ABSTRACT
Romosozumab treatment results in a transient early increase in bone formation and sustained decrease in bone resorption. Histomorphometric analyses revealed that the primary bone-forming effect of romosozumab is a transient early stimulation of modeling-based bone formation on cancellous and endocortical surfaces. Furthermore, preclinical studies have demonstrated that romosozumab may affect changes in the remodeling unit, resulting in positive bone balance. To further investigate the effects of romosozumab on bone balance, mo 12 (M12) and mo 2 (M2) (to analyze early effects) unpaired bone biopsies from the FRAME clinical trial were analyzed using remodeling site reconstruction to assess whether positive changes in bone balance on cancellous/endocortical surfaces may contribute to the progressive improvement in bone mass/structure and reduced fracture risk in osteoporotic women at high fracture risk. At M12, bone balance was higher with romosozumab vs placebo on cancellous (+6.1 vs +1.5 µm; P = .012) and endocortical (+5.2 vs -1.7 µm; P = .02) surfaces; higher bone balance was due to lower final erosion depth (40.7 vs 43.7 µm; P = .05) on cancellous surfaces and higher completed wall thickness (50.8 vs 47.5 µm; P = .037) on endocortical surfaces. At M2, the final erosion depth was lower on the endocortical surfaces (42.7 vs 50.7 µm; P = .021) and was slightly lower on the cancellous surfaces (38.5 vs 44.6 µm; P = .11) with romosozumab vs placebo. Sector analysis of early endocortical formative sites revealed higher osteoid thickness (29.9 vs 19.2 µm; P = .005) and mineralized wall thickness (18.3 vs 11.9 µm; P = .004) with romosozumab vs placebo. These evolving bone packets may reflect the early stimulation of bone formation that contributes to the increase in completed wall thickness at M12. These data suggest that romosozumab induces a positive bone balance due to its effects on bone resorption and formation at the level of the remodeling unit, contributing to the positive effects on bone mass, structure, and fracture risk.
Romosozumab treatment has a dual effect on bone, adding new bone and reducing bone loss. In the FRAME clinical trial, romosozumab increased the bone mass and strength and reduced fracture risk in postmenopausal women with osteoporosis. Addition of new bone occurs early in treatment and rapidly on cancellous and endocortical bone surfaces where bone resorption is not ongoing. However, it remains unclear if romosozumab affects bone loss or gain in areas where bone resorption is ongoing (remodeling units), contributing to a further positive bone balance. Here, we examined whether changes at the remodeling unit occur early (2 mo) and/or late (12 mo) in treatment by using bone biopsies from patients treated with romosozumab or placebo in FRAME. At M2, a combination of lower bone resorption and higher bone gain on endocortical surfaces resulted in a positive bone balance with romosozumab vs placebo. At M12, the bone balance was positive with romosozumab vs placebo due to lower bone resorption on cancellous surfaces and greater bone gain on endocortical surfaces. This demonstrates that romosozumab induces a positive bone balance at remodeling units early in treatment, leading to overall gains observed later, contributing to the positive effects of romosozumab on bone mass and structure.
Subject(s)
Antibodies, Monoclonal , Bone Remodeling , Humans , Female , Bone Remodeling/drug effects , Antibodies, Monoclonal/pharmacology , Aged , Bone Density/drug effects , Middle AgedABSTRACT
Sclerostin is an extracellular inhibitor of canonical Wnt signaling that inhibits bone formation and stimulates bone resorption. Anti-sclerostin antibodies (Scl-Ab) have been developed as bone-building agents. DKK1, another extracellular inhibitor of the pathway, is upregulated in osteocytes in response to sclerostin inhibition. To further enhance bone-forming effects, a bispecific antibody inhibiting both sclerostin and DKK1 was created (AMG 147). In nonclinical safety studies, AMG 147 resulted in novel skull findings. In the rat, there was increased thickness of skull bones of neural crest origin due to increased subperiosteal compact lamellar and intramembranous woven bone. Externally, subperiosteal fibroblastic/osteoblastic stromal cell proliferation with woven bone and hemorrhage was also observed. Scl-Ab alone resulted in increased skull thickness in the rat, like AMG 147, but without the stromal cell proliferation/woven bone formation. In contrast to embryonic flat bone development, intramembranous bone formed similar to plexiform bone. In the monkey, AMG 147 resulted in macroscopic skull thickening due to a diffuse increase in appositional lamellar bone and increased intramembranous bone on both periosteal surfaces of all skull bones. These data demonstrate that dual inhibition of sclerostin and DDK1 results in unique effects on the skull not observed with sclerostin inhibition alone.
