Inhibition Of Osteoclastic Bone Resorption Research Articles

Targeting Bone Marrow Microenvironment for Treatment of High-Risk B-Cell Acute Lymphoblastic Leukemia

Introduction B-cell acute lymphoblastic leukemia (B-ALL) is the most common form of cancer in children, accounting for approximately 20% of all pediatric cancers. Although treatment of B-ALL represents one of the success stories of modern medicine with 5-year survival rates approaching 90%, some children continue to have a dismal prognosis, particularly those with high-risk genetic alterations in their ALL cells or those who relapse. The leukemia microenvironment is an attractive novel therapeutic target, especially for children with high-risk leukemia as dose-limiting toxicities of conventional chemotherapeutic agents have prevented further improvements in survival. Aims In this study, we investigate whether therapeutically targeting the bone marrow (BM) osteoclasts can improve treatment outcome for B-ALL and the mechanisms of crosstalk between osteoclasts and leukemia cells. Methods We have established syngeneic and patient-derived xenograft (PDX) models for high-risk B- ALL that recapitulate the clinical symptoms of bone loss. For in vitro experiments, leukemia cells were cultured with osteoclast-derived conditioned media (CM) for 3 days. Results To evaluate the therapeutic potential of targeting osteoclasts during leukemia development, we administered zoledronic acid (2ug 5 days/week for 2 weeks), a bisphosphonate inhibitor of osteoclastic bone resorption, alone and in combination with imatinib (100mg/kg daily for 28 days) or dasatinib (10mg/kg daily for 28 days) in our BCR-ABL1 + B-ALL syngeneic mouse model. We showed that zoledronic acid treatment significantly reversed leukemia-induced bone loss by attenuating osteoclast activity. Zoledronic acid alone was found to enhance event-free survival in leukemia-bearing mice and further improvement was shown in combination with imatinib (p<0.05) or dasatinib (p<0.01). In addition, we administered zoledronic acid alone and in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in a relapsed B-ALL PDX model. We further demonstrated that zoledronic acid alone or in combination with VXL improved survival compared to mice treated with vehicle (p<0.001) or VXL alone (p<0.01), respectively. In the clinical setting, we assessed the feasibility and safety of incorporating zoledronic acid early into leukemia therapy in three children with B-ALL. The patients were undergoing induction or consolidation chemotherapy and were administered zoledronic acid at the same time. Zoledronic acid was well tolerated without any significant adverse effects, indicating the feasibility of safely administering zoledronic acid in combination with chemotherapy in children with ALL. To gain mechanistic insight into the effect of osteoclasts on leukemia cells, we compared the molecular profile of the leukemia cells cultured with CM and control media using RNA sequencing. There were 30 differentially expressed genes identified at the 5% false discovery rate with a log fold-change value ≥1 or ≤-1. KEGG pathway analysis revealed a significant difference in cell cycle pathway in leukemia cells cultured with CM compared to control. Biological process analysis showed a positive regulation of B-cell proliferation and negative regulation of lymphocyte apoptotic process in leukemia cells cultured with CM compared to control. To functionally examine the effect of osteoclasts on leukemia cells, we showed that osteoclast-derived CM significantly increased B-ALL cell proliferation, as demonstrated by increased cell number (p<0.001), as well as reduced cell percentages in G0/G1 phases and increased S+G2/M phases (p<0.05) via cell cycle analyses. Using annexin V and DAPI staining, we further demonstrated that there were more live cells and less apoptotic and dead cells when the leukemia cells were cultured with CM compared to control (p<0.001). Together, our findings suggest that soluble factors produced by osteoclasts promote leukemia cell proliferation and survival. Conclusion Our study reveals osteoclasts play a role in leukemogenesis and leukemia-induced bone loss, and that targeting osteoclasts can provide dual clinical benefit in terms of bone health and improved survival for children with high-risk B-ALL.

Systemic Convergent Multitarget Interactions of Plant Polyphenols Revealed by Affinity-Based Protein Profiling of Bone Cells Using C-Glucosidic Vescal(ag)in-Bearing Chemoproteomic Probes.

The ellagitannins vescalagin and vescalin, known as actin-dependent inhibitors of osteoclastic bone resorption, were mounted onto chemical probes to explore their interactions with bone cell proteins by means of affinity-based chemoproteomics and bioinformatics. The chemical reactivity of the pyrogallol units of these polyphenols toward oxidation into electrophilic ortho-quinones was exploited using NaIO4 to promote the covalent capture of target proteins, notably those expressed at lower abundance and those interacting with polyphenols at low-to-moderate levels of affinity. Different assays revealed the multitarget nature of both ellagitannins, with 100-370 statistically significant proteins captured by their corresponding probes. A much higher number of proteins were captured from osteoclasts than from osteoblasts. Bioinformatic analyses unveiled a preference for the capture of proteins having phosphorylated ligands and GTPase regulators and enabled the identification of 33 potential target proteins with systemic relevance to osteoclast differentiation and activity, as well as to the regulation of actin dynamics.

