Abaloparatide, a PTH receptor agonist with homology to PTHrP, enhances callus bridging and biomechanical properties in rats with femoral fracture.

Fractures typically heal via endochondral and intramembranous bone formation, which together form a callus that achieves union and biomechanical recovery. PTHrP, a PTH receptor agonist, plays an important physiological role in fracture healing as an endogenous stimulator of endochondral and intramembranous bone formation. Abaloparatide, a novel systemically-administered osteoanabolic PTH receptor agonist that reduces fracture risk in women with postmenopausal osteoporosis, has 76% homology to PTHrP, suggesting it may have potential to improve fracture healing. To test this hypothesis, ninety-six 12-week-old male rats underwent unilateral internally-stabilized closed mid-diaphyseal femoral fractures and were treated starting the next day with daily s.c. saline (Vehicle) or abaloparatide at 5 or 20 µg/kg/d for 4 or 6 weeks (16 rats/group/time point). Histomorphometry and histology analyses indicated that fracture calluses from the abaloparatide groups exhibited significantly greater total area, higher fluorescence scores indicating more newly-formed bone, and higher fracture bridging scores versus Vehicle controls. Callus bridging score best correlated with callus cartilage score (r = 0.64) and fluorescence score (r = 0.67) at week 4, and callus area correlated with cartilage score (r = 0.60) and fluorescence score (r = 0.89) at Week 6. By micro-CT, calluses from one or both abaloparatide groups had greater bone volume, bone volume fraction, bone mineral content, bone mineral density, and cross-sectional area at both time points versus Vehicle controls. Destructive bending tests indicated greater callus maximum load and stiffness in one or both abaloparatide groups at both time points versus Vehicle controls. These results provide preliminary preclinical evidence for improved fracture healing with systemically-administered abaloparatide. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Hypocalcaemia in patients with metastatic bone disease treated with denosumab.

BACKGROUND: This analysis was performed to further characterise treatment-emergent hypocalcaemia in patients with bone metastases receiving denosumab. METHODS: Laboratory abnormalities and adverse events of hypocalcaemia in patients with metastatic bone disease were analysed using data from three identically designed phase 3 trials of subcutaneous denosumab 120 mg (n = 2841) versus intravenous zoledronic acid 4 mg (n = 2836). RESULTS: The overall incidence of laboratory events of hypocalcaemia grade ⩾ 2 was higher with denosumab (12.4%) than with zoledronic acid (5.3%). Hypocalcaemia events were primarily grade 2 in severity and usually occurred within the first 6 months of treatment. Patients who reported taking calcium and/or vitamin D supplements had a lower incidence of hypocalcaemia. Prostate cancer or small-cell lung cancer, reduced creatinine clearance and higher baseline bone turnover markers of urinary N-telopeptide of type I collagen (uNTx; > 50 versus ⩽ 50 nmol/mmol) and bone-specific alkaline phosphatase (BSAP; > 20.77 µg/L [median] versus ⩽ 20.77 µg/L) values were important risk factors for developing hypocalcaemia. The risk associated with increased baseline BSAP levels was greater among patients who had > 2 bone metastases at baseline versus those with ⩽ 2 bone metastases at baseline. CONCLUSION: Hypocalcaemia was more frequent with denosumab versus zoledronic acid, consistent with denosumab's greater antiresorptive effect. Low serum calcium levels and potential vitamin D deficiency should be corrected before initiating treatment with a potent osteoclast inhibitor, and corrected serum calcium levels should be monitored during treatment. Adequate calcium and vitamin D intake appears to substantially reduce the risk of hypocalcaemia.

Progressive increases in bone mass and bone strength in an ovariectomized rat model of osteoporosis after 26 weeks of treatment with a sclerostin antibody.