Subject(s)
Adaptor Proteins, Signal Transducing , Antibodies , Bone and Bones , Intercellular Signaling Peptides and Proteins , Animals , Rats , Antibodies/pharmacology , Osteogenesis , Primates , Skull , Intercellular Signaling Peptides and Proteins/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Bone and Bones/drug effects , Bone and Bones/physiologyABSTRACT
Osteoarthritis (OA) is a disabling, degenerative disease characterized by progressive cartilage and bone damage. There remains a need for local therapies that, following a single injection, can provide long-term pain relief and functional improvement and potentially delay disease progression. FX201 is a novel, intra-articular (IA), interleukin-1 receptor antagonist (IL-1Ra) gene therapy in development for the treatment of OA. In this study, we assessed the efficacy, biodistribution, and safety of helper-dependent adenovirus (HDAd)-ratIL-1Ra, the rat surrogate of FX201, and the biodistribution of FX201, in the anterior cruciate ligament transection (ACLT) rat OA model. A single IA injection of HDAd-ratIL-1Ra administered 7 days post-ACLT mitigated OA-related changes to cartilage, bone, and the synovial membrane at week 12 following surgery. Furthermore, FX201 and HDAd-ratIL-1Ra persisted for at least 92 days in the injected joint and proximal tissues with minimal evidence of vector spreading peripherally. Finally, HDAd-ratIL-1Ra showed a favorable safety profile without any local or systemic adverse effects. In conclusion, HDAd-ratIL-1Ra demonstrated local therapeutic and disease-modifying effects and was well tolerated, supporting further clinical development of FX201.
Subject(s)
Interleukin 1 Receptor Antagonist Protein , Osteoarthritis , Adenoviridae/genetics , Animals , Disease Models, Animal , Genetic Therapy , Injections, Intra-Articular , Interleukin 1 Receptor Antagonist Protein/genetics , Osteoarthritis/genetics , Osteoarthritis/metabolism , Osteoarthritis/therapy , Rats , Synovial Membrane/metabolism , Tissue DistributionABSTRACT
The bone-forming agent romosozumab is a monoclonal antibody that inhibits sclerostin, leading to increased bone formation and decreased resorption. The highest levels of bone formation markers in human patients are observed in the first 2 months of treatment. Histomorphometric analysis of bone biopsies from the phase 3 FRAME trial (NCT01575834) showed an early significant increase in bone formation with concomitant decreased resorption. Preclinical studies demonstrated that most new bone formation after romosozumab treatment was modeling-based bone formation (MBBF). Here we analyzed bone biopsies from FRAME to assess the effect of 2 months of romosozumab versus placebo on the surface extent of MBBF and remodeling-based bone formation (RBBF). In FRAME, postmenopausal women aged ≥55 years with osteoporosis were randomized 1:1 to 210 mg romosozumab or placebo sc every month for 12 months, followed by 60 mg denosumab sc every 6 months for 12 months. Participants in the bone biopsy substudy received quadruple tetracycline labeling and underwent transiliac biopsies at month 2. A total of 29 biopsies were suitable for histomorphometry. Using fluorescence microscopy, bone formation at cancellous, endocortical, and periosteal envelopes was classified based on the appearance of underlying cement lines as modeling (smooth) or remodeling (scalloped). Data were compared using the Wilcoxon rank-sum test, without multiplicity adjustment. After 2 months, the median percentage of MBBF referent to the total bone surface was significantly increased with romosozumab versus placebo on cancellous (18.0% versus 3.8%; p = 0.005) and endocortical (36.7% versus 3.0%; p = 0.001), but not on periosteal (5.0% versus 2.0%; p = 0.37) surfaces, with no significant difference in the surface extent of RBBF on all three bone surfaces. These data show that stimulation of bone formation in the first 2 months of romosozumab treatment in postmenopausal women with osteoporosis is predominately due to increased MBBF on endocortical and cancellous surfaces. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Subject(s)