PSVI-8 Bisphosphonate Pharmacokinetics in Juvenile Horses

Abstract Bisphosphonates alter bone metabolism via inhibition of osteoclastic bone resorption. Although bisphosphonates are beneficial to treat bone disease, extra-label use in healthy juvenile horses may interfere with normal bone metabolism. The objective was to determine plasma pharmacokinetics of the bisphosphonate clodronate disodium (CD) following a single intramuscular dose to better define the time course of CD in juvenile horses. Ten yearling Quarter Horses (334 ± 18 kg, 504 ± 13 d of age) were used to test the hypothesis that juvenile horses would have faster clearance rate of CD compared with previously published data in adult horses. Horses were placed in individual stalls, jugular intravenous catheters were inserted, and an initial blood sample (0 h) was collected. Horses were administered CD intramuscularly at 1.8 mg/kg BW per label. Plasma samples were collected at 0.5, 1, 3, 6, 12, 24, 48, and 72 h post-administration. Plasma concentration of clodronate was determined by liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were calculated using a two-compartment model. Blood chemistry parameters [blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALKP), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), and creatine kinase (CK)] were determined via a commercial laboratory. Included blood chemistry parameters were analyzed for an effect of time using PROC Mixed of SAS. Clodronate was detected in plasma up to 72 h post-administration. Maximum plasma concentration (43,971 ± 25,920 ng/mL) was reached at 0.5 h post-administration. Analysis revealed bicompartmental kinetics with distribution and terminal half-lives of 1.9 ± 0.4 h and 24.4 ± 4.3 h, respectively. Area under the curve to last sample was 22,755 ± 11,426 ng*h/mL and extrapolated to infinity was 26,778 ± 12,465 ng*h/mL. Blood chemistry parameters varied over time within normal ranges. Markers of renal function, BUN and creatinine, decreased from h 0 to h 72 (P < 0.01). Liver function markers ALKP and AST were not affected by CD treatment (P > 0.27). However, GGT and CK changed over time (P < 0.01), with GGT greater at 48 h than other time points, and CK increasing from 0.5 to 24 h before decreasing by 72 h but remaining above baseline. Plasma clodronate concentrations were greater in juvenile horses than previously reported in mature horses (Krueger et al., 2020, Equine Vet J.; Knych et al., 2022, Equine Vet J.). Further, the observed bicompartmental kinetics differed from the linear kinetics previously reported in mature horses whereby juvenile horses from the current study had greater exposure to CD over time as indicated by a longer terminal half-life. Moreover, the single intramuscular dose of CD (1.8 mg/kg BW) did not cause markers of kidney or liver function to deviate from normal ranges in juvenile Quarter Horses.

Micheliolide prevents estrogen deficiency-induced bone loss via inhibiting osteoclast bone resorption.

Osteoporosis is one of the major health problems characterized by decreased bone density and increased risk of fractures. Nowadays, the treating strategies against osteoporosis are efficient, but still have some drawbacks. Micheliolide, a guaianolide sesquiterpene lactone isolated from Michelia compressa and Michelia champac, has been reported to have anti-inflammatory effects. Here, our data suggest that Micheliolide could protect mice from ovariectomy induced bone loss. According to the Micro-CT scan and histomorphometry quantification data, Micheliolide treatment inhibits excessive osteoclast bone resorption without affecting bone formation in estrogen deficiency mice. Consistently, our data suggest that Micheliolide could inhibit osteoclastogenesis in vitro. Additionally, we confirmed that Micheliolide inhibits osteoclasts formation via inhibiting P38 MAPK signaling pathway, and P79350 (a P38 agonist) could rescue this effect. In summary, our data suggest that Micheliolide could ameliorate estrogen deficiency-induced bone loss via attenuating osteoclastogenesis. Hence, Micheliolide could be used as a novel anti-resorptive agent against osteoporosis.

Semaphorin 3A delivered by a rapidly polymerizing click hydrogel overcomes impaired implant osseointegration in a rat type 2 diabetes model.

