Ian Pyrah

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.

Time-dependent cellular and transcriptional changes in the osteoblast lineage associated with sclerostin antibody treatment in ovariectomized rats

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP). At 50mg/kg/wk, bone volume (BV/total volume [TV]) increased through d183 and declined during the TFP. Bone formation rate (BFR/bone surface [BS]) and total OB number increased through d29, then progressively declined, coincident with a decrease in total osteoprogenitor (OP) numbers from d29 through d183. Analysis of differentially expressed genes (DEGs) from microarray analysis of mRNA isolated from laser capture microdissection samples enriched for OB, lining cells, and osteocytes (OCy) revealed modules of genes that correlated with BFR/BS, BV/TV, and osteoblastic surface (Ob.S)/BS. Expression change of canonical Wnt target genes was similar in all three cell types at d8, including upregulation of Twist1 and Wisp1. At d29, the pattern of Wnt target gene expression changed in the OCy, with Twist1 returning to VEH level, sustained upregulation of Wisp1, and upregulation of several other Wnt targets that continued into the TFP. Predicted activation of pathways recognized to integrate with and regulate canonical Wnt signaling were also activated at d29 in the OCy. The most significantly affected pathways represented transcription factor signaling known to inhibit cell cycle progression (notably p53) and mitogenesis (notably c-Myc). These changes occurred at the time of peak BFR/BS and continued as BFR/BS declined during treatment, then trended toward VEH level in the TFP. Concurrent with this transcriptional switch was a reduction in OP numbers, an effect that would ultimately limit bone formation. This study confirms that the initial transcriptional response in response to Scl-Ab is activation of canonical Wnt signaling and the data demonstrate that there is induction of additional regulatory pathways in OCy with long-term treatment. The interactions between Wnt and p53/c-Myc signaling may be key in limiting OP populations, thus contributing to self-regulation of bone formation with continued Scl-Ab administration.

Transcriptional Profiling of Laser Capture Microdissected Subpopulations of the Osteoblast Lineage Provides Insight Into the Early Response to Sclerostin Antibody in Rats

Sclerostin antibody (Scl‐Ab) increases bone formation through a process dependent on the activation of canonical Wnt signaling, although the specific signaling in the osteoblast lineage in vivo is largely unknown. To gain insight into the signaling pathways acutely modulated by Scl‐Ab, the transcriptional response of subpopulations of the osteoblast lineage was assessed by TaqMan and microarray analyses of mRNA isolated from laser capture microdissection (LCM)–enriched samples from the vertebrae of ovariectomized rats during the first week after Scl‐Ab administration. Briefly, 6‐month‐old Sprague‐Dawley rats were ovariectomized and, after 2 months, received a single dose of vehicle (VEH) or 100 mg/kg Scl‐Ab (n = 20/group). Lumbar vertebrae were collected at 6, 24, 72, and 168 hours postdose and cryosectioned for LCM. Osteocytes were captured from bone matrix, and osteoblasts and lining cells were captured from bone surfaces based on fluorochrome labeling. mRNA was isolated, amplified, and profiled by TaqMan and microarray. Expression analysis revealed that Scl‐Ab caused strikingly similar transcriptional profiles across all three cell types. Only 13 known canonical Wnt target genes, the majority with known functions in bone, showed a significant change in expression by microarray in response to Scl‐Ab, with Wisp1 and Twist1 being the most responsive. Coincident with increased expression of Wnt target genes was the upregulation of numerous extracellular matrix (ECM) genes. The acute and progressive upregulation of ECM genes in lining cells supports their activation into matrix‐producing osteoblasts, consistent with modeling‐based bone formation. A similar transcriptional profile in osteocytes may indicate that Scl‐Ab stimulates perilacunar/pericanalicular matrix deposition. Pathway analyses indicated that Scl‐Ab regulated a limited number of genes related to cell cycle arrest and B‐cell development. These data describe the acute downstream signaling in response to Scl‐Ab in vivo and demonstrate selected canonical Wnt target gene activation associated with increased bone formation in all mature osteoblast subpopulations.