Bone Density Conservation Agents , Osteoporosis, Postmenopausal , Osteoporosis , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Bone Density , Bone Density Conservation Agents/therapeutic use , Female , Humans , Middle Aged , Osteogenesis , Osteoporosis/drug therapy , Osteoporosis, Postmenopausal/drug therapyABSTRACT
Inhibition of sclerostin increases bone formation and decreases bone resorption, leading to increased bone mass, bone mineral density, and bone strength and reduced fracture risk. In a clinical study of the sclerostin antibody romosozumab versus alendronate in postmenopausal women (ARCH), an imbalance in adjudicated serious cardiovascular (CV) adverse events driven by an increase in myocardial infarction (MI) and stroke was observed. To explore whether there was a potential mechanistic plausibility that sclerostin expression, or its inhibition, in atherosclerotic (AS) plaques may have contributed to this imbalance, sclerostin was immunostained in human plaques to determine whether it was detected in regions relevant to plaque stability in 94 carotid and 50 femoral AS plaques surgically collected from older female patients (mean age 69.6 ± 10.4 years). Sclerostin staining was absent in most plaques (67%), and when detected, it was of reduced intensity compared with normal aorta and was located in deeper regions of the plaque/wall but was not observed in areas considered relevant to plaque stability (fibrous cap and endothelium). Additionally, genetic variants associated with lifelong reduced sclerostin expression were explored for associations with phenotypes including those related to bone physiology and CV risk factors/events in a population-based phenomewide association study (PheWAS). Natural genetic modulation of sclerostin by variants with a significant positive effect on bone physiology showed no association with lifetime risk of MI or stroke. These data do not support a causal association between the presence of sclerostin, or its inhibition, in the vasculature and increased risk of serious cardiovascular events. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Subject(s)
Cardiovascular Diseases , Plaque, Atherosclerotic , Aged , Aged, 80 and over , Alendronate , Bone Density , Cardiovascular Diseases/genetics , Down-Regulation , Female , Humans , Middle Aged , Plaque, Atherosclerotic/geneticsABSTRACT
Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Antibodies, Monoclonal/pharmacology , Bone Density Conservation Agents/pharmacology , Cardiovascular System/drug effects , Animals , Antibodies, Monoclonal/therapeutic use , Bone Density Conservation Agents/therapeutic use , Drug Evaluation, Preclinical , Female , Fractures, Bone/prevention & control , Humans , Macaca fascicularis , Male , Mice, Inbred C57BL , Mice, Knockout, ApoE , Osteoporosis/drug therapy , Rats, Sprague-Dawley , RiskABSTRACT
The physical fractionator is a convenient and practical solution for estimation of total cell number in a regulatory toxicology setting because it is insensitive to shrinkage allowing for paraffin processing/embedding and does not require measurement of the reference or organ volume. The principle involves sampling a known fraction of an organ in one or more steps and counting the total number of cells present in the final sample, physical disector section pairs. The total cell number in the organ is estimated by multiplying the cell count in the final fraction by the inverse of the sampling fraction(s). The key feature of the design is that tissue shrinkage due to paraffin processing occurs before the organ is uniformly sampled. Another requirement is that thermal expansion or contraction is avoided during the preparation of disector sections from the individual embedded subsamples, which ensures that the disector sections represent a known constant fraction. This vertical physical fractionator with subsampling is a simple and fast estimator to obtain precise and robust estimates of total cell number in large flat or hollow organs that do not prolong routine necropsy procedures. It is compatible with paraffin processing, avoids exhaustive sectioning, and allows for the collection of routine histopathology sections.
