endocortex (cynos only) were considered to be MBF or RBF based on characteristics of their associated cement lines. In OVX rats, Scl‐Ab increased MBF by eightfold (from 7% to 63% of bone surface, compared to vehicle). In cynos, Scl‐Ab markedly increased MBF on trabecular (from 0.6% to 34%) and endocortical surfaces (from 7% to 77%) relative to vehicle. Scl‐Ab did not significantly affect RBF in rats or cynos despite decreased resorption surface in both species. In cynos, Scl‐Ab resulted in a greater proportion of RBF and MBF containing sequential labels from week 2, indicating an increase in the lifespan of the formative site. This extended formation period was associated with robust increases in the percent of new bone volume formed. These results demonstrate that Scl‐Ab increased bone volume by increasing MBF and prolonged the formation period at both modeling and remodeling sites while reducing bone resorption. Through these unique effects on bone formation and resorption, Scl‐Ab may prove to be an effective therapeutic to rapidly increase bone mass in diseases such as osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Finite Element Analysis of Denosumab Treatment Effects on Vertebral Strength in Ovariectomized Cynomolgus Monkeys

Finite element analysis has not yet been validated for measuring changes in whole‐bone strength at the hip or spine in people after treatment with an osteoporosis agent. Toward that end, we assessed the ability of a clinically approved implementation of finite element analysis to correctly quantify treatment effects on vertebral strength, comparing against direct mechanical testing, in cynomolgus monkeys randomly assigned to one of three 16‐month‐long treatments: sham surgery with vehicle (Sham‐Vehicle), ovariectomy with vehicle (OVX‐Vehicle), or ovariectomy with denosumab (OVX‐DMAb). After treatment, T12 vertebrae were retrieved, scanned with micro‐CT, and mechanically tested to measure compressive strength. Blinded to the strength data and treatment codes, the micro‐CT images were coarsened and homogenized to create continuum‐type finite element models, without explicit porosity. With clinical translation in mind, these models were then analyzed for strength using the U.S. Food and Drug Administration (FDA)‐cleared VirtuOst software application (O.N. Diagnostics, Berkeley, CA, USA), developed for analysis of human bones. We found that vertebral strength by finite element analysis was highly correlated (R² = 0.97; n = 52) with mechanical testing, independent of treatment (p = 0.12). Further, the size of the treatment effect on strength (ratio of mean OVX‐DMAb to mean OVX‐Vehicle, as a percentage) was large and did not differ (p = 0.79) between mechanical testing (+57%; 95% CI [26%, 95%]) and finite element analysis (+51% [20%, 88%]). The micro‐CT analysis revealed increases in cortical thickness (+45% [19%, 73%]) and trabecular bone volume fraction (+24% [8%, 42%]). These results show that a preestablished clinical finite element analysis implementation—developed for human bone and clinically validated in fracture‐outcome studies—correctly quantified the observed treatment effects of denosumab on vertebral strength in cynomolgus monkeys. One implication is that the treatment effects in this study are well explained by the features contained within these finite element models, namely, the bone geometry and mass and the spatial distribution of bone mass. © 2016 American Society for Bone and Mineral Research.     

Soluble RANKL Induces High Bone Turnover and Decreases Bone Volume,Density, and Strength in Mice

Receptor activator for nuclear factor-kappa B ligand (RANKL) is an essential mediator of osteoclastogenesis. We hypothesized that administration of soluble RANKL to mice would result in high turnover and deleterious effects on both cortical and trabecular bone. For 10 days, 10-week-old C57BL/6J female mice (n = 12/group) were given twice-daily subcutaneous injections of human recombinant RANKL (0.4 or 2 mg/kg/day) or inert vehicle (VEH). Bone turnover was greatly accelerated by RANKL, as evidenced by the 49-84% greater levels of serum TRAP-5b (bone resorption marker) and 300-400% greater levels of serum alkaline phosphatase (bone formation marker). RANKL resulted in significantly greater endocortical bone erosion surface (79-83%) and periosteal bone formation rate (64-87%) vs. VEH. Microcomputed tomographic (microCT) analysis of the proximal tibia indicated a reduction in trabecular volume fraction (-84%) for both doses of RANKL. Cortical bone geometry and strength were also negatively influenced by RANKL. MicroCT analysis of the femoral diaphysis indicated significantly lower cortical bone volume (-10% to -13%) and greater cortical porosity (8-9%) relative to VEH. Biomechanical testing of the femur diaphysis revealed significantly lower maximum bending load (-19% to -25%) vs. VEH. Bone strength remained correlated with bone mass, independent of RANKL stimulation of bone turnover. These findings are consistent with the hypothesis that soluble RANKL could be an important etiologic factor in pathologic bone loss. RANKL also has potential utility as a model for studying the consequences of high bone turnover on bone quality and strength in animals.     