Romosozumab Improves Bone Mass and Strength While Maintaining Bone Quality in Ovariectomized Cynomolgus Monkeys

Romosozumab (Romo), a humanized sclerostin antibody, is a bone‐forming agent under development for treatment of osteoporosis. To examine the effects of Romo on bone quality, mature cynomolgus monkeys (cynos) were treated 4 months post‐ ovariectomy (OVX) with vehicle, 3 mg/kg, or 30 mg/kg Romo for 12 months, or with 30 mg/kg Romo for 6 months followed by vehicle for 6 months (30/0). Serum bone formation markers were increased by Romo during the first 6 months, corresponding to increased cancellous, endocortical, and periosteal bone formation in rib and iliac biopsies at months 3 and 6. Dual‐energy X‐ray absorptiometry (DXA) bone mineral density (BMD) was increased by 14% to 26% at the lumbar spine and proximal femur at month 12, corresponding to significant increases in bone strength at 3 and 30 mg/kg in lumbar vertebral bodies and cancellous cores, and at 30 mg/kg in the femur diaphysis and neck. Bone mass remained positively correlated with strength at these sites, with no changes in calculated material properties at cortical sites. These bone‐quality measures were also maintained in the 30/0 group, despite a gradual loss of accrued bone mass. Normal bone mineralization was confirmed by histomorphometry and ash analyses. At the radial diaphysis, a transient, reversible 2% reduction in cortical BMD was observed with Romo at month 6, despite relative improvements in bone mineral content (BMC). High‐resolution pQCT confirmed this decline in cortical BMD at the radial diaphysis and metaphysis in a second set of OVX cynos administered 3 mg/kg Romo for 6 months. Radial diaphyseal strength was maintained and metaphyseal strength improved with Romo as estimated by finite element modeling. Decreased radial cortical BMD was a consequence of increased intracortical remodeling, with no increase in cortical porosity. Romo resulted in marked improvements in bone mass, architecture, and bone strength, while maintaining bone quality in OVX cynos, supporting its bone efficacy and safety profile.

Carcinogenicity risk assessment of romosozumab: A review of scientific weight-of-evidence and findings in a rat lifetime pharmacology study.

Romosozumab is a humanized immunoglobulin G2 monoclonal antibody that binds and blocks the action of sclerostin, a protein secreted by the osteocyte and an extracellular inhibitor of canonical Wnt signaling. Blockade of sclerostin binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) allows Wnt ligands to activate canonical Wnt signaling in bone, increasing bone formation and decreasing bone resorption, making sclerostin an attractive target for osteoporosis therapy. Because romosozumab is a bone-forming agent and an activator of canonical Wnt signaling, questions have arisen regarding a potential carcinogenic risk. Weight-of-evidence factors used in the assessment of human carcinogenic risk of romosozumab included features of canonical Wnt signaling, expression pattern of sclerostin, phenotype of loss-of-function mutations in humans and mice, mode and mechanism of action of romosozumab, and findings from romosozumab chronic toxicity studies in rats and monkeys. Although the weight-of-evidence factors supported that romosozumab would pose a low carcinogenic risk to humans, the carcinogenic potential of romosozumab was assessed in a rat lifetime study. There were no romosozumab-related effects on tumor incidence in rats. The findings of the lifetime study and the weight-of-evidence factors collectively indicate that romosozumab administration would not pose a carcinogenic risk to humans.

Review of the Application of RNA Interference Technology in the Pharmaceutical Industry

Ribonucleic acid (RNA) interference (RNAi) is a recently discovered phenomenon whereby the introduction of double stranded (ds) RNA into the cytoplasm of the cell results in the specific and efficient degradation of complementary messenger (m) RNA and, therefore, reduced protein production. It was discovered by chance during attempts to develop flowers with increased colour intensity. The specific nature of the inhibition of protein production of cells has resulted in an explosion of research to understand and exploit RNAi. The technique is now established in in vitro systems, and much work is focussed in adapting RNAi for in vivo application. The potential of the technology in understanding physiological and pathological processes is significant, while its development as a therapeutic agent holds much promise as targeted agents. This review will describe the basic biological processes that drive RNAi, indicate current areas of areas research, and forecast future areas of development.

Differential temporal effects of sclerostin antibody and parathyroid hormone on cancellous and cortical bone and quantitative differences in effects on the osteoblast lineage in young intact rats

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague-Dawley rats were administered either vehicle, Scl-Ab (3 or 50mg/kg/week subcutaneously), or human PTH (1-34) (75 μg/kg/day subcutaneously) for 4 or 26 weeks. The 50mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume. Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.

Reproductive toxicity of denosumab in cynomolgus monkeys.