Subject(s)
Neurons , Cell Count , Paraffin EmbeddingABSTRACT
Sclerostin, a protein produced by osteocytes, inhibits bone formation. Administration of sclerostin antibody results in increased bone formation in multiple animal models. Romosozumab, a humanized sclerostin antibody, has a dual effect on bone, transiently increasing serum biochemical markers of bone formation and decreasing serum markers of bone resorption, leading to increased BMD and reduction in fracture risk in humans. We aimed to evaluate the effects of romosozumab on bone tissue. In a subset of 107 postmenopausal women with osteoporosis in the multicenter, international, randomized, double-blind, placebo-controlled Fracture Study in Postmenopausal Women with Osteoporosis (FRAME), transiliac bone biopsies were performed either after 2 (n = 34) or 12 (n = 73) months of treatment with 210 mg once monthly of romosozumab or placebo to evaluate histomorphometry and microcomputed tomography-based microarchitectural endpoints. After 2 months, compared with either baseline values assessed after a quadruple fluorochrome labeling or placebo, significant increases (P < 0.05 to P < 0.001) in dynamic parameters of formation (median MS/BS: romosozumab 1.51% and 5.64%; placebo 1.60% and 2.31% at baseline and month 2, respectively) were associated with a significant decrease compared with placebo in parameters of resorption in cancellous (median ES/BS: placebo 3.4%, romosozumab 1.8%; P = 0.022) and endocortical (median ES/BS: placebo 6.3%, romosozumab 1.6%; P = 0.003) bone. At 12 months, cancellous bone formation was significantly lower (P < 0.05 to P < 0.001) in romosozumab versus placebo and the lower values for resorption endpoints seen at month 2 persisted (P < 0.001), signaling a decrease in bone turnover (P = 0.006). No significant change was observed in periosteal and endocortical bone. This resulted in an increase in bone mass and trabecular thickness with improved trabecular connectivity, without significant modification of cortical porosity at month 12. In conclusion, romosozumab produced an early and transient increase in bone formation, but a persistent decrease in bone resorption. Antiresorptive action eventually resulted in decreased bone turnover. This effect resulted in significant increases in bone mass and improved microarchitecture. © 2019 American Society for Bone and Mineral Research.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Bone Density Conservation Agents/therapeutic use , Bone and Bones/diagnostic imaging , Bone and Bones/pathology , Osteogenesis , Osteoporosis, Postmenopausal/diagnostic imaging , Osteoporosis, Postmenopausal/drug therapy , X-Ray Microtomography , Aged , Antibodies, Monoclonal/pharmacology , Biopsy , Bone Density Conservation Agents/pharmacology , Bone Resorption/diagnostic imaging , Bone Resorption/drug therapy , Bone and Bones/drug effects , Female , Humans , Osteogenesis/drug effectsABSTRACT
Antiresorptive agents, such as bisphosphonates and denosumab, are frequently used for the management of osteoporosis. Indeed, both medications decrease the risk of osteoporotic fractures; however, these medications are associated with rare but potentially severe side effects, such as osteonecrosis of the jaw (ONJ). ONJ, defined as an area of exposed bone in the maxillofacial region that lasts for 8 weeks, often presents with significant pain and infection and can lead to serious complications. Interestingly, other treatments for osteoporosis have been developed, such as antibodies against the osteocyte-secreted protein, sclerostin. Sclerostin functions to inhibit the Wnt signaling cascade, leading to inhibition of bone formation. In clinical trials, a sclerostin antibody (romosozumab, Amgen Inc., UCB Brussels) increases bone formation and lowers the risk of osteoporotic fractures. However, in conjunction with increased osteoblastic activity, a reduction in bone resorption markers is observed. This antiresorptive effect raises the concern of possible ONJ development in patients treated with sclerostin antibodies. Here, utilizing ligature-induced experimental periodontitis (EP), we evaluated the effects of sclerostin inhibition on the development of ONJ-like lesions in ovariectomized rats. Beginning 8 weeks post-ovariectomy, rats were treated for 22 weeks with weekly injections of vehicle (Veh), 200 µg/kg zoledronic acid (ZA), a potent bisphosphonate at 100-fold the osteoporosis dose, or 5 mg/kg sclerostin antibody (Scl-Ab) at the osteoporotic dose. EP was initiated at week 12 and maintained for the remainder of the study. Scl-Ab treatment transiently increased serum P1NP, a bone formation marker, increased BV/TV, and decreased eroded surfaces in lumbar vertebrae. ZA-treated rats developed histologic features of ONJ, whereas Veh-treated controls did not. Scl-Ab animals lost less periodontal bone in sites with EP. However, these animals presented with no histologic signs of ONJ. In conclusion, sclerostin inhibition enhanced structural bone parameters, without inducing ONJ-like lesions, in ovariectomized rats with EP. © 2018 American Society for Bone and Mineral Research.
