Sclerostin-Antibody Treatment Decreases Fracture Rates in Axial Skeleton and Improves the Skeletal Phenotype in Growing oim/oim Mice.

In osteogenesis imperfecta (OI), vertebrae brittleness causes thorax deformations and leads to cardiopulmonary failure. As sclerostin-neutralizing antibodies increase bone mass and strength in animal models of osteoporosis, their administration in two murine models of severe OI enhanced the strength of vertebrae in growing female Crtap-/- mice but not in growing male Col1a1Jrt/+ mice. However, these two studies ignored the impact of antibodies on spine growth, fracture rates, and compressive mechanical properties. Here, we conducted a randomized controlled trial in oim/oim mice, an established model of human severe OI type III due to a mutation in Col1a2. Five-week-old female WT and oim/oim mice received either PBS or sclerostin antibody (Scl-Ab) for 9 weeks. Analyses included radiography, histomorphometry, pQCT, microcomputed tomography, and biomechanical testing. Though it did not modify vertebral axial growth, Scl-Ab treatment markedly reduced the fracture prevalence in the pelvis and caudal vertebrae, enhanced osteoblast activity (L4), increased cervico-sacral spine BMD, and improved the lumbosacral spine bone cross-sectional area. Scl-Ab did not impact vertebral height and body size but enhanced the cortical thickness and trabecular bone volume significantly in the two Scl-Ab groups. At lumbar vertebrae and tibial metaphysis, the absolute increase in cortical and trabecular bone mass was higher in Scl-Ab WT than in Scl-Ab oim/oim. The effects on trabecular bone mass were mainly due to changes in trabecular number at vertebrae and in trabecular thickness at metaphyses. Additionally, Scl-Ab did not restore a standard trabecular network, but improved bone compressive ultimate load with more robust effects at vertebrae than at metaphysis. Overall, Scl-Ab treatment may be beneficial for reducing vertebral fractures and spine deformities in patients with severe OI.

Sclerostin Antibody Reverses the Severe Sublesional Bone Loss in Rats After Chronic Spinal Cord Injury.

To date, no efficacious therapy exists that will prevent or treat the severe osteoporosis in individuals with neurologically motor-complete spinal cord injury (SCI). Recent preclinical studies have demonstrated that sclerostin antibody (Scl-Ab) can prevent sublesional bone loss after acute SCI in rats. However, it remains unknown whether sclerostin inhibition reverses substantial bone loss in the vast majority of the SCI population who have been injured for several years. This preclinical study tested the efficacy of Scl-Ab to reverse the bone loss that has occurred in a rodent model after chronic motor-complete SCI. Male Wistar rats underwent either complete spinal cord transection or only laminectomy. Twelve weeks after SCI, the rats were treated with Scl-Ab at 25 mg/kg/week or vehicle for 8 weeks. In the SCI group that did not receive Scl-Ab, 20 weeks of SCI resulted in a significant reduction of bone mineral density (BMD) and estimated bone strength, and deterioration of bone structure at the distal femoral metaphysis. Treatment with Scl-Ab largely restored BMD, bone structure, and bone mechanical strength. Histomorphometric analysis showed that Scl-Ab increased bone formation in animals with chronic SCI. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increased Tcf7, ENC1, and the OPG/RANKL ratio expression, and decreased SOST expression. Our findings demonstrate for the first time that Scl-Ab reverses the sublesional bone loss when therapy is begun after relatively prolonged spinal cord transection. The study suggests that, in addition to being a treatment option to prevent bone loss after acute SCI, sclerostin antagonism may be a valid clinical approach to reverse the severe bone loss that invariably occurs in patients with chronic SCI.

Romosozumab Treatment Converts Trabecular Rods into Trabecular Plates in Male Cynomolgus Monkeys.

Treatment with sclerostin antibody (romosozumab) increases bone formation while reducing bone resorption, leading to increases in bone volume and bone mineral density. Sclerostin antibody treatment may also provide beneficial changes in trabecular microarchitecture and strength that are not reflected in bone volume and density. Here we use three-dimensional dynamic histomorphometry to determine longitudinal changes in vertebral trabecular microarchitecture in adolescent male cynomolgus monkeys (4-5 years old) treated with sclerostin antibody. Animals were treated bi-weekly with either sclerostin antibody (30 mg/kg, sc, n = 6) or vehicle (n = 6) for 10 weeks. Animals were administered fluorochrome bone formation labels on days 14 and 24 (tetracycline) and on days 56 and 66 (calcein), followed by necropsy on day 70. Cylindrical specimens of cancellous bone from the 5th lumbar vertebrae were used to generate high-resolution, three-dimensional images of bone and fluorescent labels of bone formation (0.7 × 0.7 × 5.0 µm/voxel). The three-dimensional images of the bone formation labels were used to determine the bone volume formed between days 14 and 66 and the resulting alterations in trabecular microarchitecture within each bone. Treatment with sclerostin antibody resulted in a conversion of rod-like trabeculae into plate-like trabeculae at a higher rate than in vehicle-treated animals (p = 0.01). Plate bone volume fraction was greater in the sclerostin antibody group relative to vehicle (mean 43 vs. 30%, p < 0.05). Bone formation increased the thickness of trabeculae in all three trabecular orientations (axial, oblique, and transverse, p < 0.05). The volume of bone formed between days 14 to 66 was greater in sclerostin antibody-treated groups (9.0 vs. 5.4%, p = 0.02), and new bone formation due to sclerostin antibody treatment was associated with increased apparent stiffness as determined from finite element models. Our results demonstrate that increased bone formation associated with sclerostin antibody treatment increases plate-like trabecular morphology and improves mechanical performance.

