New application of zoledronic acid in ALS treatment

Abstract

The invention provides a novel application of zoledronic acid in ALS treatment, belongs to the field of biological medicine, and particularly relates to a novel application of zoledronic acid in treatment of amyotrophic lateral sclerosis, animal experiments and patient-derived cell experiments prove the treatment effect of zoledronic acid on ALS, and the application of zoledronic acid in ALS treatment is a novel application of zoledronic acid in ALS treatment. And a new treatment scheme and a new research direction are provided for treatment of amyotrophic lateral sclerosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a new application of zoledronic acid in the treatment of ALS. Background Art

[0002] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a progressive, fatal neurodegenerative disease that affects motor neurons in the brainstem, spinal cord, and motor cortex. Its clinical hallmarks are muscle atrophy and weakness. ALS patients experience rapid disease progression, with death typically occurring within 2-5 years of the onset of motor symptoms, typically due to paralysis of the respiratory muscles and diaphragm. Currently, the treatment of ALS faces significant challenges, with no known cure for the disease. Although riluzole and edaravone have been approved by the US Food and Drug Administration (FDA), there is currently no cure or effective treatment for ALS. Existing treatment strategies primarily focus on improving patients' quality of life and slowing disease progression.

[0003] In-depth exploration of the pathogenic mechanisms of ALS is crucial for the development of effective treatments. Current research shows that the pathogenesis of ALS is extremely complex, involving multiple levels of pathophysiological processes, including autophagy disorders, abnormal RNA metabolism, impaired axonal transport and cytoskeleton stability, excessive oxidative stress response, neuroinflammatory response, mitochondrial dysfunction, lipid imbalance, ectopic toxicity, and glial dysfunction. These intertwined pathogenic mechanisms jointly promote the occurrence and development of ALS, making disease intervention and treatment extremely difficult. Given the current status of amyotrophic lateral sclerosis treatment, there is an urgent need to develop new treatment strategies and methods.

[0004] Zoledronic acid is a bisphosphonate drug mainly used to treat diseases related to abnormal bone metabolism, such as osteoporosis, hypercalcemia caused by malignant tumors, and bone pain caused by bone metastasis. Summary of the Invention

[0005] To address these issues, the present invention, through animal and patient-derived cell experiments, has for the first time discovered that zoledronic acid can protect motor neurons by inhibiting inflammation in peripheral and central macrophages, significantly improving motor function and effectively prolonging survival in ALS model animals. This discovery reveals the significant value of zoledronic acid in the treatment of ALS, transcending the limitations of its original indications and providing a new treatment approach and solution for ALS, with significant potential for clinical translation.

[0006] To achieve the above objectives, the specific technical solutions provided by the present invention are as follows:

[0007] The first aspect of the present invention provides the use of zoledronic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or alleviating motor neuron disease.

[0008] Furthermore, the motor neuron disease includes amyotrophic lateral sclerosis, progressive muscular atrophy, primary lateral sclerosis, and progressive bulbar palsy.

[0009] Furthermore, the motor neuron disease is amyotrophic lateral sclerosis.

[0010] In the present invention, zoledronic acid, chemical name 1-hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-diphosphonic acid, molecular formula C5H 10 N2O7P2 is a bisphosphonate drug. Its CAS number is 118072-93-8. Zoledronic acid is primarily used to treat diseases associated with abnormal bone metabolism, such as osteoporosis, hypercalcemia caused by malignant tumors, and bone pain caused by bone metastases. Its mechanism of action is to inhibit bone resorption by inhibiting osteoclast activity and inducing osteoclast apoptosis. It can bind to hydroxyapatite in the bone and preferentially accumulate in areas of high bone turnover, such as bone metastases or areas of osteoporosis, to exert its pharmacological effects. In addition, zoledronic acid can also lower serum calcium and phosphorus levels and increase urinary calcium and phosphorus excretion, helping to regulate calcium and phosphorus metabolism.

[0011] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt of the active compound and is prepared by reacting zoledronic acid with a suitable organic or inorganic acid or acid derivative. Pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate, nitrate, sodium, calcium, potassium, zinc, and meglumine salts.

[0012] In the present invention, the term "treatment" refers to the process of intervening in or changing a specific health condition, including eliminating the cause, symptomatic treatment or supportive treatment.

