Chris Paszty

Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength.

Introduction: Sclerosteosis is a rare high bone mass genetic disorder in humans caused by inactivating mutations in SOST, the gene encoding sclerostin. Based on these data, sclerostin has emerged as a key negative regulator of bone mass. We generated SOST knockout (KO) mice to gain a more detailed understanding of the effects of sclerostin deficiency on bone.

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength in a Rat Model of Postmenopausal Osteoporosis

The development of bone‐rebuilding anabolic agents for potential use in the treatment of bone loss conditions, such as osteoporosis, has been a long‐standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation, although the magnitude and extent of sclerostins role in the control of bone formation in the aging skeleton is still unclear. To study this unexplored area of sclerostin biology and to assess the pharmacologic effects of sclerostin inhibition, we used a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl‐AbII) for testing in an aged ovariectomized rat model of postmenopausal osteoporosis. Six‐month‐old female rats were ovariectomized and left untreated for 1 yr to allow for significant estrogen deficiency‐induced bone loss, at which point Scl‐AbII was administered for 5 wk. Scl‐AbII treatment in these animals had robust anabolic effects, with marked increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. This not only resulted in complete reversal, at several skeletal sites, of the 1 yr of estrogen deficiency‐induced bone loss, but also further increased bone mass and bone strength to levels greater than those found in non‐ovariectomized control rats. Taken together, these preclinical results establish sclerostins role as a pivotal negative regulator of bone formation in the aging skeleton and, furthermore, suggest that antibody‐mediated inhibition of sclerostin represents a promising new therapeutic approach for the anabolic treatment of bone‐related disorders, such as postmenopausal osteoporosis.

Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength

The development of bone‐rebuilding anabolic agents for treating bone‐related conditions has been a long‐standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin‐neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin‐neutralizing monoclonal antibody (Scl‐AbIV) to gonad‐intact female cynomolgus monkeys. Two once‐monthly subcutaneous injections of Scl‐AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl‐AbIV treatment had clear anabolic effects, with marked dose‐dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl‐AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle‐treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest‐dose group. Taken together, the marked bone‐building effects achieved in this short‐term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.

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.

Characterization of the Structural Features and Interactions of Sclerostin MOLECULAR INSIGHT INTO A KEY REGULATOR OF Wnt-MEDIATED BONE FORMATION

The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.

Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model.

Sclerostin monoclonal antibody (Scl-Ab) has been shown to increase bone mass and bone strength by stimulating bone formation in an ovariectomy-induced bone loss rat model. The purpose of this study was to determine the effects of Scl-Ab in a rat immobilization/disuse model in which there was both a decrease in bone formation and an increase in bone resorption. Ten-month-old female Sprague Dawley rats were divided into normal weight-bearing (normal-loaded, NL) and right hindlimb-immobilization (under-loaded, UL) groups. Both NL and UL rats were treated with vehicle or Scl-Ab at 5 or 25 mg/kg, twice per week for 4 weeks. Trabecular and cortical bone histomorphometric analyses were performed on the proximal tibial metaphysis (PTM) and tibial shaft (TS). Compared to NL controls, UL rats had reduced body and muscle weights, increased bone marrow fat cells in the PTM, increased trabecular bone resorption and periosteal mineral apposition rate (MAR) as well as decreased trabecular MAR and bone formation rate (BFR/BS). In NL bones, treatment with Scl-Ab significantly increased bone formation and decreased bone resorption, resulting in increased trabecular and cortical bone mass. In UL trabecular bone, treatment with Scl-Ab at 5 or 25 mg/kg induced significant and dose-dependent increases in trabecular bone volume and thickness, mineralized surfaces (MS/BS), MAR and BFR/BS, and a significant decrease in eroded surface (Er.S/BS) compared with UL controls. In UL cortical bone, Scl-Ab treatment induced significant increases in cortical width, periosteal and endocortical MS/BS, MAR and BFR/BS, and significant decreases in endocortical Er.S/BS compared with UL controls. Taken together, these findings suggest that antibody-mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization-induced osteopenia.

Sclerostin: A gem from the genome leads to bone-building antibodies

Discovery of Sclerostin databases using either homology-based programs (eg, BLAST) or a special CxGxC-class cystine-knot search pattern Genomics technologies and DNA sequencing have had a transformative impact on biological research, giving us unprecedented access to the genomes of numerous organisms and ushering in such new fields as systems biology and, more recently, synthetic biology. In the 1990s, the advent of highthroughput sequencing spurred application of this powerful new technology to the challenging endeavor of trying to understand the genetic basis of various inherited human diseases. One such disease was sclerosteosis, a very rare, recessively inherited highbone-mass disorder that was thought to be caused primarily by excessive osteoblast-mediated bone formation rather than by defective osteoclast-mediated bone resorption. Within the realm of inherited high-bone-mass disorders, it was the hope for a discovery in the area of osteoblast biology that made sclerosteosis particularly interesting. Indeed, the identification of new bone-building pathways that might yield novel anabolic agents had been a long-standing goal in bone biology, with hopes for therapeutic application in fracture healing, orthopedic procedures, and low-bone-mass conditions such as osteoporosis. Against this backdrop came the exciting news, independently from Mary Brunkow’s group at Celltech R&D, Inc., and from Wim Van Hul’s group at the University of Antwerp, that sclerosteosis was caused by mutations in a single gene (SOST) encoding a novel secreted protein. Because these were inactivating mutations, it was clear that this protein, aptly given the name sclerostin, functioned either directly or indirectly as an inhibitor of bone formation. During this same general time period, large-scale cDNA/ expressed sequence tag (EST) and genomic DNA sequencing efforts were beingmade in academia and industry to rapidly identify new human genes. A novel secreted protein of unknown function, which turned out to be sclerostin, was discovered by computational mining of large DNA sequence

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.

Treatment with a sclerostin antibody increases cancellous bone formation and bone mass regardless of marrow composition in adult female rats.

The current report describes the skeletal effects of a sclerostin monoclonal antibody (Scl-AbIII) treatment at a yellow (fatty) marrow skeletal site in adult female rats. Ten-month-old female Sprague-Dawley rats were treated with vehicle or Scl-AbIII at 5 or 25 mg/kg, twice per week by s.c. injection for 4 weeks. Trabecular bone from a yellow (fatty) marrow site, the 5th caudal vertebral body (CVB), was processed undecalcified for quantitative bone histomorphometric analysis. Compared to vehicle controls, Scl-AbIII at both doses significantly increased bone formation parameters and trabecular bone volume and thickness and decreased bone resorption parameter in the trabecular bone of the CVB. As a reference, we also found that the Scl-AbIII at both doses significantly decreased bone resorption and increased bone formation and bone volume in a red (hematopoietic) marrow site, the 4th lumber vertebral body (LVB). It appears that the percentage of increase in trabecular bone volume induced by Scl-AbIII treatment was slightly larger in the LVB than in the CVB. In summary, these preclinical findings show that antibody-mediated sclerostin inhibition has significant bone anabolic effects at both red and yellow marrow skeletal sites.