Sclerostin Antibody Research Articles

Co-deletion of Lrp5 and Lrp6 in the skeleton severely diminishes bone gain from sclerostin antibody administration.

The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of Lrp5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when Lrp5 is suppressed), or through other yet undiscovered mechanisms independent of Lrp5/6. To distinguish between these two possibilities, we generated mice with compound deletion of Lrp5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both Lrp5 and Lrp6 (Lrp5f/f; Lrp6f/f), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre-) mice were injected with Scl-mAb or vehicle from 4.5 to 14weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre- mice, as assessed by DXA, μCT, pQCT, and histology, indicating that Lrp5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre- mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of Lrp5 alone, the bone-selective late-stage co-deletion of Lrp5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both Lrp5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody.

Sclerostin antibody treatment increases bone mass and normalizes circulating phosphate levels in growing Hyp mice

X-linked hypophosphatemia (XLH), caused by a loss-of-function mutation in the phosphate regulating gene with homology to endopeptidase located on the X chromosome (PHEX), is the most common form of vitamin D-resistant rickets. Loss of functional PHEX results in elevated fibroblast growth factor 23 (FGF23) levels, impaired phosphate reabsorption, and inhibited skeletal mineralization. Sclerostin, a protein produced primarily in osteocytes, suppresses bone formation by antagonizing Wnt signaling and is reported to be elevated in XLH patients. This study used the Hyp mouse model to investigate sclerostin's role in the pathophysiology of XLH by evaluating the use of a monoclonal antibody to sclerostin in a mouse model of XLH, the Hyp mouse. Male and female wild-type and Hyp littermates were injected with 25 mg/kg of vehicle or sclerostin antibody (Scl-Ab) twice weekly, beginning at 4 weeks of age and euthanized at 8 weeks of age. Scl-Ab treatment increased serum phosphate levels and suppressed circulating levels of intact FGF23 in treated wild-type and Hyp mice of both sexes. Cortical area, trabecular bone volume fraction (BV/TV), metaphyseal apparent density, and the peak load increased with Scl-Ab treatment in both sexes. This short-term treatment study suggests that Scl-Ab treatment can effectively improve some of the pathologies associated with XLH, including normalization of phosphate, and that sclerostin may play a role in regulating FGF23 and phosphate metabolism in XLH. © 2019 American Society for Bone and Mineral Research.

Mechanically Driven Counter-Regulation of Cortical Bone Formation in Response to Sclerostin-Neutralizing Antibodies.

Sclerostin (Scl) antibodies (Scl-Ab) potently stimulate bone formation, but these effects are transient. Whether the rapid inhibition of Scl-Ab anabolic effects is due to a loss of bone cells' capacity to form new bone or to a mechanostatic downregulation of Wnt signaling once bone strength exceeds stress remains unclear. We hypothesized that bone formation under Scl-Ab could be reactivated by increasing the dose of Scl-Ab and/or by adding mechanical stimuli, and investigated the molecular mechanisms involved in this response, in particular the role of periostin (Postn), a co-activator of the Wnt pathway in bone. For this purpose, C57Bl/6, Postn-/- and Postn+/+ mice were treated with vehicle or Scl-Ab (50 to 100 mg/kg/wk) for various durations and subsequently subjected to tibia axial compressive loading. In wild-type (WT) mice, Scl-Ab anabolic effects peaked between 2 and 4 weeks and declined thereafter, with no further increase in bone volume and strength between 7 and 10 weeks. Doubling the dose of Scl-Ab did not rescue the decline in bone formation. In contrast, mechanical stimulation was able to restore cortical bone formation concomitantly to Scl-Ab treatment at both doses. Several Wnt inhibitors, including Dkk1, Sost, and Twist1, were upregulated, whereas Postn was markedly downregulated by 2 to 4 weeks of Scl-Ab. Mechanical loading specifically upregulated Postn gene expression. In turn, Scl-Ab effects on cortical bone were more rapidly downregulated in Postn-/- mice. These results indicate that bone formation is not exhausted by Scl-Ab but inhibited by a mechanically driven downregulation of Wnt signaling. Hence, increasing mechanical loads restores bone formation on cortical surfaces, in parallel with Postn upregulation. © 2020 American Society for Bone and Mineral Research (ASBMR).

Corrigendum to: Gut epithelial impairment, microbial translocation and immune system activation in inflammatory bowel disease-associated spondyloarthritis.