[0013] In the present invention, the term "relief" refers to a reduction in the intensity or frequency of disease activity compared to before treatment, such as a reduction in inflammatory response, improvement in movement disorders, and prolonged survival. In some embodiments, administration of the active ingredient of the present invention (e.g., zoledronic acid or a pharmaceutically acceptable salt thereof) reduces the severity or duration of disease manifestations in patients by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to administration of no active ingredient of the present invention (e.g., placebo).

[0014] Motor neuron disease (MND) is a group of chronic, progressive neurodegenerative diseases that selectively affect the anterior horn cells of the spinal cord, brainstem motor neurons, cortical pyramidal cells, and the pyramidal tracts. MNDs primarily include upper motor neuron, lower motor neuron, and mixed upper and lower motor neuron types, depending on the type and location of affected neurons. Clinical manifestations include progressive muscle atrophy and weakness accompanied by pyramidal tract signs, slurred speech, coughing when drinking, and, when respiratory muscles are affected, dyspnea and respiratory failure.

[0015] Furthermore, the drug treats or alleviates motor neuron disease by inhibiting cellular inflammatory response and protecting motor neurons.

[0016] Furthermore, the drug can be used directly or in the form of a pharmaceutical composition.

[0017] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some embodiments, oral administration is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, pharmaceutically acceptable acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may be administered to the recipient by any route that reaches the target tissue.

[0018] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of zoledronic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0019] In the present invention, the term "therapeutically effective amount" refers to a dose that can treat and / or alleviate the diseases described herein, or inhibit macrophage inflammatory responses, protect neurons, enhance neuronal activity, and / or alleviate movement disorders in a subject. The amount depends on the patient, the severity of the disease, and the route and medium used for administration. Based on their expertise, those skilled in the art will be able to determine an appropriate amount of the active compound (e.g., zoledronic acid) in such a therapeutic composition.

[0020] Furthermore, the therapeutically effective amount is selected from 4 mg to 5 mg.

[0021] Furthermore, the pharmaceutical composition is administered once a year.

[0022] Furthermore, the pharmaceutical composition also includes other drugs for treating or alleviating motor neuron disease.

[0023] The pharmaceutical compositions of the present invention may also be used in combination with other drugs for treating or alleviating motor neuron disease. These other drugs may be administered simultaneously with the main active ingredient (e.g., zoledronic acid), or even administered simultaneously in the same composition. The other therapeutic compound may also be administered separately in a separate composition or in a dosage form different from that of the main active ingredient.

[0024] Furthermore, the other drugs for treating or alleviating motor neuron disease include one or more of riluzole, edaravone, and sodium phenylbutyrate combined with tauroursodeoxycholic acid.

[0025] In the present invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids such as water, saline, glycerol and ethanol. Auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in such vehicles.

[0026] Furthermore, the pharmaceutically acceptable carrier includes one or more of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant and a flavoring agent.

[0027] Furthermore, the dosage forms of the drug or pharmaceutical composition include oral dosage forms, injections, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms, and cavity administration dosage forms.

[0028] Furthermore, the oral dosage form includes ordinary tablets, sustained-release tablets, capsules, granules, drops, powders, emulsions, and pills.

[0029] Furthermore, the respiratory tract administration dosage form includes aerosol, spray, and powder spray.

[0030] Furthermore, the skin administration dosage form includes patches, ointments, and creams.

[0031] Furthermore, the mucosal administration dosage forms include drops, films, gels, patches, ointments, and creams.

[0032] Furthermore, the intracavitary administration dosage form includes suppositories and pills.

[0033] The second aspect of the present invention provides the use of zoledronic acid or a pharmaceutically acceptable salt thereof in preparing a product, wherein the use includes any of the following:

[0034] 1) Application in the preparation of products for treating or alleviating cellular inflammatory responses.

[0035] 2) Application in the preparation of products for protecting motor neurons.

[0036] 3) Use in the preparation of products for treating or alleviating motor neuron disease.

[0037] Furthermore, the motor neuron disease includes amyotrophic lateral sclerosis, progressive muscular atrophy, primary lateral sclerosis, and progressive bulbar palsy.

[0038] Furthermore, the motor neuron disease is amyotrophic lateral sclerosis.

[0039] Advantages and beneficial effects of the present invention: Through animal and patient-derived cell experiments, the present invention demonstrates for the first time that zoledronic acid can inhibit peripheral and central macrophage inflammation, exerting a protective effect on motor neurons, significantly improving motor function and effectively prolonging survival in ALS model animals. This discovery reveals the significant value of zoledronic acid in the treatment of ALS, transcending the limitations of its original indications and providing a new treatment approach and solution for ALS, with significant potential for clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Representative fluorescence images (a) and statistical graphs (b) show that zoledronic acid reduces the expression levels of inflammatory factors (F4 / 80, IL-1β, IL-6, TNF-α, IL-4, and IL-10) in macrophages differentiated from peripheral blood monocytes in ALS patients.