The effects of up to 26 weeks of sclerostin antibody (Scl-Ab) treatment were investigated in ovariectomized (OVX) rats. Two months after surgery, 6-month-old osteopenic OVX rats were treated with vehicle or Scl-Ab (25 mg/kg, sc, one time per week) for 6, 12, or 26 weeks. In vivo dual-energy x-ray absorptiometry analysis demonstrated that the bone mineral density of lumbar vertebrae and femur-tibia increased progressively through 26 weeks of Scl-Ab treatment along with progressive increases in trabecular and cortical bone mass and bone strength at multiple sites. There was a strong correlation between bone mass and maximum load at lumbar vertebra, femoral neck, and diaphysis at weeks 6 and 26. Dynamic histomorphometric analysis showed that lumbar trabecular and tibial shaft endocortical and periosteal bone formation rates (BFR/BS) increased and peaked at week 6 with Scl-Ab-treatment; thereafter trabecular and endocortical BFR/BS gradually declined but remained significantly greater than OVX controls at week 26, whereas periosteal BFR/BS returned to OVX control levels at week 26. In the tibia metaphysis, trabecular BFR/BS in the Scl-Ab treated group remained elevated from week 6 to week 26. The osteoclast surface and eroded surface were significantly lower in Scl-Ab-treated rats than in OVX controls at all times. In summary, bone mass and strength increased progressively over 26 weeks of Scl-Ab treatment in adult OVX rats. The early gains were accompanied by increased cortical and trabecular bone formation and reduced osteoclast activity, whereas later gains were attributed to residual endocortical and trabecular osteoblast stimulation and persistently low osteoclast activity.

Role of RANK ligand and denosumab, a targeted RANK ligand inhibitor, in bone health and osteoporosis: a review of preclinical and clinical data.

BACKGROUND: Postmenopausal osteoporosis results from bone loss and decreased bone strength mediated by an increased rate of bone remodeling secondary to reduced estrogen levels. Remodeling cycles are initiated by osteoclasts, the formation, function, and survival of which depend on RANK ligand (RANKL). RANKL inhibition therefore represents a novel strategy for reducing remodeling and its effects on fracture risk. OBJECTIVES: The goal of this study was to review the preclinical and clinical evidence supporting the value of RANKL inhibition in conditions of bone loss and to provide the rationale for the use of the fully human antibody denosumab, a RANKL inhibitor, in such conditions. METHODS: We searched PubMed from January 2005 to May 2011 using the following terms: RANK Ligand, RANKL, denosumab, and NOT cancer, metastatic bone, or rheumatoid in the title. RESULTS: The search method retrieved 111 articles. Preclinical evidence from several bone disease models suggests that RANKL inhibition leads to increased bone volume, density, and strength. Denosumab prevents RANKL from binding to its receptor, decreasing osteoclast activity and thereby reducing bone resorption and increasing cortical and trabecular bone mass and strength. It has consistently been reported to reduce bone turnover, increase bone density, and reduce the risk of fracture in clinical studies of postmenopausal women. Phase III head-to-head trials comparing denosumab with the bisphosphonate alendronate reported that denosumab was associated with significantly greater increases in bone density. Eczema as an adverse event and cellulitis as a serious adverse event were more common with denosumab than with placebo. CONCLUSIONS: Preclinical studies defined the role of RANKL in bone remodeling and provided evidence for the therapeutic potential of RANKL inhibition in conditions of bone loss. Clinical studies evaluating RANKL inhibition with denosumab in postmenopausal women have reported significant reductions in vertebral, nonvertebral, and hip fractures, providing evidence compatible with the use of denosumab in postmenopausal women with osteoporosis.

Local delivery of recombinant osteoprotegerin enhances postorthodontic tooth stability.

Relapse after orthodontic tooth movement is a significant problem in orthodontics. The purpose of this study was to examine the efficacy of the osteoclast inhibitor osteoprotegerin-Fc (OPG-Fc) for inhibiting postorthodontic relapse. Rat maxillary molars were moved mesially and allowed to relapse for 24 days. Low-dose (1 mg/kg) or high-dose (5 mg/kg) OPG-Fc or saline was injected adjacent to the molars during relapse. Tooth movement, micro-CT, histologic bone quality, and serum OPG and TRAP-5b were measured. OPG-Fc injections significantly diminished postorthodontic relapse from 63% (0.78/1.20 mm) of total movement in vehicle control rats to 31% (0.31/1.00 mm) in low-dose and 24% (0.28/1.16 mm) in high-dose OPG-Fc groups 24 days after appliance removal. Normalization of bone and periodontal tissues occurred as early as 8 and 16 days in the high- and low-dose OPG-Fc-treated groups, respectively, while the vehicle-treated group showed only partial tissue recovery 24 days following tooth movement. After 24 days of relapse, there was complete recovery to pre-tooth-movement values for bone volume fraction (BVF) and tissue mineral density (TMD) in both the low- and high-dose OPG-Fc groups, while BVF recovered only partially and TMD did not recover in the vehicle control group. Greatly elevated serum OPG levels and reduced serum TRAP-5b levels in OPG-Fc-treated animals indicated systemic exposure to locally injected drug. The profound decrease in postorthodontic relapse by local OPG-Fc administration indicates that osteoclasts are critical to bone maturation following tooth movement and points to the potential pharmacologic use of OPG-Fc or other RANKL inhibitors for orthodontic retention.