Raloxifene enhances vertebral mechanical properties independent of bone density

Anti-remodeling agents produce similar reductions in vertebral fracture risk despite large differences in BMD changes suggesting the mechanism of fracture risk reduction may differ among these agents. Forty-eight intact (non-ovariectomized) skeletally mature female beagle dogs were treated orally for 12 months with clinically relevant doses of risedronate (RIS, 0.10 mg/kg/day), alendronate (ALN, 0.2 mg/kg/day), raloxifene (RAL, 0.50 mg/kg/day), or saline (VEH, 1 ml/kg/day). After sacrifice, the following measurements were made on vertebral bone: areal (aBMD) and volumetric (vBMD) bone mineral densities, tissue mineralization by ash content, static and dynamic histomorphometric parameters, microdamage, and extrinsic and intrinsic measures of biomechanical strength, stiffness and energy to fracture. At these doses, RAL suppressed bone turnover (-20%) significantly less than the bisphosphonates (-66 and -71%) and did not produce significant differences in aBMD, vBMD, BV/TV or percent ash compared to VEH-treated animals. Microdamage accumulation in RAL-treated animals was not significantly different than VEH; both RIS and ALN had significantly higher crack surface density compared to VEH. Stiffness was significantly higher than VEH in all treatment groups. Ultimate load divided by aBMD, a measure of strength independent of BMD, was significantly higher only in RAL-treated animals compared to VEH (+16%, P = 0.015). Based on these data, we conclude that raloxifene produces improvements in bone mechanical properties in ways that do not involve increases in BMD.     

Effects of Daily or Cyclic Teriparatide on Bone Formation in the Iliac Crest in Women on No Prior Therapy and in Women on Alendronate

There is little information on the effects of combination therapy for osteoporosis at the tissue level. Using quadruple tetracycline‐labeled bone biopsies, we have compared the bone formation response to teriparatide (TPTD) in treatment‐naïve subjects (Rx‐Naïve) and in subjects on prior and ongoing alendronate (ALN) treatment (ALN‐Rx). Three bone envelopes were analyzed: cancellous, endocortical, and intracortical. TPTD was given as a standard, continuous daily injection or as a cyclic regimen (3 months on daily TPTD, 3 months off, 3 months on daily TPTD). Subjects were biopsied at 7 weeks and at 7 months to allow comparison of the bone formation response to the first and second cycles of TPTD. Baseline values for dynamic bone formation indices were lower in ALN‐Rx than Rx‐Naïve subjects. Both Rx‐Naïve and ALN‐RX subjects responded to TPTD with significant increases in bone formation indices at both time points. With cyclic TPTD treatment, the first and second cycles of TPTD stimulated bone formation rate in the cancellous and endocortical envelopes to a similar extent in ALN‐Rx and Rx‐Naïve subjects. However, in Rx‐Naïve patients, bone formation rate (BFR/BS) was higher in patients receiving daily treatment compared with those receiving cyclic TPTD treatment in all three envelopes in the 7‐month biopsies. This suggests that the cyclic approach does not provide a skeletal benefit in treatment‐naive patients. In the 7‐month biopsies, cortical porosity was higher in the Rx‐Naïve group receiving daily TPTD than in all other groups. These data provide supporting evidence at the tissue level for previous biochemical and densitometric data suggesting that addition of either cyclic or daily TPTD to ongoing ALN treatment may be an effective approach for patients with severe osteoporosis already treated with ALN who remain at high risk of fracture. © 2016 American Society for Bone and Mineral Research.     