Semaphorin 3A (sema3A) is an osteoprotective factor that enhances bone formation while inhibiting osteoclast bone resorption. It is produced by rat calvarial osteoblasts cultured on grit-blasted/acid-etched microtextured (SLA) titanium surfaces at higher levels than on tissue culture polystyrene, suggesting that it may improve performance of titanium implants in vivo, particularly in conditions characterized by compromised bone quality. To test this, we established a clinically relevant type 2 diabetes mellitus (T2DM) rat model and used a non-toxic click hydrogel that rapidly polymerizes in situ (GEL) to provide localized controlled delivery of sema3A. In vitro studies confirmed that sema3A released from GEL was biologically active, increasing osteoblast differentiation of a pre-osteoblast cell-line. Whereas increased sema3A production was not observed in T2DM calvarial osteoblasts cultured on SLA, exogenous sema3A enhanced surface-induced osteoblast differentiation, indicating that it would be a viable candidate for in vivo use. Delivery of sema3A either by GEL or by local injection to bone defects enhanced osseointegration of SLA implants in the T2DM rats. Trabecular bone mass and bone-to-implant contact were decreased in T2DM rats compared to normal rats; sema3A delivered locally improved both parameters. These findings suggest that reduced trabecular bone contributes to poor osseointegration in T2DM patients and support GEL as a promising treatment option for sustained release of therapeutic doses of sema3A. Moreover, using this clinically translatable T2DM model and developing a biocompatible, Cu-free click chemistry hydrogel platform for the non-invasive delivery of therapeutics has major implications for regenerative medicine as a whole. STATEMENT OF SIGNIFICANCE: Osseointegration is compromised in patients with poor bone quality due to conditions like type 2 diabetes mellitus (T2DM). Previously, we showed that semaphorin 3A (sema3A) production is increased when human bone marrow stromal cells are cultured on titanium substrates that support osseointegration in vivo, suggesting it may enhance peri-implant osteogenesis in diabetes. Here we established a spontaneously developing T2DM rat model with clinical translatability and used it to assess sema3A effectiveness. Sema3A was delivered to the implant site via a novel copper-free click hydrogel, which has minimal swelling behavior and superior rheological properties. Osseointegration was successfully restored, and enhanced compared to burst release through injections. This study provides scientific evidence for using sema3A to treat impaired osseointegration in T2DM patients.

Optimization of an Injectable, Resorbable, Bioactive Cement Able to Release the Anti-Osteoclastogenic Biomolecule ICOS-Fc for the Treatment of Osteoporotic Vertebral Compression Fractures.

Vertebral compression fractures are typical of osteoporosis and their treatment can require the injection of a cement through a minimally invasive procedure to restore vertebral body height. This study reports the development of an injectable calcium sulphate-based composite cement able to stimulate bone regeneration while inhibiting osteoclast bone resorption. To this aim, different types of strontium-containing mesoporous glass particles (Sr-MBG) were added to calcium sulphate powder to impart a pro-osteogenic effect, and the influence of their size and textural features on the cement properties was investigated. Anti-osteoclastogenic properties were conferred by incorporating into poly(lactic-co-glycolic)acid (PLGA) nanoparticles, a recombinant protein able to inhibit osteoclast activity (i.e., ICOS-Fc). Radiopaque zirconia nanoparticles (ZrO2) were also added to the formulation to visualize the cement injection under fluoroscopy. The measured cement setting times were suitable for the clinical practice, and static mechanical testing determined a compressive strength of ca. 8 MPa, comparable to that of human vertebral bodies. In vitro release experiments indicated a sustained release of ICOS-Fc and Sr2+ ions up to 28 days. Overall, the developed cement is promising for the treatment of vertebral compression fractures and has the potential to stimulate bone regeneration while releasing a biomolecule able to limit bone resorption.

Caffeic Acid Inhibits Swelling, Bone Loss, and Osteoclastogenesis in Adjuvant-induced Arthritis Rats

BACKGROUND: Increase in inflammatory cytokine levels promotes pathological osteoclast differentiation. Caffeic acid has anti-inflammatory properties and can inhibit osteoclast bone resorption. In vitro studies have reported the ability of caffeic acid in inhibiting osteoclastogenesis pathways, however the in vivo study is rarely conducted. The aim of this study is to examine the role of caffeic acid in reducing inflammation and inhibiting osteoclastogenesis in Adjuvant-Induced Arthritis (AIA) rats.METHODS: Rats were injected with Freund’s Complete Adjuvant (CFA) and mineral oil. One day after injection, various concentration (0, 5, 25, 125 mg) of caffeic acid were given gastro-intestinally. Swelling degree in rats’ ankle joints was determined by measuring height and width of each ankle joint. Bone loss level was examined with soft X-ray, and then bone density was calculated. To examine osteoclastogenesis, ankle joints were stained with Tartrate-Resistant Acid Phosphatase (TRAP) and evaluated microscopically. RESULTS: Ankle joints of AIA rats had severe swelling before treated, yet the swelling was reduced based on concentration-dependent after receiving caffeic acid. Severe bone loss in AIA rats’ ankle joints were also observed, however the treatment of 125 mg caffeic acid showed remarkable inhibition effect toward rats’ bone loss. Osteoclastogenesis in AIA rats’ ankle joints were higher than the normal ones, as indicated with high TRAP-positive Multinucleated Cells (MNCs). But low number of TRAP-positive MNCs was observed in ankle joint of AIA rats that received 125 mg caffeic acid.CONCLUSION: Administration of caffeic acid can reduce the degree of swallowing, inhibit bone loss, and inhibit osteoclastogenesis in ankle joint of arthritis-induced rats.KEYWORDS: caffeic acid, osteoclastogenesis, bone loss, swelling, inflammation, RANKL, TNF-α

Salidroside promotes osteoblast proliferation and differentiation via the activation of AMPK to inhibit bone resorption of knee osteoarthritis mice