Inhibition of sclerostin by monoclonal antibody increases bone formation, bone mass, and bone strength in aged male rats.

The purpose of this study was to evaluate the effects of sclerostin inhibition by treatment with a sclerostin antibody (Scl-AbII) on bone formation, bone mass, and bone strength in an aged, gonad-intact male rat model. Sixteen-month-old male Sprague-Dawley rats were injected subcutaneously with vehicle or Scl-AbII at 5 or 25 mg/kg twice per week for 5 weeks (9-10/group). In vivo dual-energy X-ray absorptiometry (DXA) analysis showed that there was a marked increase in areal bone mineral density of the lumbar vertebrae (L(1) to L(5) ) and long bones (femur and tibia) in both the 5 and 25 mg/kg Scl-AbII-treated groups compared with baseline or vehicle controls at 3 and 5 weeks after treatment. Ex vivo micro-computed tomographic (µCT) analysis demonstrated improved trabecular and cortical architecture at the fifth lumbar vertebral body (L(5) ), femoral diaphysis (FD), and femoral neck (FN) in both Scl-AbII dose groups compared with vehicle controls. The increased cortical and trabecular bone mass was associated with a significantly higher maximal load of L(5) , FD, and FN in the high-dose group. Bone-formation parameters (ie, mineralizing surface, mineral apposition rate, and bone-formation rate) at the proximal tibial metaphysis and tibial shaft were markedly greater on trabecular, periosteal, and endocortical surfaces in both Scl-AbII dose groups compared with controls. These results indicate that sclerostin inhibition by treatment with a sclerostin antibody increased bone formation, bone mass, and bone strength in aged male rats and, furthermore, suggest that pharmacologic inhibition of sclerostin may represent a promising anabolic therapy for low bone mass in aged men.

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.

RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFalpha or anti-IL-1 therapies.

INTRODUCTION: Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFalpha, IL-1beta, and receptor activator of NF-kappaB ligand (RANKL). Anti-IL-1 or anti-TNFalpha therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared. METHODS: Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFalpha inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E2 (PGE2), acute-phase protein alpha-1-acid glycoprotein (alpha1AGP), multiple cytokines) were measured in serum (day 14 post onset). RESULTS: Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFalpha reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFalpha and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFalpha and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum alpha1AGP, IL-1beta, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFalpha and IL-17. Anti-TNFalpha reduced systemic alpha1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic alpha1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model. CONCLUSIONS: Anti-TNFalpha or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.

RANKL is a marker and mediator of local and systemic bone loss in two rat models of inflammatory arthritis.

UNLABELLED: RANKL is an essential mediator of bone erosions, but the role of RANKL in systemic bone loss had not been studied in arthritis. RANKL protein was increased in rat joint extracts and serum at the earliest stages of arthritis. Osteoprotegerin (OPG) treatment reversed local and systemic bone loss, suggesting that RANKL is both a marker and mediator of bone loss in arthritis. INTRODUCTION: RANKL is well established as an essential mediator of bone erosions in inflammatory arthritis, but the role of RANKL in systemic bone loss in arthritis had not been studied. We hypothesized that serum RANKL could serve as both a mediator and as a novel biomarker for local and systemic bone loss in arthritis. We challenged this hypothesis in two established rat models of inflammatory arthritis. We sought to determine whether serum RANKL was elevated early in disease progression and whether RANKL suppression could prevent both local and systemic bone loss in these models. MATERIALS AND METHODS: Detailed time-course studies were conducted in animals with collagen-induced (CIA) or adjuvant-induced (AIA) arthritis to evaluate the onset and progression of inflammation (paw swelling), bone erosions, osteoclast numbers, and RANKL protein levels in arthritic joints and in serum. Additional CIA and AIA rats (n=8/group) received placebo (PBS) or recombinant OPG (3 mg/kg three times weekly) for 10 days beginning 4 days after disease onset (first macroscopic evidence of hind paw erythema and edema) to assess the role of RANKL in local and systemic bone loss. RESULTS: RANKL protein was significantly elevated in the joints and serum of CIA and AIA rats within 1-2 days of disease onset. Increased RANKL levels were associated with local (hind paw) and systemic (vertebral) osteopenia in both models. The RANKL inhibitor OPG prevented local and systemic osteopenia in both models of established disease. CONCLUSIONS: RANKL protein is significantly increased both locally and systemically during the earliest stages of inflammatory arthritis in rats, suggesting that serum RANKL might have prognostic value for bone erosions and systemic osteopenia in this condition. RANKL inhibition through OPG prevented local and systemic bone loss in these arthritis models, suggesting that RANKL inhibition is a promising new approach for treating bone loss in arthritis.