Novel EP4 Receptor Agonist‐Bisphosphonate Conjugate Drug (C1) Promotes Bone Formation and Improves Vertebral Mechanical Properties in the Ovariectomized Rat Model of Postmenopausal Bone Loss

Current treatments for postmenopausal osteoporosis aim to either promote bone formation or inhibit bone resorption. The C1 conjugate drug represents a new treatment approach by chemically linking the antiresorptive compound alendronate (ALN) with the anabolic agent prostanoid EP4 receptor agonist (EP4a) through a linker molecule (LK) to form a conjugate compound. This enables the bone‐targeting ability of ALN to deliver EP4a to bone sites and mitigate the systemic side effects of EP4a, while also facilitating dual antiresorptive and anabolic effects. In vivo hydrolysis is required to release the EP4a and ALN components for pharmacological activity. Our study investigated the in vivo efficacy of this drug in treating established bone loss using an ovariectomized (OVX) rat model of postmenopausal osteopenia. In a curative experiment, 3‐month‐old female Sprague‐Dawley rats were OVX, allowed to lose bone for 7 weeks, then treated for 6 weeks. Treatment groups consisted of C1 conjugate at low and high doses, vehicle‐treated OVX and sham, prostaglandin E₂ (PGE₂), and mixture of unconjugated ALN‐LK and EP4a to assess the effect of conjugation. Results showed that weekly administration of C1 conjugate dose‐dependently increased bone volume in trabecular bone, which partially or completely reversed OVX‐induced bone loss in the lumbar vertebra and improved vertebral mechanical strength. The conjugate also dose‐dependently stimulated endocortical woven bone formation and intracortical resorption in cortical bone, with high‐dose treatment increasing the mechanical strength but compromising the material properties. Conjugation between the EP4a and ALN‐LK components was crucial to the drug's anabolic efficacy. To our knowledge, the C1 conjugate represents the first time that a combined therapy using an anabolic agent and the antiresorptive compound ALN has shown significant anabolic effects which reversed established osteopenia. © 2014 American Society for Bone and Mineral Research.     

Overlapping and Continued Alendronate or Raloxifene Administration in Patients on Teriparatide: Effects on Areal and Volumetric Bone Mineral Density—The CONFORS Study

Nine month teriparatide (TPTD) monotherapy followed by co‐administration of raloxifene (RAL) or alendronate (ALN) for another nine 9 months resulted in incremental bone mineral density (BMD) increase. The aim of this study was to investigate the effects of continued antiresorptive treatments for 12 months in the extension phase. Postmenopausal women (n = 125) with severe osteoporosis on ongoing TPTD treatment for 9 months were randomized into three open‐label groups for another 9 months: ALN (70 mg/week, n = 41), RAL (60 mg/d, n = 37) in addition to TPTD or no additional medication (n = 47) except Ca and vitamin D. After discontinuation of TPTD the respective antiresorptives were continued for a further 12 months, while patients in the TPTD monotherapy group received Ca and vitamin D. Amino‐terminal propeptide of type I procollagen (P1NP) and cross‐linked C‐telopeptide (CTX), areal and volumetric BMD at the lumbar spine (LS) and hip were assessed. ALN resulted in continued BMD increase in LS (4.3 ± 1.5%; mean ± SD), femoral neck (4.2 ± 1.6%) and total hip (4 ± 1.6%; p < 0.001 for all), while RAL was only effective at the LS (2.4 ± 1.7%, p < 0.001) but no changes at the femoral neck (0.4 ± 1.4%) or total hip (?0.8 ± 1.5%) were observed. Cortical bone only increased in the ALN group (femoral neck 6.7 ± 2.7% and ?1.3 ± 2.5%; total hip 13.8 ± 2.9% and ?2.3 ± 2.5% for ALN and RAL, p < 0.001 for all; respectively). Analyzing the entire 30 months of therapy, the ALN group revealed the largest BMD increase in all regions. Our results suggest that the addition of ALN to ongoing TPTD and continuing ALN after TPTD was stopped may be beneficial for patients in terms of areal and volumetric BMD increase. Further research is warranted to determine the optimal timing of the initiation of the combination treatment, the respective antiresorptive medication and the potential benefit of this BMD increase regarding fracture prevention. © 2014 American Society for Bone and Mineral Research     

