Use of tetrathiomolybdate in the preparation of medicines for the prevention and / or treatment of asthma

Abstract

This invention provides the use of tetrathiomolybdate in the prevention or treatment of asthma. The invention involves administering a tetrathiomolybdate solution via gavage to asthma model mice; this drug effectively alleviates symptoms of allergic asthma by modulating the immune status. This invention not only provides a new drug option for the treatment of asthma but also has broad application prospects as it can be combined with other therapeutic drugs or methods.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more particularly to the use of tetrathiomolybdate in the preparation of medicaments for the prevention and / or treatment of asthma. Background Technology

[0002] Allergic asthma (bronchial asthma) is a chronic airway inflammation involving multiple cells, particularly mast cells, eosinophils, and T lymphocytes. In susceptible individuals, this inflammation can cause recurrent episodes of wheezing, shortness of breath, chest tightness, and / or coughing, often occurring at night and / or in the early morning. The airways exhibit increased responsiveness to various stimuli. In the past decade or so, the global prevalence and mortality rates of asthma have been on the rise, with approximately 100 million asthma patients worldwide, making it a major chronic disease that seriously threatens public health. The prevalence of asthma in China is approximately 1%, reaching up to 3% in children, with estimates suggesting over 10 million asthma patients nationwide.

[0003] Asthma attacks can cause anxiety and fear. Severe asthma attacks are life-threatening emergencies requiring urgent, skilled, and professional treatment. If not treated adequately and promptly, severe asthma attacks can be fatal. While asthma is a chronic disease that is difficult to cure, attacks can often be prevented, for example, by avoiding allergens or undergoing desensitization therapy. However, current desensitization therapies and medications for preventing secondary attacks have limited effectiveness, and long-term use can have adverse effects on the growth and development of patients, especially children.

[0004] Therefore, there is an urgent need to develop drugs that can safely and effectively prevent and treat asthma. Summary of the Invention

[0005] The purpose of this invention is to provide a medicine that can safely and effectively prevent and treat asthma.

[0006] A first aspect of the invention provides the use of tetrathiomolybdate in the preparation of a medicament for the prevention and / or treatment of asthma in a subject.

[0007] In another preferred embodiment, the asthma is allergic asthma.

[0008] In another preferred embodiment, the asthma is ovalbumin-induced sensitization asthma.

[0009] In another preferred embodiment, the drug is used for purposes selected from the group consisting of:

[0010] (a) Inhibit eosinophilic infiltration in the lungs of the subjects;

[0011] (b) Reduce the total number of white blood cells in the lungs of the subject;

[0012] (c) Inhibit the lung Th2 cytokine levels of the subjects;

[0013] (d) Reduce mucus production in the lung goblet cells of the subject; or / and

[0014] (e) Reduced collagen deposition in the lungs of the subjects.

[0015] In another preferred embodiment, the drug is used to suppress type II immune responses in the lung tissue of patients with allergic asthma.

[0016] In another preferred embodiment, the subject has characteristics selected from the following group:

[0017] (i) The total white blood cell count in the bronchoalveolar lavage fluid of the subjects was high;

[0018] (ii) The lung tissue of the subjects had elevated levels of eosinophils;

[0019] (iii) The subjects had elevated levels of eosinophilic progenitor cells in their bone marrow;

[0020] (iv) The bronchoalveolar lavage fluid of the subjects had elevated levels of Th2 cytokines;

[0021] (v) The subjects had elevated mucus secretion from goblet cells in their lungs; or / and

[0022] (vi) The subjects had elevated levels of collagen deposition in their lungs.

[0023] In another preferred embodiment, the above-mentioned high content refers to a content significantly higher than the benchmark value.

[0024] In another preferred embodiment, the benchmark value is the corresponding content in normal subjects.

[0025] In another preferred embodiment, the content is significantly higher than the reference value of ≥4 / 3, preferably ≥3 / 2, and even more preferably ≥5 / 3.

[0026] In another preferred embodiment, the Th2 cytokines include IL-4, IL-5, and IL-13.

[0027] In another preferred embodiment, the tetrathiomolybdate is selected from: alkali metal salts of tetrathiomolybdate (e.g., sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, and salts that can be prepared from physiologically acceptable organic bases.