Subject(s)
Antibodies/pharmacology , Bisphosphonate-Associated Osteonecrosis of the Jaw/metabolism , Bone Morphogenetic Proteins/antagonists & inhibitors , Osteoporosis/metabolism , Periodontitis/metabolism , Animals , Bisphosphonate-Associated Osteonecrosis of the Jaw/pathology , Female , Genetic Markers , Osteoporosis/pathology , Ovariectomy , Periodontitis/pathology , Rats , Rats, Sprague-Dawley , Zoledronic Acid/adverse effects , Zoledronic Acid/pharmacologyABSTRACT
INTRODUCTION: Single intra-articular (IA) injections of poly(lactic-co-glycolic acid) (PLGA) microsphere-based triamcinolone acetonide extended-release (TA-ER; formerly FX006) demonstrated sustained, clinically relevant benefits in patients with knee osteoarthritis. The local effects of TA-ER were assessed in normal canine knees in three nonclinical studies. METHODS: Knees were evaluated for up to 6 weeks or 9 months after a single injection of TA-ER (2.1/6.25/18.75 mg TA), or TA crystalline suspension (TAcs, 18.75 mg TA), and for up to 6 months after three injections (every 1 or 3 months) of TA-ER (6.25/18.75 mg TA) or TAcs (18.75 mg). Vehicle-diluent, blank microspheres, and untreated knees were used as controls. Plasma and synovial fluid (SF) TA concentrations and standard histopathological assessment of the synovium were conducted. Articular cartilage morphology was assessed via modified Mankin scoring. RESULTS: Plasma and SF concentrations indicated prolonged dose-dependent TA joint residency with TA-ER compared with TAcs. Effects in articular cartilage were dose- and time-dependent and consistent with known effects of corticosteroids in the normal knee. Loss of Safranin O staining occurred, indicative of a reduction in cartilage matrix proteoglycan, and recovered in a similar manner for TA-ER and TAcs across all studies. Structural lesions were infrequent and generally comparable in severity between TA-ER and TAcs but slightly higher in incidence for TA-ER. Focal/multifocal foreign-body responses (FBR) to PLGA were observed in the superficial layer of the synovium, peaking after 4-6 weeks, with significant recovery or complete resolution by month 6. CONCLUSIONS: These findings suggest that the effects of IA injections of TA-ER on cartilage are predominantly transient, and comparable to those observed with TAcs in the normal canine knee joint. These mild effects in the normal joint differ from the beneficial effects observed with TA-ER and other corticosteroids in disease models. The synovial FBR to PLGA microspheres was focal and transient. FUNDING: Flexion Therapeutics, Inc. Plain language summary available for this article.
ABSTRACT
Sclerostin antibody (Scl-Ab) stimulates bone formation, which with long-term treatment, attenuates over time. The cellular and molecular mechanisms responsible for the attenuation of bone formation are not well understood, but in aged ovariectomized (OVX) rats, the reduction in vertebral cancellous bone formation is preceded by a reduction in osteoprogenitor (OP) number and significant induction of signaling pathways known to suppress mitogenesis and cell cycle progression in the osteocyte (OCy) (Taylor et al., 2016). To determine if the reduction in OP number is associated with a decrease in proliferation, aged OVX rats were administered vehicle or Scl-Ab for 9 or 29â¯days and implanted with continuous-delivery 5-bromo-2'-deoxyuridine (BrdU) mini-osmotic pumps 5â¯days prior to necropsy. The total number of BrdU-labeled osteoblasts (OB) was quantified in vertebral cancellous bone to indirectly assess the effects of Scl-Ab treatment on OP proliferation at the time of activation of modeling-based bone formation at day 9 and at the time of maximal mineralizing surface, initial decrease in OP number, and transcriptional changes in the OCy at day 29. Compared with vehicle, Scl-Ab resulted in an increase in the total number of BrdU-positive OB (+260%) at day 9 that decreased with continued treatment (+50%) at day 29. These differences in proliferation occurred at time points when the increase in total OB number was significant and similar in magnitude. These findings suggest that reduced OP proliferation contributes to the decrease in OP numbers, an effect that would limit the OB pool and contribute to the attenuation of bone formation that occurs with long-term Scl-Ab treatment.