[0041] Figure 2 Representative fluorescence images of the changes in the levels of inflammatory factors (CD68, IL-6, IL-1β, TNF-α, NLRP3) in macrophages in the sciatic nerve (a), lung (b), and liver tissue (c) after administration of zoledronic acid.

[0042] Figure 3 Representative fluorescence images and statistical graphs of the effects of zoledronic acid on central neurons and glial cells, among which a shows the activation of microglia and astrocytes in the anterior horn of the spinal cord after zoledronic acid treatment and the significant increase in the number of motor neurons in the mice in the zoledronic acid group; b quantifies the intensity of GFAP and IBA1-positive cells in the lumbar spinal cord (n=3) and counts Neun-positive motor neurons in the anterior horn of the spinal cord in each group on day 120 (n=3).

[0043] Figure 4 Zoledronic acid for SOD1G93A Figure 3: Effects of zoledronic acid on the onset of disease (a) and survival (b) of transgenic mice. Zoledronic acid injection delayed weight loss (c) and improved motor dysfunction as shown by rotation test (d). DETAILED DESCRIPTION

[0044] The various reagents involved in the technical solutions and experimental procedures described in this invention are all commonly used or commercially available reagents that are well known and readily available to those skilled in the art based on their professional knowledge and routine practice. The description of the reagents in this invention is intended to clearly illustrate the material basis involved in the technical solutions, and those skilled in the art, based on their professional qualities and industry common sense, will be able to successfully obtain and correctly use these reagents to achieve the technical objectives of this invention.

[0045] For the purposes of this invention, the term "amyotrophic lateral sclerosis" (ALS), also known as Lou Gehrig's disease (Lou Gehrig's disease), refers to a chronic, progressive neurological disease that primarily damages upper and lower motor neurons and the muscles of the trunk, limbs, head, and face that they innervate. The primary clinical manifestations of ALS are progressive skeletal muscle weakness, muscle atrophy, fasciculations, and bulbar paralysis, which gradually worsen with disease progression and may even affect respiratory muscles, leading to dyspnea. Patients may also experience symptoms such as numbness, limb paralysis, weight loss, and cardiac arrhythmias. They are also likely to face emotional problems such as depression and anxiety.

[0046] In this study, the SOD1-G93A mouse is a transgenic model carrying a humanized mutant SOD1 gene (glycine at position 93 is mutated to alanine), using a C57BL / 6 or B6SJL hybrid background. This model is used to simulate amyotrophic lateral sclerosis (ALS), with phenotypes including movement disorders, neuronal death, and shortened lifespan. Its pathological features are highly similar to those of human ALS, making it a core model for ALS research.

[0047] As used herein, the "rotarod test," also known as the "rotarod fatigue test," is a method used to assess an animal's motor skills and motor integrity. In this test, the animal is placed on a rotating rod, and the speed of the rod is gradually increased. The animal's performance at different speeds, such as dwell time and endurance, is observed. This test can be used to assess skeletal muscle weakness, fatigue, and central nervous system depression. In specific embodiments of the present invention, the rotarod test is used to test mice for skeletal muscle weakness and evaluate motor deficits.

[0048] In this application, the terms "anti-inflammatory cytokines" and "anti-inflammatory cytokines" are used interchangeably. These cytokines have the functions of suppressing excessive inflammation, maintaining immune tolerance, and promoting repair, and include IL-4, IL-10, IL-13, IL-37, TGF-β, and the like. The term "pro-inflammatory cytokines" has the functions of activating inflammatory responses, recruiting immune cells, and clearing pathogens. Overexpression may cause tissue damage, and includes IL-6, TNF-α, IL-1β, IL-8, IL-12, IL-17, IFN-γ, and the like. The balance of inflammation is regulated by both pro-inflammatory and anti-inflammatory cytokines.

[0049] In the present invention, F4 / 80 is an orphan receptor belonging to the G protein-coupled receptor 2 family that is involved in cell adhesion and may participate in cell-cell interactions specifically involving cells of the immune system. F4 / 80 is primarily expressed on the surface of macrophages and serves as a marker for mature macrophage tissue.

