Susan Y. Smith

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.

Odanacatib reduces bone turnover and increases bone mass in the lumbar spine of skeletally mature ovariectomized rhesus monkeys

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once‐weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen‐deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle‐treated OVX monkeys. Treatment with ODN also led to dose‐dependent increases in serum 1‐CTP and maintained estrogen deficiency–elevated Trap‐5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L1 to L4) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose‐dependently increased L1 to L4 BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX‐vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX‐vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN‐treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long‐term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates.

Cathepsin K inhibitors prevent bone loss in estrogen‐deficient rabbits

Two cathepsin K inhibitors (CatKIs) were compared with alendronate (ALN) for their effects on bone resorption and formation in ovariectomized (OVX) rabbits. The OVX model was validated by demonstrating significant loss (9.8% to 12.8%) in lumbar vertebral bone mineral density (LV BMD) in rabbits at 13‐weeks after surgery, which was prevented by estrogen or ALN. A potent CatKI, L‐006235 (L‐235), dosed at 10 mg/kg per day for 27 weeks, significantly decreased LV BMD loss (p < .01) versus OVX‐vehicle control. ALN reduced spine cancellous mineralizing surface by 70%, whereas L‐235 had no effect. Similarly, endocortical bone‐formation rate and the number of double‐labeled Haversian canals in the femoral diaphysis were not affected by L‐235. To confirm the sparing effects of CatKI on bone formation, odanacatib (ODN) was dosed in food to achieve steady‐state exposures of 4 or 9 µM/day in OVX rabbits for 27 weeks. ODN at both doses prevented LV BMD loss (p < .05 and p < .001, respectively) versus OVX‐vehicle control to levels comparable with sham or ALN. ODN also dose‐dependently increased BMD at the proximal femur, femoral neck, and trochanter. Similar to L‐235, ODN did not reduce bone formation at any bone sites studied. The positive and highly correlative relationship of peak load to bone mineral content in the central femur and spine suggested that ODN treatment preserved normal biomechanical properties of relevant skeletal sites. Although CatKIs had similar efficacy to ALN in preventing bone loss in adult OVX rabbits, this novel class of antiresorptives differs from ALN by sparing bone formation, potentially via uncoupling bone formation from resorption.

Intermittent intravenous administration of the bisphosphonate ibandronate prevents bone loss and maintains bone strength and quality in ovariectomized cynomolgus monkeys.

Using a clinically relevant regimen, this study investigated the effects of treatment with ibandronate, a highly potent nitrogen-containing bisphosphonate, on bone loss, biochemical markers of bone turnover, densitometry, histomorphometry, biomechanical properties, and bone concentration in aged ovariectomized monkeys. Sixty-six female cynomolgus monkeys, aged 9 years and older, were ovariectomized (OVX) or sham operated. Intravenous (iv) bolus injections of ibandronate at 10, 30, or 150 microg/kg or placebo were administered at 30-day intervals (corresponding to intervals of 3 months in humans), starting at OVX, for 16 months. OVX significantly decreased bone mass at the lumbar spine, proximal femur, femoral neck, and radius and increased bone turnover in a time-dependent manner, as assessed by dual energy X-ray absorptiometry, peripheral quantitative computed tomography, or histomorphometry. Ibandronate iv bolus injections administered at 30 microg/kg every 30 days prevented osteopenia induced by estrogen depletion. OVX-induced increases in bone turnover (as determined by activation frequency, bone formation rate, and biochemical markers of bone turnover, including urinary N-telopeptide and deoxypyridinoline excretion and serum values for osteocalcin and bone-specific alkaline phosphatase) were suppressed on treatment, and bone mass, architecture, and strength were preserved at clinically relevant sites. Treatment with high-dose (150 microg/kg/dose) iv bolus injections of ibandronate further increased bone mass and improved bone strength at both the spine and femoral neck, without adversely affecting bone quality. In contrast, treatment with a 10 microg/kg/dose only partially prevented the OVX-induced effects. These data support the potential for the long-term administration of ibandronate by intermittent iv bolus injections in humans to prevent osteoporosis and improve bone quality at clinically relevant sites.