Denosumab is a monoclonal antibody that inhibits bone resorption by targeting RANKL, an essential mediator of osteoclast formation, function, and survival. Reproductive toxicity of denosumab was assessed in cynomolgus monkeys in an embryofetal development study (dosing GD20-50) and a pre-postnatal toxicity study (dosing GD20-parturition). In the embryofetal toxicity study, denosumab did not elicit maternal toxicity, fetal harm or teratogenicity. In the pre-postnatal toxicity study, there were increased stillbirths, and one maternal death due to dystocia. There was no effect on maternal mammary gland histomorphology, lactation, or fetal growth. In infants exposed in utero, there was increased postnatal mortality, decreased body weight gain, and decreased growth/development. Denosumab-related effects in infants were present in bones and lymph nodes. There was full recovery at 6 months of age from most bone-related changes observed earlier postpartum. The effects observed in mothers and infants were consistent with the pharmacological action of denosumab.

Infant cynomolgus monkeys exposed to denosumab in utero exhibit an osteoclast-poor osteopetrotic-like skeletal phenotype at birth and in the early postnatal period

RANKL is a key regulator of bone resorption and osteoclastogenesis. Denosumab is a fully human IgG2 monoclonal antibody that inhibits bone resorption by binding and inhibiting the activity of RANKL. To determine the effects of denosumab on pre- and postnatal skeletal growth and development, subcutaneous injections of 0 (control) or 50 mg/kg/month denosumab were given to pregnant cynomolgus monkeys from approximately gestation day (GD) 20 until parturition (up to 6 doses). For up to 6 months postpartum (birth day [BD] 180/181), evaluation of the infants included skeletal radiographs, bone biomarkers, and oral examinations for assessment of tooth eruption. Infant bones were collected at necropsy for densitometry, biomechanical testing, and histopathologic evaluation from control and denosumab-exposed infants on BD1 (or within 2 weeks of birth) and BD181, and from infants that died or were euthanized moribund from BD5 to BD69. In all denosumab-exposed infants, biomarkers of bone resorption and formation were markedly decreased at BD1 and BD14 and slightly greater at BD91 vs. control, then similar to control values by BD181. Spontaneous long bone fractures were detected clinically or radiographically in 4 denosumab-exposed infants at BD28 and BD60, with evidence of radiographic healing at ≥BD60. In BD1 infants exposed to denosumab in utero, radiographic evaluations of the skeleton revealed decreased long bone length; a generalized increased radio-opacity of the axial and appendicular skeleton and bones at the base of the skull with decreased or absent marrow cavities, widened growth plates, flared/club-shaped metaphysis, altered jaw/skull shape, and reduced jaw length; and delayed development of secondary ossification centers. Densitometric evaluations in these infants demonstrated a marked increase in bone mineral density at trabecular sites, but cortical bone mineral density was decreased. Histologically, long bone cortices were attenuated and there was an absence of osteoclasts. Bones with active endochondral ossification consisted largely of a dense network of retained primary spongiosa with reduced marrow space consistent with an osteopetrotic phenotype. A minimal increase in growth plate thickness largely due to the expansion of the hypertrophic zone was present. Retained woven bone was observed in bones formed by intramembranous ossification, consistent with absence of bone remodeling. These changes in bone tissue composition and geometry were reflected in reduced biomechanical strength and material properties of bones from denosumab-exposed infants. Material property changes were characterized by increased tissue brittleness reflected in reductions in calculated material toughness at the femur diaphysis and lack of correlation between energy and bone mass at the vertebra; these changes were likely the basis for the increased skeletal fragility (fractures). Although tooth eruption was not impaired in denosumab-exposed infants, the reduced growth and increased bone density of the mandible resulted in dental abnormalities consisting of tooth malalignment and dental dysplasia. Radiographic changes at BD1 persisted at BD28, with evidence of resumption of bone resorption and remodeling observed in most infants at BD60 and/or BD90. In 2 infants euthanized on BD60 and BD69, there was histologic and radiographic evidence of subphyseal/metaphyseal bone resorption accompanied by multiple foci of ossification in growth plates that were markedly increased in thickness. In infants necropsied at BD181, where systemic exposure to denosumab had been below limits of quantitation for approximately 3months, there was largely full recovery from all bone-related changes observed earlier postpartum, including tissue brittleness. Persistent changes included dental dysplasia, decreased bone length, reduced cortical thickness, and decreased peak load and ultimate strength at the femur diaphysis. In conclusion, the skeletal and secondary dental effects observed in infant monkeys exposed in utero to denosumab are consistent with the anticipated pharmacological activity of denosumab as a monoclonal antibody against RANKL and inhibitor of osteoclastogenesis. The resulting inhibition of resorption impaired both bone modeling and remodeling during skeletal development and growth. The skeletal phenotype of these infant monkeys resembles human infants with osteoclast-poor osteopetrosis due to inactivating mutations of RANK or RANKL.