ABSTRACT
Inhibition of sclerostin with sclerostin antibody (Scl-Ab) results in stimulation of bone formation on cancellous (Cn), endocortical (Ec), and periosteal (Ps) surfaces in rodents and non-human primates. With long-term dosing of Scl-Ab, the increase in bone formation is not sustained, attenuating first on Cn surfaces and later on Ec and Ps surfaces. In Cn bone, the attenuation in bone formation (self-regulation) is associated with transcriptional changes in the osteocyte (OCy) that would limit mitogenesis and are sustained with continued dosing. The expression changes in Cn OCy occur coincident with a decrease in osteoprogenitor (OP) numbers that may directly or indirectly be a consequence of the transcriptional changes in the OCy to limit OP proliferation. To characterize the Scl-Ab-mediated changes in cortical (Ct) bone and compare these changes to Cn bone, densitometric, histomorphometric, and transcriptional analyses were performed on femur diaphyses from aged ovariectomized rats. Animals were administered 50â¯mg/kg/wk of Scl-Ab or vehicle for up to 6â¯months (183â¯days), followed by a treatment-free period (up to 126â¯days). Scl-Ab increased Ct mass and area through day 183, which declined slightly when treatment was discontinued. Ps and Ec bone formation was sustained through the dosing on both Ct surfaces, with evidence of a decline in bone formation only at day 183 on the Ec surface. This is in contrast to Cn bone, where reduced bone formation was observed after day 29. TaqMan analysis of 60 genes with functional roles in the bone using mRNA isolated from laser capture micro-dissection samples enriched for Ec osteoblasts and Ct OCy suggest a pattern of gene expression in Ct bone that differed from Cn, especially in the OCy, and that corresponded to observed differences in the timing of phenotypic changes. Notable with Scl-Ab treatment was a "transcriptional switch" in Ct OCy at day 183, coincident with the initial decline in bone formation on the endocortex. A consistent sustained increase of expression for most genes in response to Scl-Ab was observed from day 8 through day 85 at the times of maximal bone formation on both Ct surfaces; however, at day 183, this increase was reversed, with expression of these genes generally returning to control values or decreasing compared to vehicle. Genes exhibiting this pattern included Wnt inhibitors Sost and Dkk1, though both had been up-regulated until the end of dosing in Cn OCy. Changes in cell cycle genes such as Cdkn1a and Ndrg1 in Ct OCy suggested up-regulation of p53 signaling, as observed in Cn OCy; however, unlike in Cn bone, p53 signaling was not associated with decreased bone formation and was absent at day 183, when bone formation began to decline on the Ec surface. These data demonstrate involvement of similar molecular pathways in Ct and Cn bone in response to Scl-Ab but with a different temporal relationship to bone formation and suggest that the specific mechanism underlying self-regulation of Scl-Ab-induced bone formation may be different between Cn and Ct bone.
ABSTRACT
Estimation of total number of a population of cells that are sparsely distributed in an organ or anatomically-defined region of interest represents a challenge for conventional stereological methods. In these situations, classic fractionator approaches that rely on systematic uniform random sampling are highly inefficient and, in many cases, impractical due to the intense sampling of the organ and tissue sections that is required to obtain sufficient counts for an acceptable level of precision. The proportionator, an estimator based on non-uniform sampling theory, marries automated image analysis with stereological principles and is the only estimator that provides a highly efficient and precise method to address these challenging quantification problems. In this paper, the practical considerations of the proportionator estimator and its implementation with Proportionator™ software and digital slide imaging are reviewed. The power of the proportionator as a stereological tool is illustrated in its application to the estimation of the total number of a very rare (~50/vertebrae) and sparsely distributed population of osteoprogenitor cells in mouse vertebral body. The proportionator offers a solution to neuroscientists interested in quantifying total cell number of sparse cell populations in the central and peripheral nervous system where systematic uniform random sampling-based stereological estimators are impractical.
ABSTRACT
This session was a series of presentations focused on safety considerations for late stage or currently marketed bone therapeutic agents. The first presentation was an overview of a major regulatory requirement in the nonclinical filing package for bone therapeutics, studies designed to assess the impact of an agent on bone quality. Two presentations focused on safety issues associated with drugs whose primary mechanism of action is inhibition of bone resorption. Typical findings associated with this class of agents in general and reproductive toxicology studies were reviewed, highlighting INHAND (International Harmonization of Nomenclature and Diagnostic Criteria) nomenclature. This was followed by an overview of safety issues that have been identified largely through clinical experience. Similar presentations followed emphasizing safety and regulatory issues associated with classes of drugs whose primary mechanism of action is stimulation of bone formation known broadly as bone anabolic agents. The major focus of these discussions was carcinogenicity risk assessment. The final presentation was an introduction to a rapidly evolving area in bone therapeutics, treatment of rare genetic bone diseases, and the developmental challenges associated with these indications and novel therapeutic modalities.
Subject(s)
Bone Density Conservation Agents/toxicity , Bone Diseases/drug therapy , Anabolic Agents/toxicity , Animals , Bone and Bones/drug effects , Carcinogens/toxicity , Drug Evaluation, Preclinical , Humans , Reproduction/drug effects , Risk Assessment , Terminology as Topic , ToxicologyABSTRACT
Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed "Bone CLARITY," a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato-labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue.
