New medicinal application of edaravone

Abstract

The invention belongs to the technical field of medicine research and development, and provides application of edaravone in preparation of a medicine for treating postmenopausal osteoporosis aiming at the technical problem that whether edaravone has a treatment effect on postmenopausal osteoporosis or not is not reported at present. In addition, by constructing a mouse ovariectomized model and combining with a ferroptosis-induced osteogenesis / osteoclast system, the edaravone is proved to be capable of effectively increasing calcium salt deposition and mineralization after osteoporosis, improving poor osteogenesis of osteoblasts and osteoblast inflammatory response caused by osteoporosis, effectively improving FSP1 expression and improving osteoporosis. And meanwhile, the expression of 5-LOX and ACSL4 proteins is reduced, and the intraosseous ferroptosis microenvironment of the postmenopausal osteoporosis is regulated through ways of inhibiting ROS, inhibiting lipid peroxidation and the like, so that the treatment effect of relieving the progress of the postmenopausal osteoporosis is achieved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical research and development technology, specifically relating to new pharmaceutical uses of edaravone. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by low bone mass and deterioration of bone microstructure, leading to increased fracture fragility and a significantly increased susceptibility to fractures throughout the body. It is a growing public health problem. The main mechanism of osteoporosis is an imbalance between bone formation and bone resorption, with osteoblast dysfunction and excessive osteoclast activation being the core components. Although various treatment options for osteoporosis are available clinically, due to its complex pathophysiological mechanisms, osteoporosis remains an incurable chronic disease to date.

[0003] Postmenopausal osteoporosis is a disease characterized by bone loss and microstructural damage caused by a sharp drop in estrogen levels after menopause. Current clinical treatment primarily involves basic interventions combined with medication. Basic treatment includes calcium supplementation, vitamin D supplementation, and lifestyle modifications; however, calcium supplementation alone is insufficient to reverse bone loss. Bisphosphonates (such as alendronate sodium) are first-line drugs that slow bone resorption by inhibiting osteoclast activity; however, long-term use may lead to complications such as osteonecrosis of the jaw and atypical femoral fractures, and may also cause gastrointestinal irritation and poor medication adherence. Selective estrogen receptor modulators (SERMs) such as raloxifene can mimic the protective effects of estrogen on bone, but may increase the risk of venous thrombosis and have limited effectiveness in preventing extravertebral fractures. Newer drugs such as denosumab (anti-RANKL monoclonal antibody) significantly increase bone mineral density by blocking osteoclast differentiation, but rapid bone loss and rebound effects with multiple vertebral fractures are common after discontinuation. In recent years, romozolium (antisclerosinumab), a bone-forming drug, has been introduced that bidirectionally regulates bone metabolism through the Wnt signaling pathway, showing significant short-term efficacy. However, its long-term cardiovascular safety still needs to be verified, and the treatment cost is high. While estrogen replacement therapy can effectively prevent bone loss, its application is limited by the risks of breast cancer, endometrial cancer, and thrombosis. Current treatments generally face the challenge of significant individual variability in response and the need for precise balancing of drug side effects and benefits. Some patients discontinue treatment due to poor gastrointestinal tolerance, fear of subcutaneous injections, or the requirement for frequent follow-up.