Daily Treatment of Aged Ovariectomized Rats with Human Parathyroid Hormone (1-84) for 12 Months Reverses Bone Loss and Enhances Trabecular and Cortical Bone Strength

Most studies that have investigated the anabolic effects of parathyroid hormone (1-84) (PTH) or PTH fragments on the skeleton of ovariectomized (OVX) rats have evaluated the short-term effects of high-dose PTH(1-34) in young animals. This study used densitometry, histomorphometry, and biomechanical testing to evaluate the effects of 12-month daily treatment with low-dose PTH (15 or 30 μg/kg) in rats that were 10 months old at baseline, 4 months after OVX. Bone mineral density (BMD) and bone strength were reduced substantially in control OVX rats. The 15 μg/kg dose of PTH restored BMD to levels similar to those in sham animals within 6 months at the lumbar spine, distal and central femur, and whole body and maintained the BMD gain from 6 to 12 months. The 30 μg/kg dose produced greater effects. Both PTH doses normalized the trabecular bone volume-to-total volume ratio (BV/TV) at lumbar vertebra 3 but not at the proximal tibia (where baseline BV/TV was very low), solely by increasing trabecular thickness. PTH dose-dependently increased bone formation by increasing the mineralizing surface, but only the 30 μg/kg dose increased resorption. PTH increased cortical BMD, area, and thickness, primarily by increasing endocortical bone formation, and restored all measures of bone strength to levels similar to those in sham animals at all skeletal sites. PTH increased bone mass safely; there was no osteoid accumulation, mineralization defect, or marrow fibrosis and there were no abnormal cells. Thus, long-term PTH therapy normalized bone strength in the aged OVX rat, a model of postmenopausal osteoporosis, through increased bone turnover and enhanced formation of both trabecular and cortical bone.     

Femoral Bone Strength and Its Relation to Cortical and Trabecular Changes After Treatment With PTH,Alendronate, and Their Combination as Assessed by Finite Element Analysis of Quantitative CT Scans

The “PTH and Alendronate” or “PaTH” study compared the effects of PTH(1‐84) and/or alendronate (ALN) in 238 postmenopausal, osteoporotic women. We performed finite element analysis on the QCT scans of 162 of these subjects to provide insight into femoral strength changes associated with these treatments and the relative roles of changes in the cortical and trabecular compartments on such strength changes. Patients were assigned to either PTH, ALN, or their combination (CMB) in year 1 and were switched to either ALN or placebo (PLB) treatment in year 2: PTH‐PLB, PTH‐ALN, CMB‐ALN, and ALN‐ALN (year 1‐year 2) treatments. Femoral strength was simulated for a sideways fall using nonlinear finite element analysis of the quantitative CT exams. At year 1, the strength change from baseline was statistically significant for PTH (mean, 2.08%) and ALN (3.60%), and at year 2, significant changes were seen for the PTH‐ALN (7.74%), CMB‐ALN (4.18%), and ALN‐ALN (4.83%) treatment groups but not for PTH‐PLB (1.17%). Strength increases were primarily caused by changes in the trabecular density regardless of treatment group, but changes in cortical density and mass also played a significant role, the degree of which depended on treatment. For PTH treatment at year 1 and for ALN‐ALN treatment at year 2, there were significant negative and positive strength effects, respectively, associated with a change in external bone geometry. Average changes in strength per treatment group were somewhat consistent with average changes in total hip areal BMD as measured by DXA, except for the PTH group at year 1. The relation between change in femoral strength and change in areal BMD was weak (r² = 0.14, pooled, year 2). We conclude that femoral strength changes with these various treatments were dominated by trabecular changes, and although changes in the cortical bone and overall bone geometry did contribute to femoral strength changes, the extent of these latter effects depended on the type of treatment.     