Objectives Gut microbiota has been widely reported to be involved in systemic inflammation through microbial translocation and T cell activation in several diseases. In this work we aimed to investigate bacterial infiltration and epithelial impairment in the gut of patients with IBD-associated SpA (SpA-IBD), as well as the relationship of microbial translocation with immune system activation and their putative role in the pathogenesis of joint inflammation in IBD patients. Methods Tight-junction proteins (TJPs) occludin and claudin-1/-4 and bacteria were assessed by real-time PCR analysis and immunohistochemical staining of the ileum. Intestinal fatty acid binding protein (I-FABP), lipopolysaccharides (LPS), soluble CD14 (sCD14), sclerostin and anti-sclerostin antibodies (anti-sclerostin-IgG) were assayed with ELISAs and peripheral mononuclear blood cells with flow cytometry. LPS and sCD14 were used in vitro to stimulate a human osteoblast cell line. Results Compared with IBD, ileal samples from SpA-IBD patients showed bacterial infiltration, epithelial damage and downregulation of TJPs. In sera, they showed higher serum levels of I-FABP, LPS, sCD14 (the latter correlating with sclerostin and anti-sclerostin-IgG) and higher CD80+/CD163+ and lower CD14+ mononuclear cells. In vitro experiments demonstrated that only the LPS and sCD14 synergic action downregulates sclerostin expression in osteoblast cells. Conclusion SpA-IBD patients are characterized by gut epithelium impairment with consequent translocation of microbial products into the bloodstream, immune system activation and an increase of specific soluble biomarkers. These findings suggest that gut dysbiosis could be involved in the pathogenesis of SpA-IBD and it could hopefully prompt the use of these biomarkers in the follow-up and management of IBD patients.

The Osteocyte as the New Discovery of Therapeutic Options in Rare Bone Diseases.

Osteocytes are the most abundant (~95%) cells in bone with the longest half-life (~25 years) in humans. In the past osteocytes have been regarded as vestigial cells in bone, since they are buried inside the tough bone matrix. However, during the last 30 years it has become clear that osteocytes are as important as bone forming osteoblasts and bone resorbing osteoclasts in maintaining bone homeostasis. The osteocyte cell body and dendritic processes reside in bone in a complex lacuno-canalicular system, which allows the direct networking of osteocytes to their neighboring osteocytes, osteoblasts, osteoclasts, bone marrow, blood vessels, and nerves. Mechanosensing of osteocytes translates the applied mechanical force on bone to cellular signaling and regulation of bone adaptation. The osteocyte lacuno-canalicular system is highly efficient in transferring external mechanical force on bone to the osteocyte cell body and dendritic processes via displacement of fluid in the lacuno-canalicular space. Osteocyte mechanotransduction regulates the formation and function of the osteoblasts and osteoclasts to maintain bone homeostasis. Osteocytes produce a variety of proteins and signaling molecules such as sclerostin, cathepsin K, Wnts, DKK1, DMP1, IGF1, and RANKL/OPG to regulate osteoblast and osteoclast activity. Various genetic abnormality-associated rare bone diseases are related to disrupted osteocyte functions, including sclerosteosis, van Buchem disease, hypophosphatemic rickets, and WNT1 and plastin3 mutation-related disorders. Meticulous studies during the last 15 years on disrupted osteocyte function in rare bone diseases guided for the development of various novel therapeutic agents to treat bone diseases. Studies on genetic, molecular, and cellular mechanisms of sclerosteosis and van Buchem disease revealed a role for sclerostin in bone homeostasis, which led to the development of the sclerostin antibody to treat osteoporosis and other bone degenerative diseases. The mechanism of many other rare bone diseases and the role of the osteocyte in the development of such conditions still needs to be investigated. In this review, we mainly discuss the knowledge obtained during the last 30 years on the role of the osteocyte in rare bone diseases. We speculate about future research directions to develop novel therapeutic drugs targeting osteocyte functions to treat both common and rare bone diseases.

Gene Expression Profile and Acute Gene Expression Response to Sclerostin Inhibition in Osteogenesis Imperfecta Bone.