[0028] In another preferred embodiment, the organic base includes trimethylamine, triethylamine, morpholine, pyridine, piperidine, methylpyridine, dicyclohexylamine, N,N'-diphenylethylenediamine, 2-hydroxyethylamine, di(2-hydroxyethyl)amine, tri(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabimethamine, N,N'-bisdehydroabimethamine, glucosamine, N-methylglucosamine, coliine, quinine, quinoline, and basic amino acids (such as lysine and arginine).

[0029] In another preferred embodiment, the drug is an oral preparation.

[0030] In another preferred embodiment, the dosage form of the oral preparation is selected from the group consisting of tablets, capsules, granules, pills, powders, solutions, emulsions, and suspensions.

[0031] In another preferred embodiment, the unit dose of the oral formulation is 0.1 to 30 mg / kg, preferably 0.2 to 20 mg / kg, more preferably 0.4 to 10 mg / kg, and even more preferably 0.4 to 4 mg / kg, based on the weight of the subject.

[0032] A third aspect of the present invention provides a pharmaceutical composition comprising:

[0033] (i) A first active ingredient, wherein the first active ingredient is tetrathiomolybdate:

[0034] (ii) Optionally, a second active ingredient, wherein the second active ingredient is selected from the group consisting of: β-adrenergic receptor agonists, anticholinergic drugs, theophylline, glucocorticoids, leukotriene modifiers, anti-IgE drugs, or combinations thereof;

[0035] (iii) Pharmaceutically acceptable carriers.

[0036] In another preferred embodiment, the content of the first active ingredient in the pharmaceutical composition is 0.01-99 wt%, more preferably 0.1-90 wt%, based on the total weight of the pharmaceutical composition.

[0037] In another preferred embodiment, tetrathiomolybdate is the sole and only active ingredient for treating asthma in the pharmaceutical composition.

[0038] A third aspect of the invention provides a method for preventing and / or treating asthma, comprising: administering a therapeutically effective amount of tetrathiomolybdate to a subject in need.

[0039] In another preferred embodiment, the subjects include rodents (such as mice and rats) and primates (such as humans).

[0040] In another preferred embodiment, the asthma is allergic asthma.

[0041] In another preferred embodiment, the asthma is ovalbumin-induced sensitization asthma.

[0042] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0043] Figure 1 Flowchart for OVA model mouse creation and tetrathiomolybdate treatment.

[0044] Figure 2 The total number of cells in the bronchoalveolar lavage fluid of mice in each group.

[0045] Figure 3 Results of the number of eosinophilic progenitor cells in the bone marrow of mice in each group

[0046] Figure 4 The results show the IL-4 content in the bronchoalveolar lavage fluid of mice in each group.

[0047] Figure 5 The results show the IL-5 content in the bronchoalveolar lavage fluid of mice in each group.

[0048] Figure 6 The results show the IL-13 content in the bronchoalveolar lavage fluid of mice in each group.

[0049] Figure 7 HE staining images of lung tissue from mice in each group.

[0050] Figure 8 PAS staining images of lung tissue from mice in each group. Detailed Implementation

[0051] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that tetrathiomolybdate can effectively inhibit and alleviate secondary relapse symptoms in asthma model mice, reduce eosinophil infiltration in the lungs, significantly reduce the number of cells in bronchoalveolar lavage fluid, inhibit goblet cell mucus production, and reduce pulmonary collagen deposition, demonstrating a significant therapeutic effect on the asthma phenotype. This invention provides a new approach for the clinical prevention and treatment of asthma. Based on this, the inventors completed this invention.

[0052] Tetrathiomolybdate

[0053] Tetrathiomolybdate (TTM) is a compound containing sulfur and molybdenum (MoS4). 2-Each sulfur atom is covalently bonded to a molybdenum atom, and these four sulfur atoms surround the central molybdenum atom to form a stable tetrasulfide bridge coordination structure. Tetrasulfide molybdates can be used as catalysts, antioxidants, and inhibitors, and can also be used for neurogenetic diseases such as Wilson's disease, to interfere with angiogenesis, and to slow tumor growth.