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0051] Example

[0052] 1. Materials and Methods

[0053] 1. Chemicals

[0054] Zoledronic acid concentrated solution for injection (Jingyouxin, national medicine standard number H20213707), the active ingredient is zoledronic acid, the specification is 4 mg / 5 ml.

[0055] 2. Patient Source

[0056] This study enrolled 10 ALS patients hospitalized in the Department of Neurology, Second Hospital of Hebei Medical University from January 2023 to January 2025, as well as 10 healthy subjects who underwent physical examinations during the same period. All included patients met the 2000 revised El-Escorial diagnostic criteria. All ALS patients diagnosed for the first time were included. Exclusion criteria: Those who have used riluzole and edaravone; Those who have recently used hormones or anti-inflammatory drugs; Severe illness requiring use of a ventilator, tracheotomy, or a combination; People with acute or chronic inflammatory diseases, including, for example, acute pneumonia and rheumatoid arthritis; Patients or family members who do not cooperate. This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (2022-R196).

[0057] 3. Cell culture and cell treatment

[0058] Human peripheral blood lymphocyte separation medium (LTS1077) was added to PBMCs (Tianjin Haoyang Biological Products Technology Co., Ltd., Tianjin, China, 601002), placed in a high-performance centrifuge tube, and centrifuged at 200 × g for 2 minutes at room temperature. Peripheral blood samples were then added and centrifuged at 800 × g for 30 minutes. The mononuclear cell layer was carefully removed from the tube and centrifuged at 300 × g for 13 minutes. After aspirating the supernatant, the pellet was washed multiple times with phosphate-buffered saline (PBS), and the PBMCs were resuspended in a solution containing 90% fetal bovine serum (FBS, CellMax, Beijing, China, SA211.02) and 10% dimethyl sulfoxide (DMSO, Sigma Aldrich, St. Louis, MO, USA, D2650-100mL). The solution was then transferred to a 1.8 mL cryovial and stored at −80°C overnight. The tubes were then stored in liquid nitrogen for long-term storage.

[0059] Previously prepared PBMCs were resuspended in prewarmed Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Waltham, MA, USA, C11875500BT) containing 10% FBS. After centrifugation at 500 × g for 5 minutes at room temperature, the supernatant was removed, and the cell pellet was resuspended in RPMI-1640 medium supplemented with 1% penicillin-streptomycin (P / S) and 10% FBS. The medium was supplemented with macrophage colony-stimulating factor (M-CSF) (PeproTech, East Windsor, NJ, USA, 300-25-10), and the cells were plated in 48-well plates for 7 days. The cells were cultured at 37°C in a 5% CO2 atmosphere, with the medium changed every 3 days. The cells were grouped as normal control, ALS patient group, or ALS patient plus zoledronic acid group. On day 7 of culture, the medium was removed and the cells were fixed with 4% paraformaldehyde in PBS.

[0060] 4. Animal Model and Treatment

[0061] SOD1 G93ATransgenic mice and age-matched wild-type (WT) littermates were interbred using male hemizygous carriers (B6SJL-Tg (SOD1-G93A) 1Gur / J) purchased from Jackson Laboratories and female B6SJL / F1 mice. Animals were housed in a temperature- and humidity-controlled environment with a 12 / 12 h light / dark schedule and provided with specific pathogen-free rodent chow and sterile water.

[0062] SOD1 G93A The genotype of transgenic mice was identified by PCR. G93A Vehicle group and SOD1 G93A Zoledronic acid group. G93A Transgenic mice were given subcutaneous injection of zoledronic acid at the onset stage (90 days of age), and the control group received an equal volume of normal saline as an excipient to eliminate the effect of the intervention method.

[0063] 5. Behavioral assessment

[0064] 5.1 Rotarod test

[0065] The rotarod test began at week 12, with testing frequency increasing to once a week. After week 17, testing frequency increased to twice a week. The first five days of the experiment were an acclimatization and learning phase for the transgenic mice. The mice were placed on a stationary rotarod, starting at 2 rpm and reaching 30 rpm within 3 minutes. The time the mice remained on the rotarod was recorded. Each mouse was tested three times, with 30-minute intervals between each test. The longest duration was recorded.

[0066] 5.2 Neurological score

[0067] Neurological assessments were performed starting from week 12 and were conducted once a day. The scoring criteria were as follows:

[0068] 0 points: When the mouse is suspended by the tail, the hind limbs are fully extended and away from the lateral midline, and the mouse can maintain this position for 2 seconds.