ABSTRACTSclerostin antibody (SclAb) therapy has been suggested as a novel therapeutic approach toward addressing the fragility phenotypic of osteogenesis imperfecta (OI). Observations of cellular and transcriptional responses to SclAb in OI have been limited to mouse models of the disorder, leaving a paucity of data on the human OI osteoblastic cellular response to the treatment. Here, we explore factors associated with response to SclAb therapy in vitro and in a novel xenograft model using OI bone tissue derived from pediatric patients. Bone isolates (approximately 2 mm3) from OI patients (OI type III, type III/IV, and type IV, n = 7; non‐OI control, n = 5) were collected to media, randomly assigned to an untreated (UN), low‐dose SclAb (TRL, 2.5 μg/mL), or high‐dose SclAb (TRH, 25 μg/mL) group, and maintained in vitro at 37°C. Treatment occurred on days 2 and 4 and was removed on day 5 for TaqMan qPCR analysis of genes related to the Wnt pathway. A subset of bone was implanted s.c. into an athymic mouse, representing our xenograft model, and treated (25 mg/kg s.c. 2×/week for 2/4 weeks). Implanted OI bone was evaluated using μCT and histomorphometry. Expression of Wnt/Wnt‐related targets varied among untreated OI bone isolates. When treated with SclAb, OI bone showed an upregulation in osteoblast and osteoblast progenitor markers, which was heterogeneous across tissue. Interestingly, the greatest magnitude of response generally corresponded to samples with low untreated expression of progenitor markers. Conversely, samples with high untreated expression of these markers showed a lower response to treatment. in vivo implanted OI bone showed a bone‐forming response to SclAb via μCT, which was corroborated by histomorphometry. SclAb induced downstream Wnt targets WISP1 and TWIST1, and elicited a compensatory response in Wnt inhibitors SOST and DKK1 in OI bone with the greatest magnitude from OI cortical bone. Understanding patients' genetic, cellular, and morphological bone phenotypes may play an important role in predicting treatment response. This information may aid in clinical decision‐making for pharmacological interventions designed to address fragility in OI. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Nonclinical cardiovascular safety evaluation of romosozumab, an inhibitor of sclerostin for the treatment of osteoporosis in postmenopausal women at high risk of fracture

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.

Gut epithelial impairment, microbial translocation and immune system activation in inflammatory bowel disease-associated spondyloarthritis.

Gut microbiota has been widely reported to be involved in systemic inflammation through microbial translocation and T cell activation in several diseases. In this work we aimed to investigate bacterial infiltration and epithelial impairment in the gut of patients with IBD-associated SpA (SpA-IBD), as well as the relationship of microbial translocation with immune system activation and their putative role in the pathogenesis of joint inflammation in IBD patients. Tight-junction proteins (TJPs) occludin and claudin-1/-4 and bacteria were assessed by real-time PCR analysis and immunohistochemical staining of the ileum. Intestinal fatty acid binding protein (I-FABP), lipopolysaccharides (LPS), soluble CD14 (sCD14), sclerostin and anti-sclerostin antibodies (anti-sclerostin-IgG) were assayed with ELISAs and peripheral mononuclear blood cells with flow cytometry. LPS and sCD14 were used in vitro to stimulate a human osteoblast cell line. Compared with IBD, ileal samples from SpA-IBD patients showed bacterial infiltration, epithelial damage and downregulation of TJPs. In sera, they showed higher serum levels of I-FABP, LPS, sCD14 (the latter correlating with sclerostin and anti-sclerostin-IgG) and higher CD80+/CD163+ and lower CD14+ mononuclear cells. In vitro experiments demonstrated that only the LPS and sCD14 synergic action downregulates sclerostin expression in osteoblast cells. SpA-IBD patients are characterized by gut epithelium impairment with consequent translocation of microbial products into the bloodstream, immune system activation and an increase of specific soluble biomarkers. These findings suggest that gut dysbiosis could be involved in the pathogenesis of SpA-IBD and it could hopefully prompt the use of these biomarkers in the follow-up and management of IBD patients.

Osteoporosis Treatments Affect Bone Matrix Maturation in a Rat Model of Induced Cortical Remodeling.