Comparing the incidence of bone tumors in rats chronically exposed to the selective PTH type 1 receptor agonist abaloparatide or PTH(1-34).

Prolonged treatment with human parathyroid hormone (hPTH) in rats results in development of bone tumors, though this finding has not been supported by clinical experience. The PTH type 1 receptor agonist abaloparatide, selected for its bone anabolic activity, is under clinical development to treat postmenopausal women with osteoporosis. To determine the carcinogenic potential of abaloparatide, Fischer (F344) rats were administered SC daily abaloparatide at doses of 0, 10, 25, and 50 µg/kg or 30 µg/kg hPTH(1-34) as a positive control for up to 2 years. Robust increases in bone density were achieved at all abaloparatide doses and with hPTH(1-34). Comprehensive histopathological analysis reflected a comparable continuum of proliferative changes in bone, mostly osteosarcoma, in both abaloparatide and hPTH(1-34) treated rats. Comparing the effects of abaloparatide and hPTH(1-34) at the 25 and 30 µg/kg respective doses, representing similar exposure multiples to the human therapeutic doses, revealed similar osteosarcoma-associated mortality, tumor incidence, age at first occurrence, and metastatic potential. There were no increases in the incidence of non-bone tumors with abaloparatide compared to vehicle. Thus, near life-long treatment with abaloparatide in rats resulted in dose and time dependent formation of osteosarcomas, with a comparable response to hPTH(1-34) at similar exposure.

Carcinogenicity risk assessment of romosozumab: A review of scientific weight-of-evidence and findings in a rat lifetime pharmacology study.

Romosozumab is a humanized immunoglobulin G2 monoclonal antibody that binds and blocks the action of sclerostin, a protein secreted by the osteocyte and an extracellular inhibitor of canonical Wnt signaling. Blockade of sclerostin binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) allows Wnt ligands to activate canonical Wnt signaling in bone, increasing bone formation and decreasing bone resorption, making sclerostin an attractive target for osteoporosis therapy. Because romosozumab is a bone-forming agent and an activator of canonical Wnt signaling, questions have arisen regarding a potential carcinogenic risk. Weight-of-evidence factors used in the assessment of human carcinogenic risk of romosozumab included features of canonical Wnt signaling, expression pattern of sclerostin, phenotype of loss-of-function mutations in humans and mice, mode and mechanism of action of romosozumab, and findings from romosozumab chronic toxicity studies in rats and monkeys. Although the weight-of-evidence factors supported that romosozumab would pose a low carcinogenic risk to humans, the carcinogenic potential of romosozumab was assessed in a rat lifetime study. There were no romosozumab-related effects on tumor incidence in rats. The findings of the lifetime study and the weight-of-evidence factors collectively indicate that romosozumab administration would not pose a carcinogenic risk to humans.

Dickkopf-1 is a master regulator of joint remodeling.

Degenerative and inflammatory joint diseases lead to a destruction of the joint architecture. Whereas degenerative osteoarthritis results in the formation of new bone, rheumatoid arthritis leads to bone resorption. The molecular basis of these different patterns of joint disease is unknown. By inhibiting Dickkopf-1 (DKK-1), a regulatory molecule of the Wnt pathway, we were able to reverse the bone-destructive pattern of a mouse model of rheumatoid arthritis to the bone-forming pattern of osteoarthritis. In this way, no overall bone erosion resulted, although bony nodules, so-called osteophytes, did form. We identified tumor necrosis factor-alpha (TNF) as a key inducer of DKK-1 in the mouse inflammatory arthritis model and in human rheumatoid arthritis. These results suggest that the Wnt pathway is a key regulator of joint remodeling.

Palopegteriparatide Treatment Improves Renal Function in Adults with Chronic Hypoparathyroidism: 1-Year Results from the Phase 3 PaTHway Trial.