[0004] Edaravone is a neuroprotective agent (free radical scavenger). Clinical studies suggest that N-acetylaspartate (NAA) is a specific marker of surviving nerve cells, and its content decreases sharply in the early stages of cerebral infarction. Administration of edaravone to patients in the acute phase of cerebral infarction can inhibit the reduction of local cerebral blood flow around the infarct, resulting in a significantly higher NAA content in the brain on day 28 post-infarction compared to the glycerol control group. Preclinical studies indicate that intravenous administration of edaravone to rats after ischemia / ischemia-reperfusion can prevent the progression of cerebral edema and cerebral infarction, alleviate accompanying neurological symptoms, and inhibit delayed neuronal death. Mechanistic studies suggest that edaravone can scavenge free radicals and inhibit lipid peroxidation, thereby inhibiting oxidative damage to brain cells, vascular endothelial cells, and nerve cells. However, whether edaravone has a therapeutic effect on postmenopausal osteoporosis has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a new pharmaceutical use for edaravone in the treatment of postmenopausal osteoporosis, especially the imbalance of the intraosseous microenvironment caused by osteoblast ferroptosis, which has a significant therapeutic effect on postmenopausal osteoporosis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first objective of this invention is to provide the use of edaravone in the preparation of a medicament for treating postmenopausal osteoporosis, which is caused by osteoblast ferroptosis leading to an imbalance in the bone microenvironment.

[0007] Preferably, the treatment of postmenopausal osteoporosis involves inhibiting osteoblast ferroptosis, thereby regulating the imbalance of the bone microenvironment.

[0008] Preferably, the dosage of edaravone is 10 mg / kg•d.

[0009] A second objective of this invention is to provide a medicament for treating postmenopausal osteoporosis, wherein the active ingredient of the medicament is edaravone.

[0010] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0011] Compared with the prior art, the advantages of this invention are as follows: This invention reveals for the first time the crucial role of ferroptosis in postmenopausal osteoporosis (PMOP) and proposes a technical approach for treating PMOP by inhibiting ferroptosis and regulating the imbalance of the bone microenvironment. Furthermore, by constructing a mouse ovariectomized model combined with a ferroptosis-induced osteoblast / osteoclast system, this invention demonstrates that edaravone can effectively increase calcium deposition and mineralization in postmenopausal osteoporosis, improve osteoblast dysplasia and osteoblast inflammation caused by osteoporosis, effectively increase FSP1 expression, and decrease 5-LOX and ACSL4 protein expression. By inhibiting ROS and lipid peroxidation, it regulates the intraosseous ferroptosis microenvironment in PMOP, achieving a therapeutic effect that alleviates the progression of PMOP. Attached Figure Description

[0012] Figure 1 Line graphs showing the changes in body weight of mice in each group; Figure 2 Figures showing bone calcium deposition and osteoblast calcification in mice of each group; A is a Von-Kossa staining image showing the degree of calcium deposition in the proximal tibia of mice; B is an Alizarin Red staining image showing the mineralization of osteoblasts in the proximal tibia of mice. Figure 3 The images show the micro-CT scans and data analysis of mice in each group; A is a Micro-CT sagittal image showing the proximal tibia of the mouse (bar=1mm); B is a Micro-CT coronal image showing the position 1.5mm below the proximal metaphysis of the mouse tibia (bar=1mm); C is a Micro-CT 3D reconstructed image (bar=1mm); D is a data analysis diagram of trabecular volume BV (mm³) / total bone volume TV (mm³); E is a data analysis diagram of trabecular surface area BS (mm²); F is a data analysis diagram of trabecular thickness Tb.Th (mm); G is a data analysis diagram of trabecular separation Tb.Sp (mm); H is a data analysis diagram of trabecular number Tb.N; and I is a data analysis diagram of trabecular classification dimension FD. in, ns P≥0.05,*P<0.05,**P<0.01, ***P<0.001, ****P<0.0001; Figure 4 Figure A shows the alkaline phosphatase content in osteoblasts of mice in each group; Figure B shows the alkaline phosphatase content in mouse MC3T3-E1 osteoblasts; Figure B shows the relative quantitative analysis of Figure A. Where *P<0.05, **P<0.01, ***P<0.001; Figure 5Figure A shows the ROS content of osteoblasts in each group of MC3T3-E1 mice; Figure B shows the fluorescence spectrum for identifying ROS content in osteoblasts of MC3T3-E1 mice; Figure A shows the relative fluorescence quantitative analysis. Among them, **P<0.01, ****P<0.0001; Figure 6 Figure 1 shows the quantitative analysis of ferroptosis-related protein levels; A is a representative image of Western blot of key proteins in the xCT-GPX4 pathway in the ferroptosis pathway; B and C are the results of Western blot quantitative analysis of GPX4 and xCT, respectively; D is a representative image of Western blot of key proteins in the lipid peroxidation pathway; E, G and I are the results of Western blot quantitative analysis of 5-LOX, ACLS4, and FPS1, respectively; H is a diagram illustrating the mechanism by which edaravone inhibits intraosseous ferroptosis and alleviates the progression of postmenopausal osteoporosis. Detailed Implementation

