Franklin J. Asuncion

Inhibition of sclerostin by monoclonal antibody enhances bone healing and improves bone density and strength of nonfractured bones.

Therapeutic enhancement of fracture healing would help to prevent the occurrence of orthopedic complications such as nonunion and revision surgery. Sclerostin is a negative regulator of bone formation, and treatment with a sclerostin monoclonal antibody (Scl‐Ab) results in increased bone formation and bone mass in animal models. Our objective was to investigate the effects of systemic administration of Scl‐Ab in two models of fracture healing. In both a closed femoral fracture model in rats and a fibular osteotomy model in cynomolgus monkeys, Scl‐Ab significantly increased bone mass and bone strength at the site of fracture. After 10 weeks of healing in nonhuman primates, the fractures in the Scl‐Ab group had less callus cartilage and smaller fracture gaps containing more bone and less fibrovascular tissue. These improvements at the fracture site corresponded with improvements in bone formation, bone mass, and bone strength at nonfractured cortical and trabecular sites in both studies. Thus the potent anabolic activity of Scl‐Ab throughout the skeleton also was associated with an anabolic effect at the site of fracture. These results support the potential for systemic Scl‐Ab administration to enhance fracture healing in patients.

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 (L1 to L5) 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 (L5), 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 L5, 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.

Sclerostin antibody treatment improves bone mass, bone strength, and bone defect regeneration in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus results in increased risk of fracture and delayed fracture healing. ZDF fa/fa rats are an established model of type 2 diabetes mellitus with low bone mass and delayed bone healing. We tested whether a sclerostin‐neutralizing antibody (Scl‐AbVI) would reverse the skeletal deficits of diabetic ZDF rats. Femoral defects of 3 mm were created in 11‐week‐old diabetic ZDF fa/fa and nondiabetic ZDF +/+ rats and stabilized by an internal plate. Saline or 25 mg/kg Scl‐AbVI was administered subcutaneously (s.c.) twice weekly for 12 weeks (n = 9–10/group). Bone mass and strength were assessed using pQCT, micro–computed tomography (µCT), and biomechanical testing. Bone histomorphometry was used to assess bone formation, and the filling of the bone defect was analyzed by µCT. Diabetic rats displayed lower spinal and femoral bone mass compared to nondiabetic rats, and Scl‐AbVI treatment significantly enhanced bone mass of the femur and the spine of diabetic rats (p < 0.0001). Scl‐AbVI also reversed the deficit in bone strength in the diabetic rats, with 65% and 89% increases in maximum load at the femoral shaft and neck, respectively (p < 0.0001). The lower bone mass in diabetic rats was associated with a 65% decrease in vertebral bone formation rate, which Scl‐AbVI increased by sixfold, consistent with a pronounced anabolic effect. Nondiabetic rats filled 57% of the femoral defect, whereas diabetic rats filled only 21% (p < 0.05). Scl‐AbVI treatment increased defect regeneration by 47% and 74%, respectively (p < 0.05). Sclerostin antibody treatment reverses the adverse effects of type 2 diabetes mellitus on bone mass and strength, and improves bone defect regeneration in rats.

A bispecific antibody targeting sclerostin and DKK-1 promotes bone mass accrual and fracture repair

Inhibition of the Wnt antagonist sclerostin increases bone mass in patients with osteoporosis and in preclinical animal models. Here we show increased levels of the Wnt antagonist Dickkopf-1 (DKK-1) in animals treated with sclerostin antibody, suggesting a negative feedback mechanism that limits Wnt-driven bone formation. To test our hypothesis that co-inhibition of both factors further increases bone mass, we engineer a first-in-class bispecific antibody with single residue pair mutations in the Fab region to promote efficient and stable cognate light–heavy chain pairing. We demonstrate that dual inhibition of sclerostin and DKK-1 leads to synergistic bone formation in rodents and non-human primates. Furthermore, by targeting distinct facets of fracture healing, the bispecific antibody shows superior bone repair activity compared with monotherapies. This work supports the potential of this agent both for treatment and prevention of fractures and offers a promising therapeutic approach to reduce the burden of low bone mass disorders.