INTRODUCTION: Individuals with chronic hypoparathyroidism managed with conventional therapy (active vitamin D and calcium) have an increased risk for renal dysfunction versus age- and sex-matched controls. Treatments that replace the physiologic effects of parathyroid hormone (PTH) while reducing the need for conventional therapy may help prevent a decline in renal function in this population. This post hoc analysis examined the impact of palopegteriparatide treatment on renal function in adults with chronic hypoparathyroidism. METHODS: PaTHway is a phase 3 trial of palopegteriparatide in adults with chronic hypoparathyroidism that included a randomized, double-blind, placebo-controlled 26-week period followed by an ongoing 156-week open-label extension (OLE) period. Changes in renal function over 52 weeks (26 weeks blinded + 26 weeks OLE) were assessed using estimated glomerular filtration rate (eGFR). A subgroup analysis was performed with participants stratified by baseline eGFR < 60 or ≥ 60 mL/min/1.73 m2. RESULTS: At week 52, over 95% (78/82) of participants remained enrolled in the OLE and of those, 86% maintained normocalcemia and 95% achieved independence from conventional therapy (no active vitamin D and ≤ 600 mg/day of calcium), with none requiring active vitamin D. Treatment with palopegteriparatide over 52 weeks resulted in a mean (SD) increase in eGFR of 9.3 (11.7) mL/min/1.73 m2 from baseline (P < 0.0001) and 43% of participants had an increase ≥ 10 mL/min/1.73 m2. In participants with baseline eGFR < 60 mL/min/1.73 m2, 52 weeks of treatment with palopegteriparatide resulted in a mean (SD) increase of 11.5 (11.3) mL/min/1.73 m2 (P < 0.001). One case of nephrolithiasis was reported for a participant in the placebo group during blinded treatment; none were reported through week 52 with palopegteriparatide. CONCLUSION: In this post hoc analysis of the PaTHway trial, palopegteriparatide treatment was associated with significantly improved eGFR at week 52 in addition to previously reported maintenance and normalization of serum and urine biochemistries. Further investigation of palopegteriparatide for the preservation of renal function in hypoparathyroidism is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT04701203.

Chronic hypoparathyroidism is caused by inadequate parathyroid hormone (PTH) levels. Hypoparathyroidism is managed with conventional therapy (active vitamin D and calcium), but over time the disease itself and conventional therapy can increase the risk of medical complications including kidney problems. This study looked at how a new treatment for chronic hypoparathyroidism, palopegteriparatide (approved in the European Union under the brand name YORVIPATH^®), affects kidney function in adults in the PaTHway clinical trial. Participants were randomly assigned to receive palopegteriparatide or a placebo injection once daily along with conventional therapy. For both groups, clinicians used a protocol to eliminate conventional therapy while maintaining normal blood calcium levels. After 26 weeks, participants on placebo switched to palopegteriparatide. Ninety-five percent of participants were still enrolled in the PaTHway trial after 52 weeks. Of those, 86% had normal blood calcium levels and 95% did not need conventional therapy (not taking vitamin D and not taking therapeutic doses of calcium [> 600 mg/day]). After 52 weeks of treatment with palopegteriparatide, significant improvements were seen in a measure of kidney function called estimated glomerular filtration rate (eGFR). Improvements in eGFR from the beginning of the trial to week 52 were considered clinically meaningful for over 57% of participants. In participants with impaired kidney function at the beginning of the trial, eGFR improvements were even greater, and 74% of participants had a clinically meaningful improvement. These results suggest that palopegteriparatide treatment may be beneficial for kidney function in adults with chronic hypoparathyroidism, especially those with impaired kidney function.

Dual Inhibition of the Wnt Inhibitors DKK1 and Sclerostin Promotes Fracture Healing and Increases the Density and Strength of Uninjured Bone: An Experimental Study in Nonhuman Primates.

BACKGROUND: Fracture repair involves the reactivation of developmental signaling cascades, including Wnt signaling that stimulates bone formation and bone regeneration. Rodent data indicate that dual inhibition of the Wnt signaling antagonists sclerostin and Dickkopf-1 (DKK1) increases callus bone volume and strength while increasing bone mass systemically. METHODS: We evaluated the effects of 16 weeks of subcutaneously administered carrier solution (vehicle, VEH), anti-sclerostin antibody (Scl-Ab), anti-DKK1 antibody (DKK1-Ab), or Scl-Ab plus DKK1-Ab combination therapy (COMBO) on ulnar osteotomy healing in nonhuman primates (cynomolgus monkeys; 20 to 22 per group). RESULTS: Scl-Ab and COMBO therapy increased systemic markers of bone formation versus VEH, with COMBO leading to synergistic increases versus Scl-Ab or DKK1-Ab monotherapies. The COMBO and Scl-Ab groups showed reduced serum markers of bone resorption versus VEH. The COMBO and DKK1-Ab groups exhibited greater callus bone mineral density (BMD), torsional stiffness, and torsional rigidity versus VEH. Lumbar vertebrae from the Scl-Ab and COMBO groups showed greater BMD and bone formation rate versus VEH, and the femoral mid-diaphysis of the Scl-Ab and COMBO groups showed greater periosteal and endocortical bone formation rates versus VEH. CONCLUSIONS: DKK1-Ab increased BMD and strength at the ulnar osteotomy site, Scl-Ab increased bone formation and BMD at uninjured skeletal sites, and Scl-Ab plus DKK1-Ab combination therapy induced all of these effects, in some cases to a greater degree versus 1 or both monotherapies. These results in nonhuman primates suggest that DKK1 preferentially regulates bone healing while sclerostin preferentially regulates systemic bone mass. CLINICAL RELEVANCE: Combination therapy with antibodies against sclerostin and DKK1 may offer a promising therapeutic strategy for both fracture treatment and fracture prevention.