The inhibition of RANKL causes greater suppression of bone resorption and hypercalcemia compared with bisphosphonates in two models of humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy (HHM) is mediated primarily by skeletal and renal responses to tumor-derived PTHrP. PTHrP mobilizes calcium from bone by inducing the expression of receptor activator for nuclear factor-kappaB ligand (RANKL), a protein that is essential for osteoclast formation, activation, and survival. RANKL does not influence renal calcium reabsorption, so RANKL inhibition is a rational approach to selectively block, and thereby reveal, the relative contribution of bone calcium to HHM. We used the RANKL inhibitor osteoprotegerin (OPG) to evaluate the role of osteoclast-mediated hypercalcemia in two murine models of HHM. Hypercalcemia was induced either by sc inoculation of syngeneic colon (C-26) adenocarcinoma cells or by sc injection of high-dose recombinant PTHrP (0.5 mg/kg, s.c., twice per day). In both models, OPG (0.2-5 mg/kg) caused rapid reversal of established hypercalcemia, and the speed and duration of hypercalcemia suppression were significantly greater with OPG (5 mg/kg) than with high-dose bisphosphonates (pamidronate or zoledronic acid, 5 mg/kg). OPG also caused greater reductions in osteoclast surface and biochemical markers of bone resorption compared with either bisphosphonate. In both models, hypercalcemia gradually returned despite clear evidence of ongoing suppression of bone resorption by OPG. These data demonstrate that osteoclasts and RANKL are important mediators of HHM, particularly in the early stages of the condition. Aggressive antiresorptive therapy with a RANKL inhibitor therefore might be a rational approach to controlling HHM.

Repair of local bone erosions and reversal of systemic bone loss upon therapy with anti-tumor necrosis factor in combination with osteoprotegerin or parathyroid hormone in tumor necrosis factor-mediated arthritis.

Local bone erosion and systemic bone loss are hallmarks of rheumatoid arthritis and cause progressive disability. Tumor necrosis factor (TNF) is a key mediator of arthritis and acts catabolically on bone by stimulating bone resorption and inhibiting bone formation. We hypothesized that the concerted action of anti-TNF, which reduces inflammation and parathyroid hormone (PTH), which stimulates bone formation, or osteoprotegerin (OPG), which blocks bone resorption and could lead to repair of local bone erosions and reversal of systemic bone loss. To test this, human TNF-transgenic mice with established erosive arthritis and systemic bone loss were treated with PTH, OPG, and anti-TNF, alone or in combination. Local bone erosions almost fully regressed, on combined treatment with anti-TNF and PTH and/or OPG, suggesting repair of inflammatory skeletal lesions. In contrast, OPG and anti-TNF alone led to arrest of bone erosions but did not achieve repair. Treatment with PTH alone had no influence on the progression of bone erosions. Local bone erosions all showed signs of new bone formation such as the presence of osteoblasts, osteoid formation, and mineralization. Furthermore, systemic bone loss was completely reversed on combined treatment and this effect was mediated by osteoblast stimulation and osteoclast blockade. In summary, we conclude that local joint destruction and systemic inflammatory bone loss because of TNF can regress and that repair requires a combined approach by reducing inflammation, blocking bone resorption, or stimulating bone formation.