[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0014] Unless otherwise specified, the equipment and reagents used in the embodiments and test examples are commercially available.

[0015] 1. Research Methods 1.1 Establishment of an ovariectomized (OVX) mouse model in C57 / BL6 mice. This invention utilizes the C57 / BL6 mouse ovariectomy model to simulate postmenopausal osteoporosis. Eight-week-old female C57 / BL6 mice were divided into an OVX group and a control group. The OVX group underwent bilateral ovariectomy. In simple terms, after satisfactory anesthesia with isoflurane gas, the C57 / BL6 female mice underwent routine skin preparation and disinfection, a lateral abdominal approach was made, the skin, fascia, and muscle layers were incised to expose the ovaries and fallopian tubes, which were then externally placed, ligated to achieve hemostasis, removed, and the surgical incision sutured. The control group underwent only external placement of the ovaries, without ovariectomy. Both groups received postoperative infection prevention and analgesia for 3 days. The treatment group (EDV) received intraperitoneal injection of 10 mg / kg•d of edaravone (edaravone).

[0016] 1.2 Cell Culture Mouse MC3T3-E1 (subclone 4) cells and RAW 264.7 cells were obtained from a US-type culture collection (ATCC, Manassas, VA, USA, catalog number: CRL-2593 and catalog number: TIB-71). MC3T3-E1 cells were cultured in osteoblast medium consisting of 89.9% α-MEM + 10% FBS + 0.1% penicillin / streptomycin, without ascorbic acid. MC3T3-E1 cells were passaged every 3-4 days, with passages 3-6 used for subsequent experiments. RAW 264.7 cells were cultured in DMEM medium supplemented with 10% FBS and 0.1% penicillin / streptomycin (i.e., 89.9% DMEM + 10% FBS + 0.1% penicillin / streptomycin). RAW 264.7 cells were passaged every 3-4 days, with passages 3-6 used for subsequent experiments. The cells were cultured in a humidified environment of 37°C, 95% air, and 5% CO.

[0017] 1.3 Osteoblast Differentiation MC3T3-E1 cells from passages 3-6 obtained in section 1.2 were seeded into wells containing complete culture medium and cultured for 7 days until the cells reached 70% confluence. To initiate differentiation, the cultured cells were incubated in osteoinduction medium (OIM) containing α-MEM, 10% FBS, dexamethasone (10⁻⁷ M), β-glycerophosphate (10 mM), and ascorbic acid (50 μg / ml) for 14 days. The osteoinduction medium was changed every 3 days.

[0018] 1.4 Osteoclast Differentiation RAW 264.7 cells (passages 3-6) obtained from the partial culture in section 1.2 were seeded in 24-well plates and cultured in DMEM medium with 10% FBS and 0.1% penicillin / streptomycin for 24 hours. The medium was then changed to α-MEM, 5% FBS, and 0.1% penicillin / streptomycin. An NF-κB ligand receptor activator (RANKL, 30 ng / ml) was added to induce osteoclast differentiation for 7 days. The medium was changed every 3 days.