Defining a Noncarcinogenic Dose of Recombinant Human Parathyroid Hormone 1–84 in a 2-Year Study in Fischer 344 Rats

The carcinogenic potential of human parathyroid hormone 1–84 (PTH) was assessed by daily subcutaneous injection (0, 10, 50, 150 μg/kg/day) for 2 years in Fischer 344 rats. Histopathological analyses were conducted on the standard set of soft tissues, tissues with macroscopic abnormalities, selected bones, and bones with abnormalities identified radiographically. All PTH doses caused widespread osteosclerosis and significant, dose-dependent increases in femoral and vertebral bone mineral content and density. In the mid- and high-dose groups, proliferative changes in bone increased with dose. Osteosarcoma was the most common change, followed by focal osteoblast hyperplasia, osteoblastoma, osteoma and skeletal fibrosarcoma. The incidence of bone neoplasms was comparable in control and low-dose groups providing a noncarcinogenic dose for PTH of 10 μg/kg/day at a systemic exposure to PTH that is 4.6-fold higher than for a 100 μg dose in humans. The ability of PTH to interact with and balance the effects of both the PTH-1 receptor and the putative C-terminal PTH receptor, may lead to the lower carcinogenic potential observed with PTH than reported previously for teriparatide.

Intermittent Ibandronate Preserves Bone Quality and Bone Strength in the Lumbar Spine After 16 Months of Treatment in the Ovariectomized Cynomolgus Monkey

The dose‐dependent effect of ibandronate treatment on bone mass and architecture was assessed in a large animal study of OVX monkeys using μCT for quantitative bone morphometry and biomechanical testing for measures of bone strength. The study showed that intermittent ibandronate preserved lumbar spine bone quality and strength in these animals after 16 months of treatment.

Cancellous and cortical bone architecture and turnover at the iliac crest of postmenopausal osteoporotic women treated with parathyroid hormone 1-84

Treatment with parathyroid hormone [PTH(1-84)] increases lumbar spine bone mineral density and decreases vertebral fractures, but its effects on bone microarchitecture are unknown. We obtained iliac crest biopsies from postmenopausal osteoporotic women given placebo (n=8) or 100 microg PTH(1-84) for 18 (n=8) or 24 (n=7) months to assess cancellous and cortical bone formation and structure. At 18 months, cancellous bone volume (BV/TV) measured by microcomputed tomography and histomorphometry was 45-48% higher in subjects treated with PTH(1-84) versus placebo, a result of higher trabecular number (Tb.N) and thickness. The higher Tb.N appeared to result from intratrabecular tunneling. Connectivity density was higher and structure model index was lower, indicating a better connected and more plate-like trabecular architecture. Cancellous bone formation rate (BFR) was 2-fold higher in PTH(1-84)-treated subjects, primarily because of greater mineralizing surface. Osteoblast and osteoid surfaces were a nonsignificant 58% and 35%, respectively, higher with PTH(1-84) treatment. Osteoclast and eroded surface were unaffected by PTH(1-84). There were no effects of PTH(1-84) treatment on cortical thickness, or endocortical or periosteal BFR, but cortical porosity tended to be higher. Although cancellous BFR was lower at 24 than at 18 months, measures of cancellous and cortical bone structure were similar at both timepoints. The bone produced by PTH(1-84) had normal lamellar structure and mineralization with no abnormal histology. In conclusion, when compared with placebo, treatment of osteoporotic women with PTH(1-84) was associated with higher BV/TV and trabecular connectivity, with a more plate-like architecture, all consistent with the lower vertebral fracture incidence.

Decreased bone remodeling and porosity are associated with improved bone strength in ovariectomized cynomolgus monkeys treated with denosumab, a fully human RANKL antibody☆