AimBalance between osteoclasts and osteoblasts was important for bone development and regeneration, which attracted wide attention. To investigate whether the pro-restorative effect of salidroside (SAL) on knee osteoarthritis in mice was associated with adenosine monophosphate-activated protein kinase (AMPK). MethodsMC3T3-E1 cells were used to perform CCK8, ALP measurement, alizarin red assay and western blot. Mouse model with knee osteoarthritis was constructed and received the treatment of SAL. Body weight, arthritis index, and inflammatory factors were recorded and measured. The paraffin sections of knee bone joints were performed by HE and immunohistochemical staining. Western blot was carried out. CCK8, EDU and flow cytometry were used to analyzed the inhibitory effect of salidroside on osteoclast. ResultsWe found that salidroside could promote osteoblast proliferation and differentiation, and upregulate COL1A1, RUNX2 and OCN proteins and increase ALP content and phosphorylation level of AMPK. In vivo assays showed that salidroside inhibited inflammatory reaction, improved pathological condition. Salidroside reduced TRAP and NFATc1 expression, and increased the expression of ALP, COL1A1, RUNX2 and OCN proteins. p-AMPK protein was upregulated by salidroside treatment. We also performed in vitro assay, and found salidroside could inhibit proliferation of osteoclast and increase apoptosis of osteoclast. ConclusionIn a word, salidroside promoted osteoblast proliferation and differentiation through AMPK activation to further inhibit osteoclast bone resorption, so as to achieve the purpose of relieving knee osteoarthritis.

P9. Sex differences in response to recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rat posterolateral fusion spine model

<h3>BACKGROUND CONTEXT</h3> Estrogen modulates bone activity largely through inhibition of osteoclast bone resorption and bone morphogenetic protein-2 (BMP-2) signaling pathways in osteoblasts. Minimal data exist concerning potential sex-dependent differences in BMP-2-mediated bone regeneration. Here, we aimed to quantify sex-dependent differences in the bone healing response to recombinant human BMP-2 (rhBMP-2) treatment in a rat posterolateral spinal fusion model. <h3>METHODS</h3> A total of 48 female and male Sprague-Dawley rats (N=24/group), underwent L4-L5 posterolateral fusion with bilateral placement of absorbable collagen sponges, loaded with 5 µg of rhBMP-2 (10 µg/animal). At 8 weeks postoperatively, 10 specimens of each sex were tested in flexion-extension with quantification of range of motion (ROM) and stiffness. The remaining specimens were evaluated for new bone growth and fusion via radiography, blinded manual palpation and microcomputed tomography (microCT). Laboratory microCT quantified bone microarchitecture and synchrotron microCT examined bone microstructure. On histology, the number of adipocytes within a standardized field of view was counted for both male and female fusion masses. <h3>RESULTS</h3> Manual palpation scores differed significantly between sexes, with mean fusion scores of 2.4±0.4 in females vs 3.1±0.6 in males, p < 0.001. Biomechanical stiffness did not differ between sexes, but ROM was significantly greater and more variable for females vs males (3.7°±5.6° vs 0.27°±0.15°,p < 0.005, respectively. Laboratory microCT showed significantly smaller volumes of fusion masses in females vs males (262±87 mm³ vs 732±238 mm³, respectively, p < 0.001) but significantly higher bone volume fraction (0.27±0.08 vs 0.12±0.05, respectively, p < 0.001). Mean trabecular thickness was not different, but trabecular number was significantly greater in females (3.1±0.5 mm-1vs 1.5±0.4 mm-1, respectively, p<0.001). Synchrotron microCT showed fine bone structures developing in both sexes at the 8-week time point. Adipocyte density was found to be significantly higher in male fusion masses than in female fusion masses. <h3>CONCLUSIONS</h3> This study demonstrates that sex-based factors may influence both the quantity and quality of bone formation in patients receiving rhBMP-2 for a variety of orthopedic applications. We found that male rats had significantly higher fusion scores, greater fusion-mass volume, and lower ratio of new bone volume to total volume compared to female rats. Male and female mean trabecular thicknesses were equal, but the female fusion masses were more densely filled with trabeculae (higher trabecular number) than those of males. MicroCT results suggested that the male trabeculae were more rod-like and the female more plate-like, although the methods that were used are generally considered less accurate for trabecular bone, which has a high degree of concavity. The incomplete fusions on three female rats, evidenced by high ROM and small gaps on microCT, suggest that females may have a more variable response to rhBMP-2. Investigation into whether these sex-based differences are specific to rhBMP-2-induced bone formation or are more general to bone regeneration/healing is prudent. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Blockade of Osteoclast-Mediated Bone Resorption With a RANKL-Inhibitor Enhances Bone Formation in a Rat Spinal Fusion Model.