[0069] 1 point: When hanging, the leg is extended outward to the midline and collapses or the hind limbs tremble.

[0070] 2 points: Walking 12 inches with toes curled at least twice, or any part of the foot dragging along the cage floor or table bottom.

[0071] 3 points: Rigid paralysis or minimal joint movement, the foot cannot produce forward movement.

[0072] 4 points: The mouse is placed at an angle and cannot be straightened within 30 seconds.

[0073] 5.3 Onset and Survival

[0074] Disease onset was recorded when mice failed to persist for 3 minutes in three consecutive rotarod tests or when the neurological score reached 1. Starting at 120 days of age, mice were monitored daily. The experimental endpoint was defined as a neurological score of 4 and / or a body weight loss exceeding 15%.

[0075] 6. Immunofluorescence

[0076] Cells were seeded on sterile glass coverslips and fixed with 4% paraformaldehyde for 30 minutes. The cells were then washed three times in phosphate-buffered saline (PBS), permeabilized with 0.5% Triton X-100 for 15 minutes, and washed three times with 1× PBS for 5 minutes each. The cells were blocked in donkey serum for 1 hour at room temperature and incubated with anti-CD68 (1:500, Abcam, Waltham, MA, USA, ab31630), F4 / 80 (1:200, Abcam, Waltham, MA, USA, ab6640), IL-1β (1:200, Genetex, GTX74034), TNF-α (1:200, Santa, sc-52746), and IL-6 (1:200, Genetex, GTX74034) overnight at 4°C on a shaker. The cells were then rinsed three times with 1× PBS for 5 minutes each. Sections were washed in PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies (1:1000, ThermoFisher, #A-11034), Alexa Fluor 594-conjugated goat anti-mouse secondary antibodies (1:1000, Thermo Fisher, #A-11032), and Alexa Fluor 647-conjugated donkey anti-goat secondary antibodies (1:1000, Thermo Fisher, #A211447) for 1 hour at room temperature. Sections were then washed again in PBS and nuclei were stained with DAPI Fluoromount-G (SouthernBiotech). Slides were observed using a fluorescence confocal microscope (LSM900, ZEISS, Germany). Imaging settings remained unchanged throughout.

[0077] 2. Experimental Results

[0078] 1. Zoledronic acid can inhibit the inflammatory activation of macrophages derived from peripheral blood monocytes in ALS patients.

[0079] We induced the differentiation of peripheral blood monocytes from ALS patients and healthy controls into macrophages. We observed changes in inflammatory cytokine levels and used immunofluorescence labeling to detect changes in IL-6, TNF-α, IL-1β, IL-4, and IL-10 in macrophages. We found that the expression levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β in the ALS group were significantly higher than those in the control group, while the expression levels of anti-inflammatory factors IL-4 and IL-10 were significantly lower in the ALS group than those in the control group (results shown in Figure 2). Figure 1 ). Zoledronic acid was administered to ALS patients' peripheral blood mononuclear cells to induce differentiation of macrophages. Immunofluorescence labeling was used to detect the decreased expression levels of proinflammatory cytokines IL-1β, TNF-α, and IL-6 in macrophages (the results are shown in Figure 1 (a) Zoledronic acid regulates the expression and release of pro- and anti-inflammatory factors.

[0080] 2. Zoledronic acid reduces SOD1 G93A Inflammatory response of macrophages in peripheral tissues of model mice.

[0081] Previous studies have shown that zoledronic acid can improve alveolar macrophage inflammation, and in ALS patients, lung tissue inflammation changes later than sciatic nerve inflammation changes. G93A Mice were given zoledronic acid subcutaneously at the early stage of the disease, and immunofluorescence staining was performed to observe the changes in the levels of inflammatory factors in macrophages in the sciatic nerve, lung, and liver tissues (the results are shown in Figure 2). Figure 2 The results showed that SOD1 G93A The levels of inflammatory factors in the sciatic nerve, lung and liver macrophages of mice were significantly higher than those in the WT group. After injection of zoledronic acid, the levels of inflammatory factors in the peripheral sciatic nerve, lung and liver macrophages of mice were downregulated (results shown in Figure 2 a, b, and c in the figure).

[0082] 3. Zoledronic acid promotes SOD1 G93A The study enhanced the survival of motor neurons and reduced glial cell activation in the spinal cord of model mice.

