Aurore Varela

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.

Bone and Joints

Abstract The skeleton fulfills several needs in an animal. It serves as a scaffold on which muscles can act to produce locomotion, provides protection for vital structures, supplies an internal source for minerals, and harbors an environment that supports hematopoiesis. Due to slow and regimented turnover, the current state and the history of a bone coexist. The gross and microscopic structure of each bone depends on its location in the skeleton and the resulting profile of biomechanical forces, as well as on the species, sex, and age. Knowledge of how bone physiology varies due to each of these factors is key in recognizing and understanding bone pathology. In this chapter, basic bone biology, including anatomy, physiology, and recent advances in our knowledge of cell–cell signaling, is covered in the earlier sections. Next, modalities used to fully analyze the health of the skeleton are reviewed, including biomarker evaluation of serum or urine, imaging techniques ranging from radiology to computed tomography, biomechanical testing, routine decalcified microscopic evaluation, and specialized undecalcified microscopy with bone histomorphometry. Animal models, both those found naturally and those generated by genetic or surgical manipulation, can offer great insight into a pathologic process as long as the strengths and limitations of the model are fully appreciated. A review of how bone responds to injury is used to demonstrate the potential range of responses to injurious and restorative events, and the interrelationships between bone cells, is followed by a summary of mechanisms of bone toxicity by various prototypical substances. Many effects on bone in toxicity studies reflect the desired pharmacology of a therapeutic agent, even if the skeleton is not the intended site of action.

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.

One Year of Abaloparatide, a Selective Activator of the PTH1 Receptor, Increased Bone Formation and Bone Mass in Osteopenic Ovariectomized Rats Without Increasing Bone Resorption

Abaloparatide is a novel 34‐amino acid peptide selected to be a potent and selective activator of the parathyroid hormone receptor (PTH1R) signaling pathway with 41% homology to PTH(1‐34) and 76% homology to PTHrP(1‐34). A 12‐month treatment study was conducted in osteopenic ovariectomized (OVX) rats to characterize the mechanisms by which abaloparatide increases bone mass. Sprague‐Dawley (SD) rats were subjected to OVX or sham surgery at age 6 months and left untreated for 3 months to allow OVX‐induced bone loss. Ten OVX rats were euthanized after this bone depletion period, and the remaining OVX rats received daily subcutaneous injections of vehicle (n = 18) or abaloparatide at 1, 5, or 25 μg/kg/d (n = 18/dose level) for 12 months. Sham controls (n = 18) received vehicle daily. Bone densitometry and biochemical markers of bone formation and resorption were assessed longitudinally, and L3 vertebra and tibia were collected at necropsy for histomorphometry. Abaloparatide increased biochemical bone formation markers without increasing bone resorption markers or causing hypercalcemia. Abaloparatide increased histomorphometric indices of bone formation on trabecular, endocortical, and periosteal surfaces without increasing osteoclasts or eroded surfaces. Abaloparatide induced substantial increases in trabecular bone volume and density and improvements in trabecular microarchitecture. Abaloparatide stimulated periosteal expansion and endocortical bone apposition at the tibial diaphysis, leading to marked increases in cortical bone volume and density. Whole‐body bone mineral density (BMD) remained stable in OVX‐Vehicle controls while increasing 25% after 12 months of abaloparatide (25 μg/kg). Histomorphometry and biomarker data suggest that gains in cortical and trabecular bone mass were attributable to selective anabolic effects of abaloparatide, without evidence for stimulated bone resorption.

Comparing the incidence of bone tumors in rats chronically exposed to the selective PTH type 1 receptor agonist abaloparatide or PTH(1–34)