ABSTRACTTo test how osteoporosis drugs affect bone matrix maturation during cortical bone remodeling, 72 pregnant rats were switched from a 0.4% to a 0.01% calcium diet at parturition for a 23‐day lactation period. At weaning, eight dams were sacrificed to establish baseline values, while the remaining dams were returned to 0.4% calcium and treated with vehicle (saline), sodium fluoride (NaF), zoledronic acid (ZA), or sclerostin antibody (Scl‐Ab) for either 7 or 28 days (eight animals per group per time point). Femora were examined by μCT, dynamic histomorphometry, Fourier transform infrared imaging, and three‐point bending of notched specimens. Cortical porosity decreased in all groups from baseline to day 28. Intracortical mineralizing surface (MS/BS) and mineral apposition rate (MAR), as well as the mineral‐to‐matrix ratio were unaffected by treatment, but intracortical crystallinity was increased in the ZA group at day 10 compared with vehicle. Cortical area increased in all groups over 28 days mainly because of an addition of bone at the endocortical surface. Endocortical MS/BS did not vary among the groups, but endocortical MAR was suppressed in the NaF group at day 2 and elevated in the Scl‐Ab group at day 4 compared with vehicle. Endocortical mineral‐to‐matrix ratio was increased at days 5 and 10 following NaF treatment and endocortical crystallinity was increased at day 5 following ZA treatment compared with vehicle. Fracture toughness did not differ among the groups. Thus, the treatments affected matrix maturation more strongly at the endocortical then intracortical envelope. In this model of induced remodeling, the bone formation phase is synchronized at multiple sites, facilitating study of the effects of drugs or other bone‐targeting agents on matrix maturation independent of their effects on the initiation of remodeling. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Romosozumab or alendronate for fracture prevention in East Asian patients: a subanalysis of the phase III, randomized ARCH study

SummaryRomosozumab, a sclerostin antibody, exerts dual effect to increase bone formation and decrease bone resorption. Among high-risk postmenopausal East Asian women, romosozumab followed by alendronate was associated with lower incidences of fractures vs alendronate alone. Romosozumab demonstrates potential to address an unmet need in osteoporosis management in Asia.IntroductionRomosozumab, a sclerostin antibody, exerts dual effect to increase bone formation and decrease bone resorption. The global ARCH study demonstrated superiority of romosozumab followed by alendronate in reducing fracture risk in high-risk postmenopausal osteoporotic women vs alendronate alone. We report outcomes among ARCH East Asian patients.MethodsIn ARCH, 4093 postmenopausal osteoporotic women with fragility fracture were randomized 1:1 to monthly romosozumab 210 mg or weekly alendronate 70 mg for 12 months, both followed by open-label alendronate. Primary endpoints were incidence of new vertebral fracture (VF) at 24 months and clinical fracture at primary analysis (confirmed fractures in ≥ 330 patients and all patients had opportunity to attend month 24 visit). This post hoc analysis was not powered to detect fracture-rate differences.ResultsThis analysis included 275 patients from Hong Kong, Korea, and Taiwan. Romosozumab followed by alendronate reduced risk of new VFs at 24 months by 60% (P = 0.11) and clinical fractures at primary analysis by 44% (P = 0.15) vs alendronate alone. Romosozumab followed by alendronate significantly increased mean bone mineral density at 24 months from baseline by a further 9.0%, 3.3%, and 3.0% at the lumbar spine, total hip, and femoral neck vs alendronate alone. Adverse event (AE) rates, including positively adjudicated serious cardiovascular AEs (1.6% vs 1.4% at 12 months for romosozumab vs alendronate), were similar across treatment groups.ConclusionsConsistent with the global analysis, romosozumab followed by alendronate was associated with lower incidences of new vertebral, clinical, non-vertebral, and hip fractures vs alendronate alone among East Asian patients.

Sclerostin-Antibody Treatment Decreases Fracture Rates in Axial Skeleton and Improves the Skeletal Phenotype in Growing oim/oim Mice.