[0019] 1.5 Identification and staining of osteoblasts and osteoclasts ALP alkaline phosphatase staining and Alizarin Red S staining were chosen as the identification protocols for evaluating osteoblast differentiation and function, while TRAP staining was chosen to evaluate osteoclast differentiation and function. In simple terms: For ALP alkaline phosphatase staining, MC3T3-E1 cells were seeded in 24-well plates and cultured for 14 days in complete medium or OIM. Cells were washed twice in PBS and fixed in fixation buffer at room temperature for 10 minutes, then stained with a TRAP&ALP dual staining kit. For Alizarin Red S staining, cells were washed twice in distilled water and fixed in 70% ice-cold ethanol, then stained with 2% Alizarin Red S solution to detect calcification. RAW 264.7 cells were cultured in α-MEM medium for 5 days. Then, RAW 264.7 cells were washed twice in PBS, fixed in fixation solution at room temperature for 10 minutes, and then stained with a TRAP&ALP dual staining kit according to the provided protocol.

[0020] 1.6 Establishment of an osteoblast estrogen deficiency model The 3rd to 6th generation MC3T3-E1 cells obtained from the partial culture in section 1.2 were seeded into wells containing complete culture medium and cultured for 3 days until complete cell adhesion and growth were observed. The medium was then replaced with complete culture medium containing estrogen (1 nM), and cultured for another 2 weeks, changing the estrogen-containing complete culture medium every 5 days during this period. After 2 weeks, estrogen deficiency was introduced, i.e., the medium was replaced with complete culture medium without estrogen, and the cells were cultured for another week for subsequent experiments.

[0021] 1.7 Cell viability assay CCK8 was used to assess cell viability in both groups of cells. In short, cell suspensions were seeded into 96-well plates (approximately 100 μl per well), setting up an experimental group (osteoblast estrogen-deficient model from section 1.6), a control group (osteoblasts cultured in complete medium throughout the entire process), and a blank group (cell-free), with 3-6 replicates per group, pre-cultured until adherent. Drugs or treatments were added, and cultured for a specific time (e.g., 24-72 hours). 10 μl of CCK8 solution (10% of total volume) was added to each well, gently shaken to mix, avoiding air bubbles. Incubation was performed at 37°C in the dark for 1-4 hours (adjusting time according to cell metabolism), until color development. The OD value was measured at 450 nm using a microplate reader, zeroing the reading with the blank well. Cell viability (experimental group OD / control group OD × 100%) or toxicity was calculated.

[0022] 1.8 Bone density testing Bone mineral density (BMD, g / cm³) of the lumbar vertebrae, femur, and tibia in mice was determined using dual-energy X-ray absorptiometry. 2Micro-CT scans were performed on the distal femur of isolated specimens, with a pixel size of 6 μm and an X-ray source of 60 kV. The focus was on the 0.5 mm region distal to the growth plate, and 3D reconstruction was performed after the scan. Statistical analysis was conducted on parameters such as BV / TV (bone volume fraction, %), Tb.N (trabecular number, 1 / mm), Tb.Th (trabecular thickness, μm), and Tb.Sp (trabecular interosseous distance, μm).

[0023] 1.9 Establishment of osteoblast and osteoclast ferroptosis models Mouse MC3T3-E1 and RAW264.7 cells were seeded in 24-well plates and cultured in DMEM medium with 10% FBS and 0.1% penicillin / streptomycin for 24 hours. Cells were then treated with gradient concentrations of Erastin (0.1 μM, 1 μM, 10 μM, 100 μM) for 12–24 hours, followed by cell viability assays. The IC-50 was calculated to determine the optimal concentration for the ferroptosis-induced model, which was then used in subsequent experiments.