&NA; Prolonged treatment with human parathyroid hormone (hPTH) in rats results in development of bone tumors, though this finding has not been supported by clinical experience. The PTH type 1 receptor agonist abaloparatide, selected for its bone anabolic activity, is under clinical development to treat postmenopausal women with osteoporosis. To determine the carcinogenic potential of abaloparatide, Fischer (F344) rats were administered SC daily abaloparatide at doses of 0, 10, 25, and 50 &mgr;g/kg or 30 &mgr;g/kg hPTH(1–34) as a positive control for up to 2 years. Robust increases in bone density were achieved at all abaloparatide doses and with hPTH(1–34). Comprehensive histopathological analysis reflected a comparable continuum of proliferative changes in bone, mostly osteosarcoma, in both abaloparatide and hPTH(1–34) treated rats. Comparing the effects of abaloparatide and hPTH(1–34) at the 25 and 30 &mgr;g/kg respective doses, representing similar exposure multiples to the human therapeutic doses, revealed similar osteosarcoma‐associated mortality, tumor incidence, age at first occurrence, and metastatic potential. There were no increases in the incidence of non‐bone tumors with abaloparatide compared to vehicle. Thus, near life‐long treatment with abaloparatide in rats resulted in dose and time dependent formation of osteosarcomas, with a comparable response to hPTH(1–34) at similar exposure. HighlightsThe PTH type 1 receptor agonist abaloparatide has potent bone anabolic activity.Near life‐long treatment in rats with abaloparatide resulted in the formation of osteosarcomas.Osteosarcoma formation was dose and time dependent.Osteosarcoma in rats were also seen in a PTH(1–34) treated positive control group.There were no increases in the incidence of non‐bone tumors with abaloparatide.

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.

One year of abaloparatide, a selective peptide activator of the PTH1 receptor, increased bone mass and strength in ovariectomized rats

Abaloparatide is a novel 34 amino acid peptide selected to be a potent and selective activator of the parathyroid hormone receptor 1 (PTHR1) signaling pathway. The effects of 12months of abaloparatide treatment on bone mass, bone strength and bone quality was assessed in osteopenic ovariectomized (OVX) rats. SD rats were subjected to OVX or sham surgery at 6months of age and left untreated for 3months to allow OVX-induced bone loss. Eighteen OVX rats were sacrificed after this bone depletion period, and the remaining OVX rats received daily s.c. injections of vehicle (n=18) or abaloparatide at 1, 5 or 25μg/kg/d (n=18/dose level) for 12months. Sham controls (n=18) received vehicle daily. Bone changes were assessed by DXA and pQCT after 0, 3, 6 or 12months of treatment, and destructive biomechanical testing was conducted at month 12 to assess bone strength and bone quality. Abaloparatide dose-dependently increased bone mass at the lumbar spine and at the proximal and diaphyseal regions of the tibia and femur. pQCT revealed that increased cortical bone volume at the tibia was a result of periosteal expansion and endocortical bone apposition. Abaloparatide dose-dependently increased structural strength of L4-L5 vertebral bodies, the femur diaphysis, and the femur neck. Increments in peak load for lumbar spine and the femur diaphysis of abaloparatide-treated rats persisted even after adjusting for treatment-related increments in BMC, and estimated material properties were maintained or increased at the femur diaphysis with abaloparatide. The abaloparatide groups also exhibited significant and positive correlations between bone mass and bone strength at these sites. These data indicate that gains in cortical and trabecular bone mass with abaloparatide are accompanied by and correlated with improvements in bone strength, resulting in maintenance or improvement in bone quality. Thus, this study demonstrated that long-term daily administration of abaloparatide to osteopenic OVX rats led to dose-dependent improvements in bone mass, geometry and strength.

Nonhuman Primate Models of Osteoporosis

This chapter reviews the use of the nonhuman primate (NHP) as an animal model for osteoporosis research. While the previous chapters have extensively covered the use of lower species, the use of a higher species such as the NHP is likely to be the ultimate, or in some cases, the only relevant species to study bone in osteoporosis research. NHPs are used extensively in osteoporosis research in the pharmaceutical industry to evaluate new drug targets and in this context have contributed much to our current understanding of the human disease. This chapter focuses on the use of the NHP in drug development, presenting practical information on model selection and current techniques used to derive the primary end points of interest. Most of the practices described are considered relevant even in a basic research laboratory setting and will hopefully prove useful to researchers outside the pharmaceutical industry.

Finite Element Analysis of Denosumab Treatment Effects on Vertebral Strength in Ovariectomized Cynomolgus Monkeys.