In osteogenesis imperfecta (OI), vertebrae brittleness causes thorax deformations and leads to cardiopulmonary failure. As sclerostin-neutralizing antibodies increase bone mass and strength in animal models of osteoporosis, their administration in two murine models of severe OI enhanced the strength of vertebrae in growing female Crtap-/- mice but not in growing male Col1a1Jrt/+ mice. However, these two studies ignored the impact of antibodies on spine growth, fracture rates, and compressive mechanical properties. Here, we conducted a randomized controlled trial in oim/oim mice, an established model of human severe OI type III due to a mutation in Col1a2. Five-week-old female WT and oim/oim mice received either PBS or sclerostin antibody (Scl-Ab) for 9weeks. Analyses included radiography, histomorphometry, pQCT, microcomputed tomography, and biomechanical testing. Though it did not modify vertebral axial growth, Scl-Ab treatment markedly reduced the fracture prevalence in the pelvis and caudal vertebrae, enhanced osteoblast activity (L4), increased cervico-sacral spine BMD, and improved the lumbosacral spine bone cross-sectional area. Scl-Ab did not impact vertebral height and body size but enhanced the cortical thickness and trabecular bone volume significantly in the two Scl-Ab groups. At lumbar vertebrae and tibial metaphysis, the absolute increase in cortical and trabecular bone mass was higher in Scl-Ab WT than in Scl-Ab oim/oim. The effects on trabecular bone mass were mainly due to changes in trabecular number at vertebrae and in trabecular thickness at metaphyses. Additionally, Scl-Ab did not restore a standard trabecular network, but improved bone compressive ultimate load with more robust effects at vertebrae than at metaphysis. Overall, Scl-Ab treatment may be beneficial for reducing vertebral fractures and spine deformities in patients with severe OI.

Sclerostin antibody treatment rescues the osteopenic bone phenotype of TGFβ inducible early gene-1 knockout female mice.

Deletion of TGFβ inducible early gene-1 (TIEG) in mice results in an osteopenic phenotype that exists only in female animals. Molecular analyses on female TIEG knockout (KO) mouse bones identified increased expression of sclerostin, an effect that was confirmed at the protein level in serum. Sclerostin antibody (Scl-Ab) therapy has been shown to elicit bone beneficial effects in multiple animal model systems and human clinical trials. For these reasons, we hypothesized that Scl-Ab therapy would reverse the low bone mass phenotype of female TIEG KO mice. In this study, wildtype (WT) and TIEG KO female mice were randomized to either vehicle control (Veh, n = 12/group) or Scl-Ab therapy (10 mg/kg, 1×/wk, s.c.; n = 12/group) and treated for 6 weeks. Following treatment, bone imaging analyses revealed that Scl-Ab therapy significantly increased cancellous and cortical bone in the femur of both WT and TIEG KO mice. Similar effects also occurred in the vertebra of both WT and TIEG KO animals. Additionally, histomorphometric analyses revealed that Scl-Ab therapy resulted in increased osteoblast perimeter/bone perimeter in both WT and TIEG KO animals, with a concomitant increase in P1NP, a serum marker of bone formation. In contrast, osteoclast perimeter/bone perimeter and CTX-1 serum levels were unaffected by Scl-Ab therapy, irrespective of mouse genotype. Overall, our findings demonstrate that Scl-Ab therapy elicits potent bone-forming effects in both WT and TIEG KO mice and effectively increases bone mass in female TIEG KO mice.

Effects of Long-Term Sclerostin Deficiency on Trabecular Bone Mass and Adaption to Limb Loading Differ in Male and Female Mice.

A new therapeutic option to treat osteoporosis is focused on Wnt signaling and its inhibitor sclerostin, a product of the Sost gene. In this work, we study the effect of sclerostin deficiency on trabecular bone formation and resorption in male and female mice and whether it affects mechano-responsiveness. Male and female 10- and 26-week-old Sost knockout (KO) and littermate controls (LCs) were subjected to in vivo mechanical loading of the left tibia for 2weeks. The right tibia served as internal control. The mice were imaged using in vivo micro-computed tomography at days 0, 5, 10, and 15 and tibiae were collected for histomorphometric analyses after euthanasia. Histomorphometry and micro-CT-based 3D time-lapse morphometry revealed an anabolic and anti-catabolic effect of Sost deficiency although increased trabecular bone resorption accompanied by diminished trabecular bone formation occurred with age. Loading led to diminished resorption in adult female but not in male mice. A net gain in bone volume could be achieved with mechanical loading in Sost KO adult female mice, which occurred through a further reduction in resorbed bone volume. Our data show that sclerostin deficiency has a particularly positive effect in adult female mice. Sclerostin antibodies are approved to treat postmenopausal women with high risk of osteoporotic fractures. Further studies are required to clarify whether both sexes benefit equally from sclerostin inhibition.

Romosozumab: anovel bone anabolic treatment option for osteoporosis?