[0024] 1.10. Mouse Von-Kossa staining Mouse tibiae were collected and fixed with 4% paraformaldehyde for 24 hours, followed by gradient dehydration with ethanol and clearing with xylene. MMA embedding was then used to prepare 5µm hard tissue sections. Dewaxing and hydration: Sections were dewaxed with xylene and then hydrated with a gradient of ethanol to distilled water. Silver nitrate treatment: 1% silver nitrate solution was added dropwise (…). Cover the tissue and irradiate it under ultraviolet light or strong light for 30-60 minutes (silver ions react with calcium phosphate to form black metallic silver). Clean with sodium thiosulfate: After rinsing with running water, use 5% sodium thiosulfate ( Process for 2 minutes to remove unreacted silver ions. Counterstain: Counterstain cell nuclei with hematoxylin or nuclear solid red for 1-2 minutes, rinse with running water to regain blue color. Dehydrate and mount: Dehydrate with graded ethanol, clear with xylene, and mount with neutral resin. Calcium salt deposits appear as black or dark brown granules, and cell nuclei appear blue (hematoxylin) or red (nuclear solid red). Quantitative analysis was performed using ImageJ software to calculate the percentage of Von Kossa positive areas in the trabecular bone region.

[0025] 1.11. Alizarin Red staining in mice For example, in section 1.10, bone tissue sections are prepared.

[0026] Alizarin Red staining: Add 2% Alizarin Red-S solution (pH 4.1-4.3, adjusted with NaOH) and incubate at room temperature in the dark for 30 minutes. Gently rinse three times with distilled water to remove non-specific staining. Counterstain cell nuclei with hematoxylin for 1 minute. Tissue sections are dehydrated with graded ethanol, cleared with xylene, and then mounted; cell samples can be directly mounted with glycerol. Calcium deposition areas appear as orange-red patches or nodules (tissue sections). Measure the percentage of Alizarin Red-positive area in the trabecular bone region (%).

[0027] 2. Experimental Results 2.1 Establish a C57 / BL6 mouse model of postmenopausal osteoporosis and verify the success of the model. 2.1.1 Changes in body weight after menopause in C57 / BL6 mice Postmenopausal women often experience abnormal fat accumulation and overweight due to hormonal imbalances and lipid metabolism disorders. Based on clinical patterns, this invention quantitatively analyzed the body weight of mice in the OVX group, control group, and treatment group, as shown in the results. Figure 1 As shown, through Figure 1 The results showed that the control group mice had stable body weight, consistent with the weight change trend of conventionally fed mice. However, the ovariectomy (OVX) mice gradually increased in weight one week post-surgery, stabilizing at 22-23g after two weeks, consistent with the clinical signs of postmenopausal osteoporosis. The treatment group (OVX mice received intraperitoneal injection of 10mg / kg•d edaravone), i.e., the EDV group, reached its peak body weight two weeks post-surgery, but gradually decreased to normal levels (by the fourth week). These results indicate that the OVX model can simulate the changes in body weight and lipid metabolism in postmenopausal osteoporosis, and that edaravone can effectively regulate body weight in mice.

[0028] 2.1.2 Detection of Calcium Salt Deposition and Mineralization The results of Von-Kossa staining and alizarin red staining are as follows: Figure 2 As shown, through Figure 2 It can be seen that, compared with the control group, the OVX group showed a significant decrease in calcium salt deposition and osteoblast mineralization levels in the proximal tibia after surgery, while the EDV group mice showed a significant increase in calcium salt deposition and osteoblast mineralization after edaravone treatment, proving that edaravone can effectively increase calcium salt deposition and mineralization after osteoporosis.

[0029] 2.1.3 Micro-CT Scan Postmenopausal osteoporosis causes a significant decrease in cancellous bone mineral density. This invention uses Micro-CT scanning to observe the microstructure of the proximal tibia and analyzes parameters such as bt / tv, BS, Tb.Th, Tb.Sp, Tb.N, and FD. The results are as follows: Figure 3 As shown, through Figure 3It can be seen that, with the Control group (corresponding to) Figure 3 Compared to the Sham group, the postmenopausal mouse model (OVX) showed significantly reduced levels of bt / tv, BS, Tb.Th, Tb.Sp, Tb.N, and FD, exhibiting signs of osteoporosis. Edaravone treatment (EDV) effectively improved these osteoporotic changes. These results indicate that edaravone can improve postmenopausal osteoporosis.