Subject(s)
Bone Marrow Cells/cytology , Stem Cells/cytology , Animals , Cell Differentiation/physiology , Cells, Cultured , Mice , Mice, Transgenic , Osteoblasts/cytology , Osteogenesis/physiology , SOX9 Transcription Factor/metabolism , Stem Cells/metabolismABSTRACT
Romosozumab, a humanized monoclonal sclerostin antibody under development for the treatment of osteoporosis, has a unique mechanism of action on bone-increasing bone formation and decreasing bone resorption. The effects on bone formation are transient, eliciting a rapid increase in bone formation that attenuates with continued treatment. Although bone formation attenuates, bone mineral density (BMD) continues to increase. To explore potential tissue-level mechanisms that could contribute to a progressive increase in spine BMD, we used kinetic reconstruction techniques to examine the effects of romosozumab on modeling and remodeling units in vertebral cancellous bone from adult cynomolgus monkeys administered romosozumab for 10 and 28weeks. The 10-week study duration captured a period of high modeling-based bone formation, and the 28-week study duration followed the self-regulation or attenuation of bone formation in cancellous bone that occurs with long-term treatment. Sequential fluorochrome labels applied for the kinetic reconstruction were also used to evaluate treatment effects on osteoblast function as early as 3weeks, and on bone formation and bone accrual in the vertebral cortex over 28weeks. Kinetic reconstruction of remodeling and modeling formation sites in vertebral cancellous bone revealed that romosozumab effected significant transient increases in mineral apposition rate in remodeling sites at week 3 that was not sustained with continued treatment. However, romosozumab treatment caused sustained improvement in fractional labeling of osteoid, an index of osteoblast efficiency, at remodeling formative sites at both weeks 10 and 28 that was the major contributor to significant increases in final wall thickness (W.Th) of remodeling packets. Remodeling W.Th matched the final W.Th of modeling packets at week 10. At both weeks 10 and 28, romosozumab significantly decreased eroded surface (ES/BS). At week 28, romosozumab also significantly reduced resorption period (Rs.P) and final resorption depth (Rs.De). The reduced final Rs.De combined with the increased W.Th resulted in a significant increase in bone balance (BB) at the level of the remodeling unit. Assessment of bone formation on the vertebral periosteal and endocortical surfaces following 28weeks of treatment revealed that romosozumab significantly increased bone formation on these surfaces, which had attenuated by week 28, resulting in significant increases in new periosteal and endocortical bone by week 28. These data suggest that multiple factors potentially contribute to the increase in spine BMD with romosozumab treatment. In the early period of treatment, increased modeling-based bone formation, increased W.Th at remodeling sites, a decrease in remodeling space secondary to decreased ES/BS in vertebral cancellous bone, and increased periosteal and endocortical bone formation in the vertebral cortex contribute to the early increase in spine BMD. Following the self-regulation of bone formation when modeling-based bone formation has attenuated, a decrease in remodeling space secondary to reduced ES/BS and a positive BB secondary to decreased final Rs.De and increased W.Th contribute to the progressive increase in spine BMD with long-term treatment.
Subject(s)
Antibodies, Monoclonal/pharmacology , Bone Density/drug effects , Animals , Bone Remodeling/drug effects , Cancellous Bone/cytology , Cancellous Bone/drug effects , Cortical Bone/cytology , Cortical Bone/drug effects , Macaca fascicularis , Male , Osteoblasts/cytology , Osteoblasts/drug effects , Osteogenesis/drug effects , Osteoporosis/metabolism , Wnt Signaling Pathway/drug effectsABSTRACT
Results of prior studies suggest that fibroblast growth factor 21 (FGF21) may be involved in bone turnover and in the actions of peroxisome proliferator-activated receptor (PPAR) α and γ in mice. We have conducted independent studies to examine the effects of FGF21 on bone homeostasis and the role of FGF21 in PPARα and γ actions. High-fat-diet-induced obesity (DIO) mice were administered vehicle or recombinant human FGF21 (rhFGF21) intraperitoneally at 0 (vehicle), 0.1, 1, and 3 mg/kg daily for 2 weeks. Additional groups of DIO mice received water or 10 mg/kg rosiglitazone daily. Mice treated with rhFGF21 or rosiglitazone showed expected metabolic improvements in glucose, insulin, and lipid levels. However, bone loss was not detected in rhFGF21-treated mice by dual-energy X-ray absorptiometry (DXA), micro-CT, and histomorphometric analyses. Mineral apposition rate, a key bone formation parameter, was unchanged by rhFGF21, while significantly decreased by rosiglitazone in DIO mice. Bone resorption markers, OPG/RANKL mRNA expression, and histological bone resorption indices were unchanged by rhFGF21 or rosiglitazone. Bone marrow fat was unchanged by rhFGF21, while increased by rosiglitazone. Furthermore, FGF21 knockout mice did not show high bone mass phenotype. Treatment with PPARα or PPARγ agonists caused similar metabolic effects in FGF21 knockout and wild-type mice. These results contrast with previous findings and suggest that FGF21 is not critical for bone homeostasis or actions of PPARα and PPARγ. © 2016 American Society for Bone and Mineral Research.