[0054] Allergic asthma

[0055] Allergic asthma is a chronic inflammatory airway disease primarily caused by the infiltration of inflammatory cells such as eosinophils, mast cells, and T lymphocytes. It is characterized by airway hyperresponsiveness, reversible airway obstruction, and dyspnea. The disease is closely related to a type 2 helper T cell (Th2)-mediated immune response; cytokines such as IL-4, IL-5, and IL-13 secreted by Th2 cells promote airway inflammation, remodeling, and eosinophil activation. Asthma is triggered by a wide range of factors, including dust mites, pollen, and air pollution, and manifests as recurrent episodes of wheezing, shortness of breath, chest tightness, and cough.

[0056] Combination of active ingredients, pharmaceutical composition

[0057] The present invention provides a combination of active ingredients, comprising a first active ingredient: tetrathiomolybdate; and a second active ingredient: a β-adrenergic receptor agonist, an anticholinergic drug, theophylline, a glucocorticoid, a leukotriene modifier, an anti-IgE drug, or a combination thereof.

[0058] Based on the above combination, the present invention provides a pharmaceutical composition that can be used to (a) inhibit eosinophilic infiltration in the lungs of the subject; (b) reduce the total number of white blood cells in the lungs of the subject; (c) inhibit the level of Th2 cytokines in the lungs of the subject; (d) reduce mucus production in goblet cells in the lungs of the subject; and / or (e) reduce collagen deposition in the lungs of the subject.

[0059] The present invention also provides a pharmaceutical composition comprising a combination of active ingredients within a safe and effective range, and a pharmaceutically acceptable carrier.

[0060] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers. Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, saline, buffer solutions, glucose, water, glycerol, polysorbate, ethanol, etc.

[0061] There are no particular limitations on the administration of the active ingredient combination or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0062] As used herein, the term “effective amount” or “effective dose” refers to an amount that is functional or active in humans and / or animals and / or cells and is acceptable to humans and / or animals.

[0063] When using pharmaceutical preparations, safe and effective amounts of active ingredients are combined and administered to mammals.

[0064] It should be understood that the effective amount of the active ingredient described in this invention may vary depending on the administration method and the severity of the disease. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, and half-life; disease severity; patient weight; patient immune status; and route of administration.

[0065] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0066] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0067] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0068] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0069] The main advantages of this invention include:

[0070] (1) This invention found that tetrathiomolybdate has significant preventive and therapeutic effects on asthma models.

[0071] (2) The tetrathiomolybdate of the present invention is stable, mature in application, convenient to administer, safe, and has few side effects on the human body, making it suitable for large-scale industrialization.

[0072] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0073] Example

[0074] Example 1: Therapeutic effect of tetrathiomolybdate on an asthma model

[0075] Preparation of tetrathiomolybdate gavage solution

[0076] Calculate and weigh tetrathiomolybdate (0.4 mg / kg) and dissolve it in 0.9% physiological saline to obtain tetrathiomolybdate gavage solution.

[0077] laboratory animals

[0078] Twenty healthy SPF-grade C57BL / 6 female mice, aged 8-10 weeks and weighing 17-20g, were provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and housed in the animal facility of the International Institute of Health and Medicine, Zhejiang University.

[0079] Experimental methods

[0080] This invention establishes a mouse asthma model using intraperitoneal injection of ovalbumin (OVA). Four groups were established: a blank control group, a model group, and a TM treatment group. The model and TM treatment groups were sensitized twice, on days 0 and 14, by intraperitoneal injection of 0.2 mL of OVA suspension [containing 80 μg of OVA]. The blank control group received saline solution instead of the sensitizing solution, with the same injection time, site, and dosage as the treated groups. From day 18, the blank control and model groups were administered 0.2 mL of 0.9% saline by gavage, while the TM treatment group was administered 0.2 mL of tetrathiomolybdate by gavage, for one week. From day 21, mice were placed in a self-made 20cm×20cm×20cm nebulizer and inhaled 1.5% OVA solution via ultrasonic nebulizer once daily for 1 hour each time, for 4 days. The control group received an equal volume of saline solution for nebulization challenge. Analysis of the mice was performed on day 25. The overall procedure is as follows: Figure 1 As shown.