[0083] The main pathological changes in the spinal cord of ALS patients are the degeneration and loss of motor neurons and the proliferation of related glial cells. Therefore, we observed the changes in neurons and glial cells after zoledronic acid injection. Figure 3 As shown in Figure 3, the activation and proliferation of glial cells were significantly reduced after administration of zoledronic acid. In addition, the number of motor neurons in the lumbar spinal cord sections of each group (n = 5) was quantified by immunofluorescence staining at the end stage. G93ACompared with mice, mice treated with zoledronic acid showed a significant increase in the number of motor neurons in the anterior horn of the spinal cord.

[0084] 4. Zoledronic acid improves SOD1 G93A Motor dysfunction and prolonged survival of transgenic mice.

[0085] To investigate whether zoledronic acid has therapeutic effects on ALS mouse models by reducing peripheral inflammatory responses, mice were divided into the following groups: SOD1 G93A Vehicle group, SOD1 G93A Zoledronic acid group, WT group, and WT zoledronic acid group (15 mice each). Mice were subcutaneously injected at the onset stage (90 days old). The control group used an equal amount of normal saline as an excipient to eliminate the effect of the intervention method. Figure 4 As shown in Figure 2, zoledronic acid injection did not delay the onset of disease (median 109 vs 105, P=0.07). However, analysis of the survival time of mice in each group found that subcutaneous injection of zoledronic acid significantly prolonged the SOD1 G93A The survival of mice was significantly delayed (median 141 vs 129, P = 0.0003). As one of the most important symptoms for evaluating the progression of ALS, the weight loss of mice injected with zoledronic acid was also significantly delayed. In addition, the SOD1 G93A Mice injected with zoledronic acid performed better on the rotarod test compared to mice injected with zoledronic acid. These results suggest that SOD1 G93A Compared with the mice, the motor function decline of the zoledronic acid-injected group was delayed and the survival period was extended to a certain extent.

[0086] Taken together, these results demonstrate that zoledronic acid reduces the inflammatory response in macrophages derived from ALS patients, inhibits inflammation in peripheral tissue macrophages from ALS mice, enhances neuronal viability and protects neurons, thereby improving motor dysfunction and prolonging survival in ALS mice. Zoledronic acid can slow the progression of ALS and represents a novel therapeutic option for the treatment of ALS.

[0087] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Use of zoledronic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or alleviating motor neuron disease; Preferably, the motor neuron disease includes amyotrophic lateral sclerosis, progressive muscular atrophy, primary lateral sclerosis, progressive bulbar palsy; Preferably, the motor neuron disease is amyotrophic lateral sclerosis.

2. The use according to claim 1, characterized in that The drug treats or alleviates motor neuron disease by inhibiting cellular inflammatory response and protecting motor neurons.

3. The use according to claim 1, characterized in that The drug can be used directly or in the form of a pharmaceutical composition.

4. The use according to claim 3, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of zoledronic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; Preferably, the therapeutically effective amount is selected from 4 mg to 5 mg; Preferably, the pharmaceutical composition is administered once a year.

5. The use according to claim 4, characterized in that The pharmaceutical composition also includes other drugs for treating or alleviating motor neuron disease; Preferably, the other drugs for treating or alleviating motor neuron disease include one or more of riluzole, edaravone, sodium phenylbutyrate combined with tauroursodeoxycholic acid.

6. The use according to claim 3, characterized in that The pharmaceutically acceptable carrier includes one or more of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant and a flavoring agent.

7. The use according to claim 3, characterized in that The dosage forms of the medicine or pharmaceutical composition include oral dosage forms, injections, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms, and cavity administration dosage forms.

8. The use according to claim 7, characterized in that The oral preparations include ordinary tablets, sustained-release tablets, capsules, granules, drops, powders, emulsions, and pills; Preferably, the respiratory tract administration dosage form includes aerosol, spray, and powder; Preferably, the skin administration dosage form includes a patch, an ointment, or a cream; Preferably, the mucosal administration dosage form includes drops, films, gels, patches, ointments, and creams; Preferably, the intracavitary administration dosage form includes suppositories and pellets.

9. Use of zoledronic acid or a pharmaceutically acceptable salt thereof in preparing a product, characterized in that: The application includes any of the following: 1) Application in the preparation of products for treating or alleviating cellular inflammatory responses; 2) Application in the preparation of products for protecting motor neurons; 3) Use in the preparation of products for treating or alleviating motor neuron disease.

10. The use according to claim 9, characterized in that The motor neuron diseases include amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, and progressive bulbar palsy; Preferably, the motor neuron disease is amyotrophic lateral sclerosis.

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