Selective Modification of Bone Quality by PTH,Pamidronate, or Raloxifene

Bone strength, a determinant of resistance to fracture, depends on BMD, geometry, microarchitecture, bone turnover rates, and properties of the bone at the material level. Despite comparable antifracture efficacy, anti‐catabolics and bone anabolic agents are likely to modify the various determinants of bone strength in very different ways. Eight weeks after ovariectomy (OVX), 8‐mo‐old osteoporotic rats received pamidronate (APD; 0.6 mg/kg, 5 days/mo, SC), raloxifene (3 mg/kg, 5/7 days, tube feeding), PTH(1–34) (10 μg/kg, 5/7 days, SC), or vehicle for 16 wk, and we measured vertebral BMD, maximal load, stiffness and energy, microarchitecture, and material properties by nanoindentation, which allows the calculation of the elastic modulus, tissue hardness, and working energy. Markers of bone turnover, plasma osteocalcin, and urinary deoxypyridinoline (Dpd) were also determined. PTH induced greater maximal load than APD or raloxifene, as well as greater absorbed energy, BMD, and increased bone turnover markers. PTH markedly increased trabecular bone volume and connectivity to values higher than sham. Animals treated with APD had BV/TV values significantly higher than OVX but lower than sham, whereas raloxifene had no effect. Tissue hardness was identical in PTH‐treated and OVX untreated controls. In contrast, APD reversed the decline in strength to levels not significantly different to sham, reduced bone turnover, and increased hardness. Raloxifene markedly increased material level cortical hardness and elastic modulus. These results show the different mechanisms by which anti‐catabolics and bone anabolics reduce fracture risk. PTH influences microarchitecture, whereas bisphosphonates alter material‐level bone properties, with probable opposite effects on remodeling space. Raloxifene primarily improved the material stiffness at the cortical level.     

Odanacatib Restores Trabecular Bone of Skeletally Mature Female Rabbits With Osteopenia but Induces Brittleness of Cortical Bone: A Comparative Study of the Investigational Drug With PTH,Estrogen, and Alendronate

Cathepsin K (CK), a lysosomal cysteine protease, is highly expressed in mature osteoclasts and degrades type 1 collagen. Odanacatib (ODN) is a selective and reversible CK inhibitor that inhibits bone loss in preclinical and clinical studies. Although an antiresorptive, ODN does not suppress bone formation, which led us to hypothesize that ODN may display restorative effect on the osteopenic bones. In a curative study, skeletally mature New Zealand rabbits were ovarectomized (OVX) and after induction of bone loss were given a steady‐state exposure of ODN (9 mM/d) for 14 weeks. Sham‐operated and OVX rabbits treated with alendronate (ALD), 17b‐estradiol (E2), or parathyroid hormone (PTH) served as various controls. Efficacy was evaluated by assessing bone mineral density (BMD), bone microarchitecture (using micro‐computed tomography), fluorescent labeling of bone, and biomechanical strength. Skeletal Ca/P ratio was measured by scanning electron microscopy (SEM) with X‐ray microanalysis, crystallinity by X‐ray diffraction, and bone mineral density distribution (tissue mineralization) by backscattered SEM. Between the sham and ODN‐treated osteopenic groups, lumbar and femur metaphyseal BMD, Ca/P ratio, trabecular microstructure and geometric indices, vertebral compressive strength, trabecular lining cells, cortical parameters (femoral area and thickness and periosteal deposition), and serum P1NP were largely comparable. Skeletal improvements in ALD‐treated or E2‐treated groups fell significantly short of the sham/ODN/PTH group. However, the ODN group displayed reduced ductility and enhanced brittleness of central femur, which might have been contributed by higher crytallinity and tissue mineralization. Rabbit bone marrow stromal cells expressed CK and when treated with ODN displayed increased formation of mineralized nodules and decreased apoptosis in serum‐deficient medium compared with control. In vivo, ODN did not suppress remodeling but inhibited osteoclast activity more than ALD. Taken together, we show that ODN reverses BMD, skeletal architecture, and compressive strength in osteopenic rabbits; however, it increases crystallinity and tissue mineralization, thus leading to increased cortical bone brittleness. © 2015 American Society for Bone and Mineral Research.