Finite element analysis has not yet been validated for measuring changes in whole‐bone strength at the hip or spine in people after treatment with an osteoporosis agent. Toward that end, we assessed the ability of a clinically approved implementation of finite element analysis to correctly quantify treatment effects on vertebral strength, comparing against direct mechanical testing, in cynomolgus monkeys randomly assigned to one of three 16‐month‐long treatments: sham surgery with vehicle (Sham‐Vehicle), ovariectomy with vehicle (OVX‐Vehicle), or ovariectomy with denosumab (OVX‐DMAb). After treatment, T12 vertebrae were retrieved, scanned with micro‐CT, and mechanically tested to measure compressive strength. Blinded to the strength data and treatment codes, the micro‐CT images were coarsened and homogenized to create continuum‐type finite element models, without explicit porosity. With clinical translation in mind, these models were then analyzed for strength using the U.S. Food and Drug Administration (FDA)‐cleared VirtuOst software application (O.N. Diagnostics, Berkeley, CA, USA), developed for analysis of human bones. We found that vertebral strength by finite element analysis was highly correlated (R2 = 0.97; n = 52) with mechanical testing, independent of treatment (p = 0.12). Further, the size of the treatment effect on strength (ratio of mean OVX‐DMAb to mean OVX‐Vehicle, as a percentage) was large and did not differ (p = 0.79) between mechanical testing (+57%; 95% CI [26%, 95%]) and finite element analysis (+51% [20%, 88%]). The micro‐CT analysis revealed increases in cortical thickness (+45% [19%, 73%]) and trabecular bone volume fraction (+24% [8%, 42%]). These results show that a preestablished clinical finite element analysis implementation—developed for human bone and clinically validated in fracture‐outcome studies—correctly quantified the observed treatment effects of denosumab on vertebral strength in cynomolgus monkeys. One implication is that the treatment effects in this study are well explained by the features contained within these finite element models, namely, the bone geometry and mass and the spatial distribution of bone mass.

Instilled or injected purified natural capsaicin has no adverse effects on rat hindlimb sensory-motor behavior or osteotomy repair.

BACKGROUND: A novel formulation of ≥98% pure capsaicin (4975) is currently undergoing clinical investigation using novel routes of delivery to provide selective analgesia lasting weeks to months with a single dose. We conducted this study to assess the safety and effects of instilled and injected 4975 in rat models of wound healing osteotomy repair and sensory-motor nerve function. METHODS: Adult male and female Sprague-Dawley rats were used. To assess the effects of 4975 on nerve or muscle, 0.0083 or 0.025 mg 4975 or vehicle (25% polyethylene glycol-300) was applied to exposed sciatic nerve, or 0.1 mg 4975 or vehicle was injected into the surrounding muscle (Group 1). To assess the effect of 4975 on bone healing, an osteotomy was made in one femur and 0.5 mg of 4975 or vehicle was instilled into the site (Group 2). Behavioral testing was performed on both groups of rats and histological evaluation of the sciatic nerve, and surrounding soft tissue and bone was done at days 3, 14, and 28 after surgery. Femurs from osteotomy rats were assessed using peripheral quantitative computed tomography and biomechanical testing. Standard statistical tests were used to compare groups. RESULTS: Rats with direct application of 4975 to the sciatic nerve and surrounding muscle were no different from the controls in nociceptive sensory responses (F = 0.910, P = 0.454), grip strength (F = 0.550, P = 0.654), or histology of the muscle or sciatic nerve. In osteotomy rats, there were no statistical differences between 4975 and vehicle-treated rats for bone area (H = 2.858, P = 0.414), bone mineral content (F = 0.945, P = 0.425), or bone mineral density (F = 0.87, P = 0.462) and no difference in soft tissue healing. There were neither differences in bone stiffness (F = 1.369, P = 0.268) nor were there noticeable differences in the macro- or microscopic appearance of the right femur osteotomy healing site and surrounding soft tissues between the control group and the 4975-treated animals. CONCLUSION: A single, clinically relevant application of instilled or injected 4975 has no observable adverse effect on wound and bone healing after osteotomy or on the structural integrity of exposed muscle and nerve.