SummaryResearch into the drug romosozumab began with the investigation of patients with excess bone formation. The understanding of the wingless-type mouse mammary tumor virus integration site (Wnt) signaling pathway in bone metabolism identified the negative regulator of bone mass sclerostin as a potential target for the treatment of osteoporosis. Preclinical studies confirmed this idea because they showed that sclerostin antibodies have the potential to increase bone formation. Biochemical analyses of clinical studies showed a significant increase in bone formation markers, which then slowly decreased within a year. This was accompanied by a particularly initially pronounced decrease in bone resorption. This dual mechanism of action led to an increase in bone mineral density and a significant reduction in fracture risk. Clinical vertebral fractures decreased by between 28 and 36%, nonvertebral fractures shown in a post hoc analysis by 42%. Romosozumab is administered once a month in the form of two injections. At the puncture site, reactions occur in about 5%. The most significant side effects are cardiovascular. In phase III studies, the number of serious cardiovascular complications was not significantly, albeit numerically, higher than in the control group. In Japan, South Korea, Canada, Australia, and the USA, osteoporosis patients at a high risk of fracture may already be treated with romosozumab (Evenity). Approval in the European Union was granted by 2019-12-12.

Sclerostin Antibody Treatment Increases Bone Mass and Normalizes Circulating Phosphate Levels in Growing Hyp Mice.

X-linked hypophosphatemia (XLH), caused by a loss-of-function mutation in the phosphate regulating gene with homology to endopeptidase located on the X chromosome (PHEX), is the most common form of vitamin D-resistant rickets. Loss of functional PHEX results in elevated fibroblast growth factor 23 (FGF23) levels, impaired phosphate reabsorption, and inhibited skeletal mineralization. Sclerostin, a protein produced primarily in osteocytes, suppresses bone formation by antagonizing Wnt signaling and is reported to be elevated in XLH patients. This study used the Hyp mouse model to investigate sclerostin's role in the pathophysiology of XLH by evaluating the use of a monoclonal antibody to sclerostin in a mouse model of XLH, the Hyp mouse. Male and female wild-type and Hyp littermates were injected with 25 mg/kg of vehicle or sclerostin antibody (Scl-Ab) twice weekly, beginning at 4 weeks of age and euthanized at 8 weeks of age. Scl-Ab treatment increased serum phosphate levels and suppressed circulating levels of intact FGF23 in treated wild-type and Hyp mice of both sexes. Cortical area, trabecular bone volume fraction (BV/TV), metaphyseal apparent density, and the peak load increased with Scl-Ab treatment in both sexes. This short-term treatment study suggests that Scl-Ab treatment can effectively improve some of the pathologies associated with XLH, including normalization of phosphate, and that sclerostin may play a role in regulating FGF23 and phosphate metabolism in XLH. © 2019 American Society for Bone and Mineral Research.

A xenograft model to evaluate the bone forming effects of sclerostin antibody in human bone derived from pediatric osteogenesis imperfecta patients.

Osteogenesis imperfecta (OI) is a rare and severe skeletal dysplasia marked by low bone mass and poor bone quality which is especially burdensome during childhood. Since clinical trials for pediatric OI are difficult, there is a widespread reliance on genetically modified murine models to understand the skeletal effects of emerging therapeutics. However a common model does not yet exist to understand how patient-specific genotype may influence treatment efficacy. Recently, sclerostin antibody (SclAb) has been introduced as a novel putative anabolic therapy for diseases of low bone mass, but effects in pediatric patients remain unexplored. In this study, we aim to establish a direct xenograft approach using OI patient-derived bone isolates which retain patient-specific genetic defects and cells residing in their intrinsic extracellular environment to evaluate the bone-forming effects of SclAb as a bridge to clinical trials. OI and age matched non-OI patient bone typically discarded as surgical waste during corrective orthopaedic procedures were collected, trimmed and implanted subcutaneously (s.c.) on the dorsal surface of 4-6-week athymic mice. A subset of implanted mice were evaluated at short (1 week), intermediate (4 week), and long-term (12 week) durations to assess bone cell survival and presence of donor bone cells in order to determine an appropriate treatment duration. Remaining implanted mice were randomly assigned to a two or four-week SclAb-treated (25mg/kg s.c. 2QW) or untreated control group. Immunohistochemistry determined osteocyte and osteoblast donor/host relationship, TRAP staining quantified osteoclast activity, and TUNEL assay was used to understand rates of bone cell apoptosis at each implantation timepoint. Longitudinal changes of in vivo μCT outcomes and dynamic histomorphometry were used to assess treatment response and ex vivo μCT and dynamic histomorphometry of host femora served as a positive internal control to confirm a bone forming response to SclAb. Human-derived osteocytes and lining cells were present up to 12 weeks post-implantation with nominal cell apoptosis in the implant. Sclerostin expression remained donor-derived throughout the study. Osterix expression was primarily donor-derived in treated implants and shifted in favor of the host when implants remained untreated. μCT measures of BMD, TMD, BV/TV and BV increased with treatment but response was variable and impacted by bone implant morphology (trabecular, cortical) which was corroborated by histomorphometry. There was no statistical difference between treated and untreated osteoclast number in the implants. Host femora confirmed a systemic bone forming effect of SclAb. Findings support use of the xenograft model using solid bone isolates to explore the effects of novel bone-targeted therapies. These findings will impact our understanding of SclAb therapy in pediatric OI tissue through establishing the efficacy of this treatment in human cells prior to extension to the clinic.