Rat spine fusion model. The present study aimed to determine whether administration of osteoprotegerin (OPG) in a rat model of spinal fusion increases bone volume, bone density, and decreases osteoclasts in the fusion mass. OPG is a soluble RANK-ligand inhibitor that blocks osteoclast differentiation and activation. This makes it a potential agent to control the remodeling process and enhance bone mass during spinal fusion. Forty-eight male Sprague-Dawley rats received a one-level spinal fusion of L4-L5 with bone allograft. Rats were then divided into four groups according to initiation of treatment: (1) saline on day 0 (saline), (2) OPG on day 0 (OPG D0), (3) OPG on day 10 (OPG D10), and (4) OPG on day 21 (OPG D21) postsurgery. After their initial injection, rats received weekly subcutaneous injections of OPG (10mg/kg) and were euthanized six weeks postsurgery. MicroCT analysis of the fusion site and histological analysis of bone surface for quantification of osteoclast lining was performed. Increased bone volume in the fusion site and around the spinous process was seen in OPG D0 and OPG D10 when compared with saline. Mean trabecular thickness was greater in all groups receiving OPG compared with saline, with OPG D0 and OPG D10 having significantly greater mean trabecular thickness than OPG D21. All OPG groups had less bone surface lined with osteoclasts when compared with Saline, with OPG D0 and OPG D10 having fewer than OPG D21. This study indicates that OPG inhibited osteoclast bone resorption, which led to greater bone at the fusion site. Future studies investigating OPG on its own or in combination with an osteogenic factor to improve spinal fusion outcomes are warranted to further elucidate its potential therapeutic effect.

Onc201 reduces osteoclastogenesis and prevents ovariectomy-induced bone loss via inhibiting RANKL-induced NFATc1 activation and the integrin signaling pathway

Osteoporosis is an osteolytic disease with a disrupted balance between the resorption and formation of bone as well as bone microstructure degeneration, leading to bone loss and increased fracture risk, which greatly affects patients’ quality of life. Currently, inhibition of osteoclast bone resorption remains the mainstream treatment for osteoporosis. Onc201, a new compound, induces the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and has an efficient anticancer effect in clinical trials. However, its effects on osteolytic disease and the mechanism of action are unclear. We examined the effect of Onc201 on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts via Cell Counting Kit-8, bone resorption assay, luciferase reporter assay, immunofluorescence staining, calcium ion intensity assay and employed an ovariectomy model to investigate the effect of Onc201 on osteoporosis in the mice. Results showed that Onc201 inhibited the function and formation of osteoclasts induced by RANKL in a manner that was dependent on time and concentration, and did not cause cytotoxicity. Mechanistically, Onc201 inhibited osteoclast-relevant genes and NFATc1 expression, the main transcriptional regulatory factor of the formation of osteoclasts induced by RANKL; meanwhile, downregulating the expressions of the osteoclast cytoskeleton key signal molecules integrin αvβ3, focal adhesion kinase (FAK), c-Src, and spleen-associated tyrosine kinase (SYK). In addition, Onc201 had a protective effect on the mouse model of bone loss caused by ovariectomy-induced estrogen deficiency, which is consistent with the in vitro results. Our findings suggest that the new small-molecular compound Onc201 has the potential to prevent osteoclast-related osteolytic diseases.

Engineered osteoclasts resorb necrotic alveolar bone in anti-RANKL antibody-treated mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of antiresorptive medications such as denosumab (humanized anti-RANKL antibody), yet its pathophysiology remains elusive. It has been posited that inhibition of osteoclastic bone resorption leads to the pathological sequelae of dead bone accumulation, impaired new bone formation, and poor wound healing in MRONJ, but this hypothesis has not been definitively tested. We previously engineered myeloid precursors with a conditional receptor activator of nuclear factor kappa-Β intracellular domain (iRANK cells), which differentiate into osteoclasts in response to a chemical inducer of dimerization (CID) independently of RANKL. In this study, we showed that CID-treated iRANK cells differentiated into osteoclasts and robustly resorbed mineralized surfaces even in the presence of anti-RANKL antibody in vitro. We then developed a tooth extraction-triggered MRONJ model in nude mice using anti-RANKL antibody to deplete osteoclasts. This model was used to determine whether reconstitution of engineered osteoclasts within sockets could prevent specific pathological features of MRONJ. Locally delivered iRANK cells successfully differentiated into multinucleated osteoclasts in response to CID treatment in vivo as measured by green fluorescent protein (GFP), tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, matrix metallopeptidase 9 (MMP-9), and cathepsin K staining. Sockets treated with iRANK cells + CID had significantly more osteoclasts and less necrotic bone than those receiving iRANK cells alone. These data support the hypothesis that osteoclast deficiency leads to accumulation of necrotic bone in MRONJ.