Are osteoclasts needed for the bone anabolic response to parathyroid hormone? A study of intermittent parathyroid hormone with denosumab or alendronate in knock-in mice expressing humanized RANKL.

PTH stimulates osteoblastic cells to form new bone and to produce osteoblast-osteoclast coupling factors such as RANKL. Whether osteoclasts or their activity are needed for PTH anabolism remains uncertain. We treated ovariectomized huRANKL knock-in mice with a human RANKL inhibitor denosumab (DMAb), alendronate (Aln), or vehicle for 4 weeks, followed by co-treatment with intermittent PTH for 4 weeks. Loss of bone mass and microarchitecture was prevented by Aln and further significantly improved by DMAb. PTH improved bone mass, microstructure, and strength, and was additive to Aln but not to DMAb. Aln inhibited biochemical and histomorphometrical indices of bone turnover,--i.e. osteocalcin and bone formation rate (BFR) on cancellous bone surfaces-, and Dmab inhibited them further. However Aln increased whereas Dmab suppressed osteoclast number and surfaces. PTH significantly increased osteocalcin and bone formation indices, in the absence or presence of either antiresorptive, although BFR remained lower in presence of Dmab. To further evaluate PTH effects in the complete absence of osteoclasts, high dose PTH was administered to RANK(-/-) mice. PTH increased osteocalcin similarly in RANK(-/-) and WT mice. It also increased BMD in RANK(-/-) mice, although less than in WT. These results further indicate that osteoclasts are not strictly required for PTH anabolism, which presumably still occurs via stimulation of modeling-based bone formation. However the magnitude of PTH anabolic effects on the skeleton, in particular its additive effects with antiresorptives, depends on the extent of the remodeling space, as determined by the number and activity of osteoclasts on bone surfaces.

Bone quality is partially recovered after the discontinuation of RANKL administration in rats by increased bone mass on existing trabeculae: an in vivo micro-CT study.

UNLABELLED: Bone loss and recovery in a receptor activator for nuclear factor κ B ligand (RANKL)-administered rat model was assessed. Microarchitecture, mineralization and strength deteriorated faster than ovariectomy (OVX). Recovery was dependent on the loss of trabecular elements and connections. Early recovery suggests a natural mechanism in rats to overcome excess RANKL, and may have implications for long-term bone loss. PURPOSE: To compare a model for experimental osteoporosis that induces bone loss by injecting RANKL into rats to an OVX rat model, and measure subsequent recovery of bone architecture, mineralization, and mechanics after stopping injections. METHODS: Mature, healthy, female Wistar rats were divided into high-dose RANKL, low-dose RANKL, OVX, and vehicle control groups. The right proximal tibiae were micro-computed tomography (micro-CT) scanned in vivo every 2 weeks from week 0 to week 12 and every 4 weeks from week 12 to week 20. Bone architectural, mineralization, and mechanical changes were determined. Serum calcium, RANKL, anti-RANKL, and osteoprotegerin were measured at weeks 0, 6, and 20. RESULTS: High-dose RANKL administration resulted in severe deterioration of the trabecular architecture (39% of baseline BV/TV), and modest decreases in tissue mineralization, bone mass, and stiffness. Bone loss occurred more rapidly than in the OVX and low-dose RANKL group, and recovery occurred prior to stopping RANKL injections. Full recovery of trabecular thickness, tissue mineralization, and cortical bone mass, partial recovery of trabecular bone volume (55% of baseline), structural model index, bone mass (69% of baseline), and stiffness (90% of baseline) but no improvement in connectivity density or trabecular number was observed. CONCLUSION: RANKL administration resulted in rapid and dose-dependent bone loss. The recovery of trabecular bone volume and stiffness appeared to be dependent on the number of remaining trabecular elements and their interconnections. Uncontrolled recovery suggests that further investigation into the RANKL-injected rat as a model of bone loss is required.

Effects of systemically administered abaloparatide, an osteoanabolic PTHrP analog, as an adjuvant therapy for spinal fusion in rats.

BACKGROUND: Abaloparatide is a parathyroid hormone receptor agonist that increases bone formation and reduces vertebral and nonvertebral fracture risk in women with postmenopausal osteoporosis. Animal studies indicate abaloparatide stimulates vertebral bone formation and enhances bony bridging and biomechanical stability of fracture calluses. AIMS: The current study is evaluating the potential utility for abaloparatide as an adjunct therapy for spinal fusions. MATERIAL AND METHODS: The effects of 14 or 28 days of daily subcutaneous injections of abaloparatide (20 µg/kg/d) or vehicle were evaluated in 32 male Sprague-Dawley rats starting 1 day after noninstrumented posterolateral fusion (PLF) with bone autograft. Fusion mass microarchitecture was analyzed by micro-computed tomography (micro-CT) and serum markers of bone formation and bone resorption were evaluated. Motion segments were scored in a blinded manner as fused or unfused by postmortem radiography and manual palpation. RESULTS: Abaloparatide-treated rats showed higher bone formation (serum osteocalcin) at day 14 and 28 compared with vehicle controls, without increases in the bone resorption marker serum TRACP-5b. Micro-CT showed greater trabecular number in fusion masses from the abaloparatide group vs vehicle controls at day 14. Manual palpation and radiography indicated no fusions in either group at day 14, whereas 25% of vehicle-treated rats and 50% of abaloparatide-treated rats had bilateral fusion at day 28. DISCUSSION AND CONCLUSION: In summary, this rat PLF model showed that abaloparatide treatment was associated with higher levels of the bone formation marker osteocalcin, improved fusion mass architecture, and a non- significant 2-fold higher fusion rate compared with vehicle.