Increased Bone Formation and Bone Mass Induced by Sclerostin Antibody Is Not Affected by Pretreatment or Cotreatment with Alendronate in Osteopenic, Ovariectomized Rats

Clinical studies have revealed a blunting of the bone anabolic effects of parathyroid hormone treatment in osteoporotic patients in the setting of pre- or cotreatment with the antiresorptive agent alendronate (ALN). Sclerostin monoclonal antibody (Scl-Ab) is currently under clinical investigation as a new potential anabolic therapy for postmenopausal osteoporosis. The purpose of these experiments was to examine the influence of pretreatment or cotreatment with ALN on the bone anabolic actions of Scl-Ab in ovariectomized (OVX) rats. Ten-month-old osteopenic OVX rats were treated with ALN or vehicle for 6 wk, before the start of Scl-Ab treatment. ALN-pretreated OVX rats were switched to Scl-Ab alone or to a combination of ALN and Scl-Ab for another 6 wk. Vehicle-pretreated OVX rats were switched to Scl-Ab or continued on vehicle to serve as controls. Scl-Ab treatment increased areal bone mineral density, volumetric bone mineral density, trabecular and cortical bone mass, and bone strength similarly in OVX rats pretreated with ALN or vehicle. Serum osteocalcin and bone formation rate on trabecular, endocortical, and periosteal surfaces responded similarly to Scl-Ab in ALN or vehicle-pretreated OVX rats. Furthermore, cotreatment with ALN did not have significant effects on the increased bone formation, bone mass, and bone strength induced by Scl-Ab in the OVX rats that were pretreated with ALN. These results indicate that the increases in bone formation, bone mass, and bone strength with Scl-Ab treatment were not affected by pre- or cotreatment with ALN in OVX rats with established osteopenia.

Increased callus mass and enhanced strength during fracture healing in mice lacking the sclerostin gene.

Humans with inherited sclerostin deficiency have high bone mass. Targeted deletion of the sclerostin gene in mice (SOST-KO) causes increases in bone formation, bone mass and bone strength. Inhibition of sclerostin by a monoclonal antibody increases bone formation and enhances fracture healing in rodent and primate models. In this study, we describe the temporal progression of femoral fracture healing in SOST-KO mice compared with wild type (WT) control mice to further characterize the role of sclerostin in fracture healing. Sixty-seven male 9-10 week-old SOST-KO (N=37) and WT (N=30) mice underwent a closed femoral fracture. Weekly radiography was used to monitor the progress of healing. Histologic sections were used to characterize callus composition, evaluate callus bridging, and quantify lamellar bone formation on days 14 and 28. Densitometry and biomechanical testing were utilized to characterize bone mass and strength at the fractured and contralateral femurs on day 45. A significant improvement in time to radiographic healing (no discernible fracture line) was observed in SOST-KO mice, which corresponded to an increase in histologic bony bridging at 14 days (38% versus 0% in WT). Both genotypes appeared to be nearly fully bridged at 28 days post-fracture. The increased bridging at 14 days was associated with 97% greater bone area and 40% lower cartilage area in the callus of SOST-KO mice as compared to WT mice. Bone formation-related endpoints were higher in SOST-KO mice at both 14 and 28 days. At 45 days post-fracture, peak load and bone mass were significantly greater in the fractured femurs of SOST-KO mice as compared to WT mice. In conclusion, fractures in mice lacking sclerostin showed accelerated bridging, greater callus maturation, and increased bone formation and strength in the callus.

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.

One Year of Transgenic Overexpression of Osteoprotegerin in Rats Suppressed Bone Resorption and Increased Vertebral Bone Volume, Density, and Strength

RANKL is an essential mediator of bone resorption, and its activity is inhibited by osteoprotegerin (OPG). Transgenic (Tg) rats were engineered to continuously overexpress OPG to study the effects of continuous long‐term RANKL inhibition on bone volume, density, and strength. Lumbar vertebrae, femurs, and blood were obtained from 1‐yr‐old female OPG‐Tg rats (n = 32) and from age‐matched wildtype (WT) controls (n = 23). OPG‐Tg rats had significantly greater serum OPG (up to 260‐fold) and significantly lower serum TRACP5b and osteocalcin compared with WT controls. Vertebral histomorphometry showed significant reductions in osteoclasts and bone turnover parameters in OPG‐Tg rats versus WT controls, and these reductions were associated with significantly greater peak load in vertebrae tested through compression. No apparent differences in bone material properties were observed in OPG‐Tg rat vertebrae, based on their unchanged intrinsic strength parameters and their normal linear relationship between vertebral bone mass and strength. Femurs from OPG‐Tg rats were of normal length but showed mild osteopetrotic changes, including reduced periosteal perimeter (−6%) and an associated reduction in bending strength. Serum OPG levels in WT rats showed no correlations with any measured parameter of bone turnover, mass, or strength, whereas the supraphysiological serum OPG levels in OPG‐Tg rats correlated negatively with bone turnover parameters and positively with vertebral bone mass and strength parameters. In summary, low bone turnover after 1 yr of OPG overexpression in rats was associated with increased vertebral bone mass and proportional increases in bone strength, with no evidence for deleterious effects on vertebral material properties.