Development of a Pure Small Molecule αVβ3 Antagonist

# Contributed equallyαVβ3 has been implicated in the pathophysiology of important disorders, including osteoporosis, sickle cell disease, tumor angiogenesis, metastasis, virus invasion, glomerulonephritis, dermal and hepatic fibrosis, acute myelogenous leukemia, supravalvular aortic stenosis, bowel strictures, and T-cell lymphoma, but no αVβ3 antagonists have been approved for human therapy. The RGD-based αVβ3 antagonist MK-0429 (MK429) had favorable results in osteoporosis patients in Phase 2, but was not advanced, and the RGD-based antagonist cilengitide was studied in patients with malignancies, but did not demonstrate efficacy. Since RGD-based antagonists can under certain circumstances activate αVβ3, prime it to bind ligand, induce apoptosis, increase tumor angiogenesis and growth, and increase hepatic fibrosis, we sought to develop a pure αVβ3 antagonist that does not induce high affinity ligand binding or exposure of the epitope for mAb AP5, both of which depend on the swing-out of the β3 PSI domain. We built on Arnaout's group's studies of a variant fibronectin fragment with high affinity for αVβ3 (hFN10) that did not induce the β3 swing-out motion because a Trp substitution on the ligand formed a π-π interaction with β3 Y122 on the β1-α1 loop, thus preventing the latter's movement toward the MIDAS, which is the trigger for β3 swing-out. To develop a small-molecule that mimics the effect of hFN10, we used inferences from a predicted docking pose of MK429 into the hFN10 binding site of the αVβ3 crystal structure to rationally design a compound with an aromatic ring that directly engages β3 Y122. Based on the results of molecular dynamics simulations, the aromatic group of this compound, TDI-4161, is only 4.4 Å from Y122 (and forms a π-π interaction), which compares with 4.7 Å for hFN10 and 7.0 Å for MK429 (which does not form a π-π interaction). We analyzed compounds in 2 assays: 1. Adhesion of HEK293 cells expressing human αVβ3 (HEK-αVβ3) to fibrinogen (reported as the concentration producing 50% inhibition; IC50). 2. Exposure of the epitope for mAb AP5 (the concentration producing 50% exposure; EC50). The racemate of MK429 (rMK429) has an IC50=0.0036 µM and an EC50=0.019 µM, and cilengitide has an IC50=0.050 µM and an EC50=0.034 µM, indicating that both are potent αVβ3 antagonists and potent inducers of the αVβ3 active conformation. In sharp contrast, TDI-4161 has an IC50=0.022 µM and an EC50>10 µM, indicating that it does not induce the β3 conformational change even at 400X the IC50. TDI-4161 inhibited the binding of: purified αVβ3 to adenovirus 2 penton base (IC50 = 0.028 µM) and purified αVβ5 to vitronectin (IC50 =2.09 µM), but not purified αVβ6 to TGF-β1 Latency Associated Peptide even at 10 µM. TDI-4161inhibited adhesion of mouse endothelial cells containing murine αVβ3 to fibrinogen (IC50 = 0.437 µM). We tested TDI-4161's ability to 'prime“ αVβ3 by: incubating it with HEK-αVβ3 at 10 µM, fixing the cells with paraformaldehyde, washing, incubating with fluorescent fibrinogen, and measuring bound fibrinogen by flow cytometry. The peptide RGDS (100 µM) increased fibrinogen binding from 8.7 ± 1.5 to 24.3 ± 1.4 GMFI (p <0.001; n=3), but TDI-4161 did not (7.0 ± 2.6 GMFI; p<0.001 vs RGDS). After oral administration of TDI-4161 to mice at 100 mg/kg, the Cmax was 7.5 µM at the Tmax of 0.94 hours; the T1/2 was 3.6 hours. Once-daily oral dosing of TDI-4161 for 28 days at 10, 30, and 100 mg/kg was well tolerated in male CD-1 mice (n=3). Day 1 and day 28 plasma levels 1 hour after dosing were similar. We tested the effect of TDI-4161 on osteoclast function by adding it at 10 µM (0.1% DMSO) to bone marrow-derived murine macrophages and cultured them either on plastic or bovine bone slices with RANKL and a source of M-CSF. After 5 days, cells incubated with TDI-4161 demonstrated an osteoclast phenotype similar to that of β3 null mice; osteoclast numbers were better preserved than with rMK429, but less well than with the DMSO control. Supernatants from bone slices were assayed for cross-linked collagen degradation products (CrossLaps). When compared to the DMSO control (32.7 nM), rMK429 (10 µM) produced 82% inhibition (5.9 nM) and TDI-4161 produced 74% inhibition (8.3 nM). In summary, using inferences from structural data derived from hFN10, we have synthesized an orally bioavailable pure αVβ3 antagonist that reacts with both human and murine αVβ3, does not prime αVβ3 to bind fibrinogen, and inhibits osteoclast bone resorption. DisclosuresFilizola:Icahn School of Medicine at Mount Sinai: Patents & Royalties. Foley:Tri-Institutional Therapeutics Discovery Institute: Patents & Royalties.

Zoledronic Acid Is Not Equally Potent on Osteoclasts Generated From Different Individuals.