Gender-Related Impact of Sclerostin Antibody on Bone in the Osteogenesis Imperfecta Mouse.

Osteogenesis imperfecta (OI), which is most often due to a collagen type 1 gene mutation, is characterized by low bone density and bone fragility. In OI patients, gender-related differences were reported, but data in the literature are not convergent. We previously observed that sclerostin antibody (Scl-Ab), which stimulates osteoblast Wnt pathway via sclerostin inactivation, improved spine and long-bone parameters and biomechanical strength in female oim/oim mice, a validated model of human type 3 OI. Here, we wanted to highlight the effect of Scl-Ab on male oim/oim bones in order to identify a possible distinct therapeutic effect from that observed in females. According to the same protocol as our previous study with female mice, male wild-type (Wt) and oim/oim mice received vehicle or Scl-Ab from 5 to 14 weeks of age. Clinimetric and quantitative bone parameters were studied using X-rays, peripheral quantitative computed tomography, microradiography, and dynamic histomorphometry and compared to those of females. Contrary to Wt mice, male oim/oim had significantly lower weight, snout-sacrum length, and bone mineral content than females at 5 weeks. No significant difference in these clinimetric parameters was observed at 14 weeks, whereas male oim showed significantly more long-bone fractures than females. Scl-Ab improved bone mineral density and bone volume/total volume ratio (BV/TV) of vertebral body in Wt and oim/oim, without significant difference between male and female at 14 weeks. Male vehicle oim/oim had a significantly lower cortical thickness (Ct.Th) and BV/TV of tibial diaphysis than female and showed a higher number of fractures at 14 weeks. Scl-Ab increased midshaft periosteal apposition rate in such a way that tibial Ct.Th of male oim/oim was not significantly different from the female one at 14 weeks. The number of fractures was lower in male than female oim/oim after 14 weeks of Scl-Ab treatment, but this difference was not significant. Nevertheless, Scl-Ab-treated oim/oim male and female mice remained smaller than the Wt ones. In conclusion, our results highlighted differences between male and female oim/oim at 4 and 14 weeks of age, as well as some male-specific response of cortical bone to Scl-Ab. These gender-related particularities of oim/oim should be considered when testing experimental treatments.

Early Effects of Abaloparatide on Bone Formation and Resorption Indices in Postmenopausal Women With Osteoporosis.

Anabolic osteoporosis drugs improve bone mineral density by increasing bone formation. The objective of this study was to evaluate the early effects of abaloparatide on indices of bone formation and to assess the effect of abaloparatide on modeling-based formation (MBF), remodeling-based formation (RBF), and overflow MBF (oMBF) in transiliac bone biopsies. In this open-label, single-arm study, 23 postmenopausal women with osteoporosis were treated with 80 µg abaloparatide daily. Subjects received double fluorochrome labels before treatment and before biopsy collection at 3 months. Change in dynamic histomorphometry indices in four bone envelopes were assessed. Median mineralizing surface per unit of bone surface (MS/BS) increased to 24.7%, 48.7%, 21.4%, and 16.3% of total surface after 3 months of abaloparatide treatment, representing 5.5-, 5.2-, 2.8-, and 12.9-fold changes, on cancellous, endocortical, intracortical, and periosteal surfaces (p < .001 versus baseline for all). Mineral apposition rate (MAR) was significantly increased only on intracortical surfaces. Bone formation rate (BFR/BS) was significantly increased on all four bone envelopes. Significant increases versus baseline were observed in MBF on cancellous, endocortical, and periosteal surfaces, for oMBF on cancellous and endocortical surfaces, and for RBF on cancellous, endocortical, and intracortical surfaces. Overall, modeling-based formation (MBF + oMBF) accounted for 37% and 23% of the increase in bone-forming surface on the endocortical and cancellous surfaces, respectively. Changes from baseline in serum biomarkers of bone turnover at either month 1 or month 3 were generally good surrogates for changes in histomorphometric endpoints. In conclusion, treatment with abaloparatide for 3 months stimulated bone formation on cancellous, endocortical, intracortical, and periosteal envelopes in transiliac bone biopsies obtained from postmenopausal women with osteoporosis. These increases reflected stimulation of both remodeling- and modeling-based bone formation, further elucidating the mechanisms by which abaloparatide improves bone mass and lowers fracture risk. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Effects of abaloparatide and teriparatide on bone resorption and bone formation in female mice.