[0030] 2.2 Screening for drugs that regulate ferroptosis 2.2.1 Edaravone effectively improves osteoblast dysfunction caused by osteoporosis. Osteogenesis disorder is a key cause of postmenopausal osteoporosis, and osteoblasts play a crucial role in this process. This invention establishes an in vitro estrogen-deficient osteoblast culture-induced differentiation model and investigates osteogenic capacity by detecting the alkaline phosphatase content of osteoblasts. The results are as follows: Figure 4 As shown. (Through) Figure 4 The results showed that the alkaline phosphatase content in the MC3T3-E1 mouse osteoblast line was significantly reduced after estrogen deficiency, indicating poor osteogenic development. Edaravone treatment resulted in a significant increase in alkaline phosphatase content in osteoblasts. These results indicate that osteoblasts can significantly enhance the osteogenic capacity of osteoblasts.

[0031] 2.2.2 Edaravone improves osteoblast inflammatory response caused by osteoporosis To further elucidate the mechanism by which edaravone improves postmenopausal osteoporosis, this invention, based on domestic and international research progress, explored the regulatory effect of edaravone on oxidative stress in osteoblasts. The results are as follows: Figure 5 As shown. (Through) Figure 5 The results showed that the ROS content of the MC3T3-E1 mouse osteoblast cell line increased significantly after estrogen deficiency, indicating an increased level of oxidative stress. Edaravone reduced the ROS content and showed a good reversal effect.

[0032] 2.2.3 Edaravone alleviates the progression of postmenopausal osteoporosis by inhibiting intraosseous ferroptosis.

[0033] To elucidate the mechanism by which edaravone reverses the progression of postmenopausal osteoporosis, this invention quantitatively analyzed the levels of ferroptosis-related proteins in mice. The results are as follows: Figure 6 As shown, through Figure 6The results showed that ovariectomized mice had reduced xCT protein levels, indicating impaired glycine transport into cells, leading to reduced GPX4 synthesis, a key factor in inducing osteoblast ferroptosis. Edaravone treatment significantly increased xCT and GPX4 expression, indicating that edaravone reverses the progression of postmenopausal osteoporosis via the glutathione-GPX4 pathway. Furthermore, in vivo studies revealed significant changes in another ferroptosis pathway in postmenopausal osteoporosis. The expression of the ferroptosis inhibitor FPS1 was significantly reduced, while the levels of lipid peroxidation, such as ACSL4 and 5-LOX, were significantly increased, further demonstrating ferroptosis in osteoblasts during postmenopausal osteoporosis. Edaravone effectively increases FSP1 expression while decreasing 5-LOX and ACSL4 protein expression, regulating the intraosseous ferroptosis microenvironment in postmenopausal osteoporosis by inhibiting ROS and lipid peroxidation, thus achieving a therapeutic effect in alleviating the progression of postmenopausal osteoporosis.

[0034] In summary, this invention demonstrates that ferroptosis leads to osteoblast dysfunction through pathways such as xCT-GPX4 pathway dysregulation and lipid peroxidation accumulation, and that edaravone can inhibit ROS generation and lipid peroxidation while activating FSP1-dependent non-canonical ferroptosis resistance pathways. Therefore, edaravone can provide a novel targeted strategy for the clinical treatment of osteoporosis.

[0035] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. The use of edaravone in the preparation of a medicament for the treatment of postmenopausal osteoporosis, characterized in that, Postmenopausal osteoporosis is caused by an imbalance in the bone microenvironment due to osteoblast ferroptosis.

2. The use according to claim 1, characterized in that, Treatment of postmenopausal osteoporosis involves inhibiting osteoblast ferroptosis, thereby regulating the imbalance of the bone microenvironment.

3. The use according to claim 2, characterized in that, The dosage of edaravone is 10 mg / kg·d.

4. A drug for treating postmenopausal osteoporosis, characterized in that, The active ingredient of the drug is edaravone.

5. The drug according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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