ABSTRACTZoledronic acid is a bisphosphonate commonly used to treat bone diseases such as osteoporosis and cancer‐induced bone disease. Patients exhibit a variable sensitivity to zoledronic acid; the underlying explanation for this remains unclear. The objective of this study was to obtain more knowledge in this regard. We hypothesized that osteoclasts generated from different individuals would show a variable sensitivity to zoledronic acid in vitro. Osteoclasts were generated using monocytes from 46 healthy female blood donors (40 to 66 years). Matured osteoclasts were reseeded onto bone slices precoated with different concentrations of zoledronic acid. IC50 values were determined based on total eroded bone surface after 3 days of resorption. The IC50 for inhibition of osteoclastic bone resorption varied from 0.06 to 12.57μM zoledronic acid; thus, a more than 200‐fold difference in sensitivity to zoledronic acid among osteoclasts from different individuals was observed. Multiple linear regression analyses showed that the determined IC50 correlated with smoking status, and the average number of nuclei per osteoclast in vitro. Further analyses showed that: (i) increasing protein levels of mature cathepsin K in osteoclast cultures rendered the osteoclasts less sensitive to zoledronic acid; (ii) surprisingly, neither the gene nor the protein expression of farnesyl diphosphate synthase was found to correlate with the IC50; and (iii) trench‐forming osteoclasts were found to be more sensitive to zoledronic acid than pit‐forming osteoclasts within the same cell culture. Thus, we conclude that there indeed is a high degree of variation in the potency of zoledronic acid on osteoclasts when generated from different individuals. We propose that our findings can explain some of the varying clinical efficacy of zoledronic acid therapy observed in patients, and may therefore be of clinical importance, which should be investigated in a clinical trial combining in vitro and in vivo investigations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Antiosteoclastic bone resorption activity of osteoprotegerin via enhanced AKT/mTOR/ULK1‐mediated autophagic pathway

Autophagy plays a critical role in the maintenance of bone homeostasis. Osteoprotegerin (OPG) is an inhibitor of osteoclast-mediated bone resorption. However, whether autophagy is involved in the antiosteoclastogenic effects of OPG remains unclear. The present study aimed to investigate the potential mechanism of autophagy during OPG-induced bone resorption via inhibition of osteoclasts differentiated from bone marrow-derived macrophages in BALB/c mice. The results showed that after treatment with receptor activator of nuclear factor-κΒ ligand and macrophage colony-stimulating factor for 3 days, TRAP+ osteoclasts formed, representing the resting state of autophagy. These osteoclasts were treated with OPG and underwent autophagy, as demonstrated by LC3-II accumulation, acidic vesicular organelle formation, and the presence of autophagosomes. The levels of autophagy-related proteins, LC3-II increased and P62 decreased at 3 hr in OPG-treated osteoclasts. The viability, differentiation, and bone resorption activity of osteoclasts declined after OPG treatment. Treatment with OPG and chloroquine, an autophagy inhibitor, attenuated OPG-induced inhibition of osteoclastic bone resorption, whereas rapamycin (RAP), an autophagy inducer, enhanced OPG-induced inhibition of differentiation, survival, and bone resorption activity of osteoclasts. Furthermore, OPG reduced the amount of phosphorylated(p) protein kinase B (AKT) and pmTOR and increased the level of pULK, in a dose-dependant manner. LY294002, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT pathway inhibitor, attenuated the decline in pAKT, but enhanced the decline in pmTOR and the increase in pULK1 following OPG treatment. RAP enhanced the OPG-induced increase in pULK1. The PI3K inhibitor 3-methyladenine partly blocked OPG-induced autophagy. Thus, the results revealed that OPG inhibits osteoclast bone resorption by inducing autophagy via the AKT/mTOR/ULK1 signaling pathway.

Proanthocyanidins inhibit osteoclast formation and function by inhibiting the NF-κB and JNK signaling pathways during osteoporosis treatment

Osteoporosis is a metabolic bone lesion in which the bone mass is reduced per unit volume due to increased bone resorption. Its main characteristics are bone pain and increasing danger of fragility fracture. Excessive osteoclast activation is known to be responsible for extensive bone resorption. Thus, inhibition of osteoclastic bone resorption and regulation of the bone microenvironment are vital treatment strategies for osteoporosis. For the first time, we investigated the effect of proanthocyanidins (PACs) extracted from grape seed, which significantly inhibited osteoclast formation and differentiation from bone marrow macrophages (BMMs) and the RAW264.7 cell line and efficiently attenuated osteoclastic bone resorption without toxicity. These findings were confirmed by changes in the NF-κB and JNK/mitogen-activated protein kinase (MAPK) signaling pathways, which are major and classical signaling pathways involved in RANKL-mediated osteoclastogenesis in vitro. The PACs inhibited osteoclast formation and differentiation by inhibiting the NF-κB and JNK signaling pathways and might be useful for the treatment of osteoporosis.

Artemisinin inhibits breast cancer-induced osteolysis by inhibiting osteoclast formation and breast cancer cell proliferation.