Intermittent administration of PTH type 1 receptor (PTH1R) agonists increases bone remodeling, with greater stimulation of bone formation relative to bone resorption causing net gains in bone mass. This pharmacodynamic feature underlies the bone-building effects of teriparatide and abaloparatide, the only PTH1R agonists approved to reduce osteoporotic fracture risk in postmenopausal women. This study in 8-week-old female mice compared bone resorption and formation responses to these agents delivered at the same 10 µg/kg dose, and a 40 µg/kg abaloparatide dose was also included to reflect its 4-fold higher approved clinical dose. Peptides or vehicle were administered by daily supra-calvarial subcutaneous injection for 12 days, and local (calvarial) and systemic (L5 vertebral and tibial) responses were evaluated by histomorphometry. Terminal bone histomorphometry data indicated that calvarial resorption cavities were similar in both abaloparatide groups versus vehicle controls, whereas the teriparatide group had more calvarial resorption cavities compared with the vehicle or abaloparatide 40 µg/kg groups. The bone resorption marker serum CTX was significantly lower in the abaloparatide 40 µg/kg group and similar in the other two active treatment groups compared with vehicle controls. Both peptides increased trabecular bone formation rate (BFR) in L5 and proximal tibia versus vehicle, and L5 BFR was higher with abaloparatide 40 µg/kg versus teriparatide. At the tibial diaphysis, periosteal BFR was higher with abaloparatide 40 µg/kg versus vehicle or teriparatide, and endocortical BFR was higher with teriparatide but not with abaloparatide 10 or 40 µg/kg versus vehicle. Few differences in structural or microarchitectural bone parameters were observed with this brief duration of treatment. In summary, calvarial bone resorption cavity counts were higher in the teriparatide group versus the vehicle and abaloparatide 40 µg/kg groups, and the abaloparatide 40 µg/kg group had lower serum CTX versus vehicle. L5 and tibial trabecular bone formation indices were higher in all three active treatment groups versus vehicle. The abaloparatide 40 µg/kg group had higher L5 trabecular BFR and tibial periosteal BFR versus teriparatide, whereas tibial endocortical BFR was higher with teriparatide but not abaloparatide. Together, these findings in female mice indicate that an improved balance of bone formation versus bone resorption is established shortly after initiating treatment with abaloparatide.

Sclerostin antibody reduces long bone fractures in the oim/oim model of osteogenesis imperfecta.

Osteogenesis imperfecta type III (OI) is a serious genetic condition with poor bone quality and a high fracture rate in children. In a previous study, it was shown that a monoclonal antibody neutralizing sclerostin (Scl-Ab) increases strength and vertebral bone mass while reducing the number of axial fractures in oim/oim, a mouse model of OI type III. Here, we analyze the impact of Scl-Ab on long bones in OI mice. After 9 weeks of treatment, Scl-Ab significantly reduced long bone fractures (3.6 ±â€¯0.3 versus 2.1 ±â€¯0.8 per mouse, p < 0.001). In addition, the cortical thickness of the tibial midshaft was increased (+42%, p < 0.001), as well as BMD (+28%, p < 0.001), ultimate load (+86%, p < 0.05), plastic energy (+184%; p < 0.05) and stiffness (+172%; p < 0.01) in OI Scl-Ab mice compared to OI vehicle controls. Similar effects of Scl-Ab were observed in Wild type (Wt) mice. The plastic energy, which reflects the fragility of the tissue, was lower in the OI than in the Wt and significantly improved with the Scl-Ab treatment. At the tissue level by nanoindentation, Scl-Ab slightly increased the elastic modulus in bones of both OI and Wt, while moderately increasing tissue hardness (+13% compared to the vehicle; p < 0.05) in Wt bones, but not in OI bones. Although it did not change the properties of the OI bone matrix material, Scl-Ab reduced the fracture rate of the long bones by improving its bone mass, density, geometry, and biomechanical strength. These results suggest that Scl-Ab can reduce long-bone fractures in patients with OI.

Decreased osteoprogenitor proliferation precedes attenuation of cancellous bone formation in ovariectomized rats treated with sclerostin antibody.

Sclerostin antibody (Scl-Ab) stimulates bone formation, which with long-term treatment, attenuates over time. The cellular and molecular mechanisms responsible for the attenuation of bone formation are not well understood, but in aged ovariectomized (OVX) rats, the reduction in vertebral cancellous bone formation is preceded by a reduction in osteoprogenitor (OP) number and significant induction of signaling pathways known to suppress mitogenesis and cell cycle progression in the osteocyte (OCy) (Taylor et al., 2016). To determine if the reduction in OP number is associated with a decrease in proliferation, aged OVX rats were administered vehicle or Scl-Ab for 9 or 29 days and implanted with continuous-delivery 5-bromo-2'-deoxyuridine (BrdU) mini-osmotic pumps 5 days prior to necropsy. The total number of BrdU-labeled osteoblasts (OB) was quantified in vertebral cancellous bone to indirectly assess the effects of Scl-Ab treatment on OP proliferation at the time of activation of modeling-based bone formation at day 9 and at the time of maximal mineralizing surface, initial decrease in OP number, and transcriptional changes in the OCy at day 29. Compared with vehicle, Scl-Ab resulted in an increase in the total number of BrdU-positive OB (+260%) at day 9 that decreased with continued treatment (+50%) at day 29. These differences in proliferation occurred at time points when the increase in total OB number was significant and similar in magnitude. These findings suggest that reduced OP proliferation contributes to the decrease in OP numbers, an effect that would limit the OB pool and contribute to the attenuation of bone formation that occurs with long-term Scl-Ab treatment.