Subject(s)
Bone Density , Fibroblast Growth Factors , Gene Expression Regulation/drug effects , Homeostasis , PPAR alpha , PPAR gamma , Animals , Bone Density/drug effects , Bone Density/genetics , Dietary Fats/adverse effects , Dietary Fats/pharmacology , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/pharmacology , Glucose/metabolism , Homeostasis/drug effects , Homeostasis/genetics , Humans , Insulin/genetics , Insulin/metabolism , Male , Mice , Mice, Knockout , Obesity/chemically induced , Obesity/metabolism , Osteoprotegerin/biosynthesis , Osteoprotegerin/genetics , PPAR alpha/agonists , PPAR alpha/biosynthesis , PPAR alpha/genetics , PPAR gamma/agonists , PPAR gamma/biosynthesis , PPAR gamma/genetics , RANK Ligand/biosynthesis , RANK Ligand/genetics , Rosiglitazone , Thiazolidinediones/pharmacologyABSTRACT
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. © 2016 American Society for Bone and Mineral Research.
Subject(s)
Absorptiometry, Photon , Antibodies, Monoclonal/pharmacology , Bone Density/drug effects , Femur Neck , Ovariectomy , Radius , Animals , Diaphyses/diagnostic imaging , Diaphyses/metabolism , Female , Femur Neck/diagnostic imaging , Femur Neck/metabolism , Macaca fascicularis , Radius/diagnostic imaging , Radius/metabolismABSTRACT
There is an unmet need for therapies that can restore bone strength and reduce fracture risk among patients at high risk of osteoporotic fracture. To address this need, bone-forming therapies that increase osteoblast activity are required to help restore bone structure and strength. Sclerostin is now recognized as a target for osteoporosis therapy. Sclerostin is predominantly secreted by the osteocyte and acts as an extracellular inhibitor of canonical Wnt signaling by binding to the receptors lipoprotein receptor-related protein-4, 5 and 6. Monoclonal antibodies to sclerostin (Scl-Ab) have been used in both clinical and in preclinical studies of osteoporosis with beneficial outcomes for bone density, structure, strength and fracture risk reduction. In this review paper, we summarize the current literature describing the effects of Scl-Ab in animal models of osteoporosis. In addition, we report new pharmacologic data from three animal studies of Scl-Ab: 1) a 12-month study evaluating bone quality in ovariectomized (OVX) rats; 2) a 6-month study evaluating bone structure and strength in adolescent cynomolgus monkeys; and 3) the effects of transition from Scl-Ab to vehicle or the RANKL inhibitor osteoprotegerin-Fc in OVX rats. Together, these results demonstrate that inhibition of sclerostin by Scl-Ab increased bone formation, and decreased bone resorption, leading to improved bone structure, bone mass and bone strength while maintaining bone quality in multiple animal models of osteoporosis. Further, gains in bone mass induced by Scl-Ab treatment were preserved by antiresorptive agents such as a RANKL inhibitor as a follow-on therapy. The bone-forming effects of Scl-Ab were unaffected by pre- or co-treatment with a bisphosphonate, and were restored following a treatment-free period after initial dosing. These data support the clinical development of Scl-Ab for treatment of conditions with low bone mass such as postmenopausal and male osteoporosis.
Subject(s)
Antibodies/therapeutic use , Bone Morphogenetic Proteins/immunology , Osteoporosis/drug therapy , Animals , Bone Resorption/drug therapy , Bone and Bones/pathology , Bone and Bones/physiopathology , Disease Models, Animal , Humans , Osteoporosis/physiopathologyABSTRACT
The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is an initiative of the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the skeletal tissues and teeth of laboratory rats and mice, with color photomicrographs illustrating examples of many common lesions. The standardized nomenclature presented in this document is also available on the internet (http://www.goreni.org/). Sources of material were databases from government, academic and industrial laboratories throughout the world.