This study examined the effects of denosumab, an anti-RANKL antibody that inhibits bone resorption, on bone histomorphometry in adult ovariectomized cynomolgus monkeys (OVX cynos). A month after surgery, OVX cynos were treated with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50mg/kg/month) for 16months (n=14-20/group). Sham controls were treated with vehicle (Sham-Veh; n=17). Areal and volumetric BMD, urine NTx, and serum osteocalcin were measured at baseline and months 3, 6, 12, and 16. Double fluorochrome labels were injected prior to iliac and rib biopsies at month 6 and month 12, and prior to sacrifice at month 16. Histomorphometry was performed on these biopsies, the tibial diaphysis, the L2 vertebra, and the proximal femur. Strength of humeral cortical beams, femur diaphysis, femur neck, and trabecular cores of L5-L6 vertebrae was determined by destructive biomechanical testing. There was no evidence of woven bone, osteomalacia, or other bone histopathologic changes with OVX or with denosumab. OVX-Veh animals exhibited significantly greater bone remodeling at all skeletal sites relative to Sham-Veh controls. Both doses of denosumab markedly inhibited bone remodeling at all sites, including significant reductions in trabecular eroded surfaces (48-86% lower than OVX-Veh controls), cortical porosity (28-72% lower), and dynamic parameters of bone formation (81-100% lower). Decreased fluorochrome labeling with denosumab was related to reductions in cortical porosity and trabecular eroded surfaces, and regression analyses suggested that these reductions contributed to denosumab-related increments in BMD and bone strength. Denosumab-treated animals with the lowest levels of fluorescent labeling exhibited the greatest structural bone strength values at each site. Intracortical remodeling had no relationship with material properties including ultimate strength, elastic modulus or toughness (r(2)=0.00-0.01). These data suggest that remodeling inhibition with denosumab improved structural strength without altering material properties under these experimental conditions. Greater structural strength in the denosumab-treated animals can be primarily explained by the combined effects of increased trabecular and cortical bone mass, and reductions in trabecular eroded surfaces and cortical porosity.

Denosumab, a fully human RANKL antibody, reduced bone turnover markers and increased trabecular and cortical bone mass, density, and strength in ovariectomized cynomolgus monkeys☆ , ☆☆

Denosumab is a fully human monoclonal antibody that inhibits RANKL, a protein essential for osteoclast formation, function, and survival. Osteoclast inhibition with denosumab decreased bone resorption, increased bone mineral density (BMD), and reduced fracture risk in osteoporotic women. The effects of 16months of continuous osteoclast inhibition on bone strength parameters were examined in adult ovariectomized (OVX) cynomolgus monkeys (cynos). One month after surgery, OVX cynos (n=14-20/group) were treated monthly with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50mg/kg). Sham-operated controls were treated with vehicle (n=17). OVX-Veh exhibited early and persistent increases in the resorption marker CTx, followed by similar increases in the formation marker BSAP, consistent with increased bone remodeling. Denosumab reduced CTx and BSAP throughout the study to levels significantly lower than in OVX-Veh or Sham-Veh, consistent with reduced remodeling. Increased remodeling in OVX-Veh led to absolute declines in areal BMD of 4.3-7.4% at the lumbar spine, total hip, femur neck, and distal radius (all p<0.05 vs baseline). Denosumab significantly increased aBMD at each site to levels exceeding baseline or OVX-Veh controls, and denosumab significantly increased cortical vBMC of the central radius and tibia by 7% and 14% (respectively) relative to OVX-Veh. Destructive biomechanical testing revealed that both doses of denosumab were associated with significantly greater peak load for femur neck (+19-34%), L3-L4 vertebral bodies (+54-55%), and L5-L6 cancellous cores (+69-82%) compared with OVX-Veh. Direct assessment of bone tissue material properties at cortical sites revealed no significant changes with denosumab. For all sites analyzed biomechanically, bone mass (BMC) and strength (load) exhibited strong linear correlations (r(2)=0.59-0.85 for all groups combined). Denosumab did not alter slopes of load-BMC regressions at any site, and denosumab groups exhibited similar or greater load values at given BMC values compared with OVX-Veh or Sham. In summary, denosumab markedly reduced biochemical markers of bone remodeling and increased cortical and trabecular bone mass in adult OVX cynos. Denosumab improved structural bone strength parameters at all sites analyzed, and strength remained highly correlated with bone mass. There was no evidence for reduced material strength properties of cortical bone with denosumab over this time period, which approximates to 4years of remodeling in the slower-remodeling adult human skeleton. These data indicate that denosumab increased bone strength by increasing bone mass and preserving bone quality.