William Scott Simonet

Denosumab, a Fully Human Monoclonal Antibody to RANKL, Inhibits Bone Resorption and Increases BMD in Knock‐In Mice That Express Chimeric (Murine/Human) RANKL

RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG‐Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG‐Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock‐in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting “huRANKL” mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG‐Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.

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.

RANKL inhibition with osteoprotegerin increases bone strength by improving cortical and trabecular bone architecture in ovariectomized rats.

Introduction: Ovariectomy (OVX) results in bone loss caused by increased bone resorption. RANKL is an essential mediator of bone resorption. We examined whether the RANKL inhibitor osteoprotegerin (OPG) would preserve bone volume, density, and strength in OVX rats.

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.