In addition to being used to treat malaria, artemisinin (Art) can be used as an anti-inflammatory and antitumor agent. In this study, we evaluated the effects of Art on osteoclast formation and activation and on the development of breast cancer cells in bone. To evaluate the effect of Art on osteoclast differentiation in vitro, we treated bone marrow-derived macrophages (BMMs) with various concentrations of Art and evaluated the expression of genes and proteins involved in osteoclast formation. We also performed cell counting kit-8 assays to evaluate the toxicity of Art in BMMs and MDA-MB-231 cells. We also performed Transwell assays, wound-healing assays, colony formation assays, and cell apoptosis assays to evaluate the effect of Art in MDA-MB-231 cells. We also evaluated the effect of Art in an in vivo osteoclast bone resorption assay using a nude mouse model. We demonstrated that Art inhibits the differentiation and establishment of osteoclasts even though Art is not toxic to osteoclasts. In addition, Art reduced expression of genes involved in osteoclast formation and inhibited osteoclast bone resorption in a concentration-dependent manner. Based on our data, we believe that Art can inhibit proliferation of breast cancer cells by activating apoptosis pathways, and inhibit osteoclast formation and differentiation by inhibiting activation of cathepsin K, ATPase H+ transporting V0 subunit D2, nuclear factor of activated T cells 1, calcitonin receptor, and tartrate-resistant acid phosphatase and by inhibiting nuclear factor-κB activation.

Cellular and Molecular Mediators of Bone Metastatic Lesions.

Bone is the preferential site of metastasis for breast and prostate tumor. Cancer cells establish a tight relationship with the host tissue, secreting factors that stimulate or inhibit bone cells, receiving signals generated from the bone remodeling activity, and displaying some features of bone cells. This interplay between tumor and bone cells alters the physiological bone remodeling, leading to the generation of a vicious cycle that promotes bone metastasis growth. To prevent the skeletal-related events (SRE) associated with bone metastasis, approaches to inhibit osteoclast bone resorption are reported. The bisphosphonates and Denosumab are currently used in the treatment of patients affected by bone lesions. They act to prevent or counteract the SRE, including pathologic fractures, spinal cord compression, and pain associated with bone metastasis. However, their primary effects on tumor cells still remain controversial. In this review, a description of the mechanisms leading to the onset of bone metastasis and clinical approaches to treat them are described.

Dental management of patients treated with intravenous bisphosphonates. A case report

Bisphosphonates are drugs used for the inhibition of osteoclastic bone resorption. Their therapeutic use is indicated for Paget’s disease, hypercalcemia of cancer, bone metastases, and osteoporosis. The main side effect of these drugs is osteonecrosis of the jaw, which is difficult to control. The aim of the present study was to report a patient, who was referred to the Special Needs Clinics of the Ribeirão Preto Dental School, University of São Paulo, for dental treatment. His medical history revealed a diagnosis of multiple myeloma with bone marrow transplantation in November 2007. Since then, he has been using zometa every 3 months. After physical examination and complementary exams, a diagnosis of angular cheilitis, erythematous candidiasis, caries, localized chronic periodontitis, pulp necrosis and partial edentulousness was established. The treatment plan was based on control of angular cheilitis candidiasis, basic periodontal therapy, supportive periodontal therapy, endodontic treatment, and partial mandibular and maxillary dentures. All invasive procedures were performed with antibiotic prophylaxis. The patient is under treatment and care is being taken to avoid osteonecrosis of the jaws. The authors highlight the need for knowledge of the side effects of bisphosphonates to prevent such effects and to maintain the oral health of the patients.

The biochemical and histological analysis of subcutaneous calcitonin and intramedullary methylprednisolone on bone repair after bone marrow ablation: an experimental comparative study in rats

BackgroundAlthough, glucocorticoid (GC) and calcitonin-induced changes in bone repair have been studied previously, the exact effects of these on fracture healing remain controversial. Hence, the purpose of this experimental study is to determine biochemical and histological effects of locally administrated GC and systemically administrated calcitonin on the kinetics of healing response after bone marrow ablation in rats.MethodsAfter having undergone marrow ablation, a steroid-treated group of rats (n = 24) received a single dose of intramedullary methylprednisolone (2 mg/kg), a calcitonin-treated group (n = 24) received intermittently administrated subcutaneous salmon calcitonin (16 IU/kg), and a control group (n = 24) received intramedullary saline (25 μl).ResultsBlood samples taken on days 1, 3, 7, 9, and 15 after ablation showed an increase in serum calcium, alkaline phosphatase (ALP), and phosphate levels in the Calcitonin and Control groups. Levels of calcium and ALP peaked on day 7 after ablation. However, an increase in phosphate levels indicated a biphasic reaction that peaked on the third and ninth day after ablation. Hypercalcemia was not observed in Steroid group because of the inhibition of osteoclastic bone resorption. In that group, the serum levels of ALP and phosphate were lower than baseline levels. The levels of urinary calcium excretion peaked 3 to 7 days after marrow ablation in the control group and 7 to 9 days after that procedure in the steroid group. Histologic evaluation showed that the rats in the control group demonstrated the expected healing period according to the histological grades and that a delay in healing occurred in the calcitonin group after day 9 because of the inhibition of osteoclastic bone resorption. All rats in the steroid group exhibited a decrease and delayed healing response.ConclusionTotal serum calcium, phosphate, and ALP levels increased after bilateral tibial bone marrow ablation and urine calcium and hydroxyproline excretion also increased as a factor of bone resorption. Subcutaneously administrated salmon calcitonin did not affect biochemical changes after marrow ablation. Single-dose intramedullary methylprednisolone inhibited extra-tibial bone resorption induced by cytokines after bone marrow ablation.