Twist1 Inactivation in Dmp1-Expressing Cells Increases Bone Mass but Does Not Affect the Anabolic Response to Sclerostin Neutralization.

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism—the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1—a gene know to regulate skeletal development—is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization.

Sclerostin Antibody-Induced Changes in Bone Mass Are Site Specific in Developing Crania.

Sclerostin antibody (Scl-Ab) is an anabolic bone agent that has been shown to increase bone mass in clinical trials of adult diseases of low bone mass, such as osteoporosis and osteogenesis imperfecta (OI). Its use to decrease bone fragility in pediatric OI has shown efficacy in several growing mouse models, suggesting translational potential to pediatric disorders of low bone mass. However, the effects of pharmacologic inhibition of sclerostin during periods of rapid growth and development have not yet been described with respect to the cranium, where lifelong deficiency of functioning sclerostin leads to patterns of excessive bone growth, cranial compression, and facial palsy. In the present study, we undertook dimensional and volumetric measurements in the skulls of growing Brtl/+ OI mice treated with Scl-Ab to examine whether therapy-induced phenotypic changes were similar to those observed clinically in patients with sclerosteosis or Van Buchem disorder. Mice treated between 3 and 14 weeks of age with high doses of Scl-Ab show significant calvarial thickening capable of rescuing OI-induced deficiencies in skull thickness. Other changes in cranial morphology, such as lengths and distances between anatomic landmarks, intracranial volume, and suture interdigitation, showed minimal effects of Scl-Ab when compared with growth-induced differences over the treatment duration. Treatment-induced narrowing of foramina was limited to sites of vascular but not neural passage, suggesting patterns of local regulation. Together, these findings reveal a site specificity of Scl-Ab action in the calvaria with no measurable cranial nerve impingement or brainstem compression. This differentiation from the observed outcomes of lifelong sclerostin deficiency complements reports of Scl-Ab treatment efficacy at other skeletal sites with the prospect of minimal cranial secondary complications. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research.

Bone Quality and Fracture-Healing in Type-1 and Type-2 Diabetes Mellitus.

➤ Patients with type-1 or type-2 diabetes have an increased risk of fracture, which is secondary to early bone loss in type-1 diabetes whereas, in type 2, the development of abnormal osseous architecture results in paradoxically increased or normal bone mineral density.➤ Diabetic patients diagnosed with osteoporosis should receive the same standard antiresorptive therapies as non-diabetic patients in conjunction with appropriate glycemic control.➤ Medical management of type-2 diabetes can affect bone health and fracture risk. Thiazolidinediones have been correlated with an increased facture risk, whereas metformin can be protective of the bone health of diabetic patients.➤ Diabetes increases the likelihood of nonunion and delayed union of fractures as well as other perioperative complications such as infection.➤ Perioperative glycemic control may decrease the risk of perioperative complications, especially surgical site infection. Methods to directly improve diabetic fracture-healing such as the use of parathyroid hormone and sclerostin antibody are in the experimental phase.➤ Prior to proceeding with operative intervention for fractures, it is necessary to consider the additional risk posed by diabetes, which must be taken into account when planning the approach, fixation, and postoperative management in this population.

Bone-Forming and Antiresorptive Effects of Romosozumab in Postmenopausal Women With Osteoporosis: Bone Histomorphometry and Microcomputed Tomography Analysis After 2 and 12 Months of Treatment.

ABSTRACTSclerostin, 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 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).