Sclerostin Antibody Treatment Improves the Bone Phenotype of Crtap(-/-) Mice, a Model of Recessive Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is characterized by low bone mass, poor bone quality, and fractures. Standard treatment for OI patients is limited to bisphosphonates, which only incompletely correct the bone phenotype, and seem to be less effective in adults. Sclerostin‐neutralizing antibodies (Scl‐Ab) have been shown to be beneficial in animal models of osteoporosis, and dominant OI resulting from mutations in the genes encoding type I collagen. However, Scl‐Ab treatment has not been studied in models of recessive OI. Cartilage‐associated protein (CRTAP) is involved in posttranslational type I collagen modification, and its loss of function results in recessive OI. In this study, we treated 1‐week‐old and 6‐week‐old Crtap–/– mice with Scl‐Ab for 6 weeks (25 mg/kg, s.c., twice per week), to determine the effects on the bone phenotype in models of “pediatric” and “young adult” recessive OI. Vehicle‐treated Crtap–/– and wild‐type (WT) mice served as controls. Compared with control Crtap–/– mice, micro–computed tomography (μCT) analyses showed significant increases in bone volume and improved trabecular microarchitecture in Scl‐Ab–treated Crtap–/– mice in both age cohorts, in both vertebrae and femurs. Additionally, Scl‐Ab improved femoral cortical parameters in both age cohorts. Biomechanical testing showed that Scl‐Ab improved parameters of whole‐bone strength in Crtap–/– mice, with more robust effects in the week 6 to 12 cohort, but did not affect the increased bone brittleness. Additionally, Scl‐Ab normalized the increased osteoclast numbers, stimulated bone formation rate (week 6 to 12 cohort only), but did not affect osteocyte density. Overall, our findings suggest that Scl‐Ab treatment may be beneficial in the treatment of recessive OI caused by defects in collagen posttranslational modification.

THU0397 Increased Bone Mass and Bone Strength by Sclerostin Antibody (Scl-Ab) is Maintained by a RANKL Inhibitor in OVX Rats with Established Osteopenia

Background Some Scl-Ab-induced BMD gains gradually reversed after treatment discontinuation in rats. Objectives To examine if follow-up treatment with a RANKL inhibitor, OPG-Fc, would maintain Scl-Ab-induced bone mass gains in OVX rats. Methods Six-month-old OVX rats (2 months post-OVX) were treated with Scl-Ab (Scl-Ab VI, 25 mg/kg, SC, 1x/wk) for 6 wks and then transitioned to vehicle (Veh) or OPG-Fc (10 mg/kg, SC, 2x/wk) for another 6 or 20 wks (n=12-14/group). Veh-treated Sham and OVX control rats were euthanized at wks 0, 6, 12, and 26, and a group of Scl-Ab-treated OVX rats were euthanized at wk 6 (n=10-12/group). Results In vivo DXA analysis showed that after 6 wks, Scl-Ab-treated OVX rats increased lumbar spine and femur-tibia BMD to levels significantly above OVX and Sham controls. These treatment-related increases were maintained by transition to OPG-Fc, and gradually reversed by transition to Veh (Fig.). Histomorphometry revealed significant increases in vertebral trabecular bone volume (BV/TV) and tibial cortical thickness (Ct.Th) after 6 wks of Scl-Ab treatment. While transition to OPG-Fc maintained BV/TV and Ct.Th at the peak levels achieved with Scl-Ab, transition to Veh led to gradual declines in these parameters to levels that remained above OVX controls at wk 26. Significant increases in trabecular, endocortical, and periosteal bone formation rates and significant decreases in trabecular and endocortical eroded surfaces were also associated with Scl-Ab treatment (6 wks). Transition to OPG-Fc led to reductions in each of these bone resorption and formation parameters, however transition to Veh reversed these changes. At wk 26, maximum load of lumbar vertebral bodies was significantly greater in the OPG-Fc vs Veh transitioned group. Conclusions After discontinuing Scl-Ab treatment, transitioning to the RANKL inhibitor OPG-Fc effectively inhibited bone resorption and maintained Scl-Ab-induced bone mass and bone strength gains. Disclosure of Interest M. Ominsky Shareholder of: Amgen Inc., Employee of: Amgen Inc., X. Li Shareholder of: Amgen Inc., Employee of: Amgen Inc., K. Warmington Shareholder of: Amgen Inc., Employee of: Amgen Inc., Q.-T. Niu Shareholder of: Amgen Inc., Employee of: Amgen Inc., F. Asuncion Shareholder of: Amgen Inc., Employee of: Amgen Inc., D. Dwyer Shareholder of: Amgen Inc., Employee of: Amgen Inc., M. Grisanti Shareholder of: Amgen Inc., Employee of: Amgen Inc., C.-Y. Han Shareholder of: Amgen Inc., Employee of: Amgen Inc., P. Kostenuik Shareholder of: Amgen Inc., Employee of: Amgen Inc., M. Stolina Shareholder of: Amgen Inc., Employee of: Amgen Inc., H. Ke Shareholder of: Amgen Inc., Employee of: Amgen Inc.