Differential time-dependent transcriptional changes in the osteoblast lineage in cortical bone associated with sclerostin antibody treatment in ovariectomized rats.

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) results in stimulation of bone formation on cancellous (Cn), endocortical (Ec), and periosteal (Ps) surfaces in rodents and non-human primates. With long-term dosing of Scl-Ab, the increase in bone formation is not sustained, attenuating first on Cn surfaces and later on Ec and Ps surfaces. In Cn bone, the attenuation in bone formation (self-regulation) is associated with transcriptional changes in the osteocyte (OCy) that would limit mitogenesis and are sustained with continued dosing. The expression changes in Cn OCy occur coincident with a decrease in osteoprogenitor (OP) numbers that may directly or indirectly be a consequence of the transcriptional changes in the OCy to limit OP proliferation. To characterize the Scl-Ab-mediated changes in cortical (Ct) bone and compare these changes to Cn bone, densitometric, histomorphometric, and transcriptional analyses were performed on femur diaphyses from aged ovariectomized rats. Animals were administered 50 mg/kg/wk of Scl-Ab or vehicle for up to 6 months (183 days), followed by a treatment-free period (up to 126 days). Scl-Ab increased Ct mass and area through day 183, which declined slightly when treatment was discontinued. Ps and Ec bone formation was sustained through the dosing on both Ct surfaces, with evidence of a decline in bone formation only at day 183 on the Ec surface. This is in contrast to Cn bone, where reduced bone formation was observed after day 29. TaqMan analysis of 60 genes with functional roles in the bone using mRNA isolated from laser capture micro-dissection samples enriched for Ec osteoblasts and Ct OCy suggest a pattern of gene expression in Ct bone that differed from Cn, especially in the OCy, and that corresponded to observed differences in the timing of phenotypic changes. Notable with Scl-Ab treatment was a "transcriptional switch" in Ct OCy at day 183, coincident with the initial decline in bone formation on the endocortex. A consistent sustained increase of expression for most genes in response to Scl-Ab was observed from day 8 through day 85 at the times of maximal bone formation on both Ct surfaces; however, at day 183, this increase was reversed, with expression of these genes generally returning to control values or decreasing compared to vehicle. Genes exhibiting this pattern included Wnt inhibitors Sost and Dkk1, though both had been up-regulated until the end of dosing in Cn OCy. Changes in cell cycle genes such as Cdkn1a and Ndrg1 in Ct OCy suggested up-regulation of p53 signaling, as observed in Cn OCy; however, unlike in Cn bone, p53 signaling was not associated with decreased bone formation and was absent at day 183, when bone formation began to decline on the Ec surface. These data demonstrate involvement of similar molecular pathways in Ct and Cn bone in response to Scl-Ab but with a different temporal relationship to bone formation and suggest that the specific mechanism underlying self-regulation of Scl-Ab-induced bone formation may be different between Cn and Ct bone.

Sclerostin Antibody Reverses Bone Loss by Increasing Bone Formation and Decreasing Bone Resorption in a Rat Model of Male Osteoporosis.

Sclerostin antibody (Scl-Ab) restored bone mass and strength in the ovariectomized rat model of postmenopausal osteoporosis. Increased bone mineral density (BMD) and decreased skeletal fragility fracture risk have been reported in postmenopausal osteoporotic women receiving Scl-Ab. In males, loss of androgen leads to rapid decreases in BMD and an increased risk of fragility fractures. We hypothesized that Scl-Ab could reverse the loss of bone mass and strength caused by androgen ablation in the orchiectomized (ORX) rat model of male osteoporosis. We treated 9-month-old ORX Sprague Dawley rats (3 months after ORX) subcutaneously twice weekly with vehicle or Scl-Ab (5 or 25 mg/kg) for 6 weeks (n = 10 per group). Both doses of Scl-Ab fully reversed the BMD deficit in the lumbar spine and femur and tibia in ORX rats. Microcomputed tomography showed that the bone mass in the fifth lumbar vertebral body, femur diaphysis, and femoral neck were dose-dependently restored by Scl-Ab. The bone strength at these sites increased significantly with Scl-Ab to levels matching those of sham-operated controls and correlated positively with improvements in bone mineral content, demonstrating bone quality maintenance. Dynamic histomorphometry of the tibial diaphysis and second lumbar vertebral body demonstrated that Scl-Ab significantly increased bone formation on periosteal, endocortical, and trabecular surfaces and significantly decreased bone resorption on endocortical and trabecular surfaces. The effects of Scl-Ab on increasing bone formation and decreasing bone resorption led to restoration of bone mass and strength in androgen-deficient rats. These findings support the ongoing evaluation of Scl-Ab as a potential therapeutic agent for osteoporosis in men.

Kinetic reconstruction reveals time-dependent effects of romosozumab on bone formation and osteoblast function in vertebral cancellous and cortical bone in cynomolgus monkeys.

Romosozumab, a humanized monoclonal sclerostin antibody under development for the treatment of osteoporosis, has a unique mechanism of action on bone-increasing bone formation and decreasing bone resorption. The effects on bone formation are transient, eliciting a rapid increase in bone formation that attenuates with continued treatment. Although bone formation attenuates, bone mineral density (BMD) continues to increase. To explore potential tissue-level mechanisms that could contribute to a progressive increase in spine BMD, we used kinetic reconstruction techniques to examine the effects of romosozumab on modeling and remodeling units in vertebral cancellous bone from adult cynomolgus monkeys administered romosozumab for 10 and 28weeks. The 10-week study duration captured a period of high modeling-based bone formation, and the 28-week study duration followed the self-regulation or attenuation of bone formation in cancellous bone that occurs with long-term treatment. Sequential fluorochrome labels applied for the kinetic reconstruction were also used to evaluate treatment effects on osteoblast function as early as 3weeks, and on bone formation and bone accrual in the vertebral cortex over 28weeks. Kinetic reconstruction of remodeling and modeling formation sites in vertebral cancellous bone revealed that romosozumab effected significant transient increases in mineral apposition rate in remodeling sites at week 3 that was not sustained with continued treatment. However, romosozumab treatment caused sustained improvement in fractional labeling of osteoid, an index of osteoblast efficiency, at remodeling formative sites at both weeks 10 and 28 that was the major contributor to significant increases in final wall thickness (W.Th) of remodeling packets. Remodeling W.Th matched the final W.Th of modeling packets at week 10. At both weeks 10 and 28, romosozumab significantly decreased eroded surface (ES/BS). At week 28, romosozumab also significantly reduced resorption period (Rs.P) and final resorption depth (Rs.De). The reduced final Rs.De combined with the increased W.Th resulted in a significant increase in bone balance (BB) at the level of the remodeling unit. Assessment of bone formation on the vertebral periosteal and endocortical surfaces following 28weeks of treatment revealed that romosozumab significantly increased bone formation on these surfaces, which had attenuated by week 28, resulting in significant increases in new periosteal and endocortical bone by week 28. These data suggest that multiple factors potentially contribute to the increase in spine BMD with romosozumab treatment. In the early period of treatment, increased modeling-based bone formation, increased W.Th at remodeling sites, a decrease in remodeling space secondary to decreased ES/BS in vertebral cancellous bone, and increased periosteal and endocortical bone formation in the vertebral cortex contribute to the early increase in spine BMD. Following the self-regulation of bone formation when modeling-based bone formation has attenuated, a decrease in remodeling space secondary to reduced ES/BS and a positive BB secondary to decreased final Rs.De and increased W.Th contribute to the progressive increase in spine BMD with long-term treatment.

Sclerostin Antibody Treatment Enhances Rotator Cuff Tendon-to-Bone Healing in an Animal Model.

BACKGROUND: Rotator cuff tears are a common source of pain and disability, and poor healing after repair leads to high retear rates. Bone loss in the humeral head before and after repair has been associated with poor healing. The purpose of the current study was to mitigate bone loss near the repaired cuff and improve healing outcomes. METHODS: Sclerostin antibody (Scl-Ab) treatment, previously shown to increase bone formation and strength in the setting of osteoporosis, was used in the current study to address bone loss and enhance rotator cuff healing in an animal model. Scl-Ab was administered subcutaneously at the time of rotator cuff repair and every 2 weeks until the animals were sacrificed. The effect of Scl-Ab treatment was evaluated after 2, 4, and 8 weeks of healing, using bone morphometric analysis, biomechanical evaluation, histological analysis, and gene expression outcomes. RESULTS: Injury and repair led to a reduction in bone mineral density after 2 and 4 weeks of healing in the control and Scl-Ab treatment groups. After 8 weeks of healing, animals receiving Scl-Ab treatment had 30% greater bone mineral density than the controls. A decrease in biomechanical properties was observed in both groups after 4 weeks of healing compared with healthy tendon-to-bone attachments. After 8 weeks of healing, Scl-Ab-treated animals had improved strength (38%) and stiffness (43%) compared with control animals. Histological assessment showed that Scl-Ab promoted better integration of tendon and bone by 8 weeks of healing. Scl-Ab had significant effects on gene expression in bone, indicative of enhanced bone formation, and no effect on the expression of genes in tendon. CONCLUSIONS: This study provides evidence that Scl-Ab treatment improves tendon-to-bone healing at the rotator cuff by increasing attachment-site bone mineral density, leading to improved biomechanical properties. CLINICAL RELEVANCE: Scl-Ab treatment may improve outcomes after rotator cuff repair.