[0081] Bronchoalveolar lavage fluid (BALF)

[0082] After blood was collected from the mouse heart, the right three lobes of the lung were ligated. The fur was cut open with surgical scissors, and the trachea was exposed by separating the tissue. A surgical suture was passed under the trachea with forceps, and a small incision was made in the trachea. A BALF needle was inserted, and 0.4 mL of PBS was injected. The rinsing fluid was collected back into an EP tube. This rinsing was repeated three times, with each mouse rinsed with 1.2 mL of PBS, ensuring a recovery rate of over 80%. The cells were centrifuged at 400g for 5 min, and the BALF supernatant was carefully aspirated and transferred to a clean EP tube for subsequent cytokine assays (IL-4, IL-5, IL-13). Rhodopsin was added to the cell pellet, resuspended for 15 s, neutralized, centrifuged, resuspended, and counted.

[0083] Treatment of lung tissue

[0084] After BALF collection, the largest lobe of the right lung was taken and placed in a 2mL EP tube. The tube was then minced with surgical scissors, and 1.5mL of digestion solution was added. The cells were digested on a shaker at 37°C for 1 hour. The mixture was filtered through a membrane, ground to obtain a cell suspension, centrifuged at 400g for 5 minutes, the supernatant was discarded, and 300μL of lysing red blood cell solution was added for lysing for 2 minutes. The suspension was neutralized, centrifuged again, resuspended, and counted for subsequent flow cytometry.

[0085] Tissue specimen collection

[0086] After the BALF was completed and the right lung was harvested, 400 μL of formaldehyde fixative was injected into the left lung through the BALF needle. The bronchial surgical suture was tied, and the inflated left lung was cut off along the trachea and placed in a test tube containing formaldehyde fixative. The tissue was then handed over to Hangzhou Haoke Biotechnology Co., Ltd. for paraffin embedding, paraffin sectioning, HE staining, and PAS staining.

[0087] Experimental results

[0088] In the observation of the appearance and behavior of mice, after each nebulization inhalation for 3 to 5 minutes, the asthmatic mice showed symptoms such as scratching their ears and noses, restlessness, rapid breathing, abdominal muscle spasms, and urinary and fecal incontinence. Throughout the nebulization cycle, the mice gradually showed reactions such as hair standing on end and loss of luster.

[0089] Total cell count in bronchoalveolar lavage fluid of mice

[0090] After the bronchoalveolar lavage fluid was refracted, a modified hemocytometer was used to perform a total white blood cell count under a microscope. Figure 2 As shown in Table 1 below, the total number of BALF white blood cells was significantly increased in the model group compared to the control group, while the total number of BALF white blood cells was significantly decreased in the TM group compared to the model group. The difference between the TM group and the model group was statistically significant. White blood cells are a crucial type of cell in the blood, key cells in the fight against disease, and their number reflects the development and infiltration of inflammation during the asthma process. In the asthma modeling process of this invention, the drug group can significantly reduce the total number of white blood cells, thereby reducing inflammatory infiltration and alleviating asthma.

[0091] Table 1

[0092] Group <![CDATA[White blood cell count (10 6 / mL, n = 3) <!-- 5 -->]]> Comparison 0.1 Model group 2.81 TM Group 0.33

[0093] Results of the number of eosinophils in mouse lung tissue and eosinophil progenitor cells in bone marrow

[0094] Lung tissue was digested and ground to obtain a single-cell suspension. After staining, the content of target cells was analyzed by flow cytometry. The results are as follows: Figure 3 As shown, the number of eosinophils in the lung tissue of the model group was significantly higher than that of the control group, while the number of eosinophils in the lung tissue of the TM group was decreasing compared to that of the model group.

[0095] Results of mouse bone marrow eosinophil progenitor cell count

[0096] Bone marrow was collected from the femur and tibia of mice, stained with erythropoietin, and analyzed by flow cytometry for the content of target cells. The results are as follows: Figure 3 As shown, the model group had a significantly increased number of bone marrow eosinophil progenitor cells compared to the control group, while the TM group had a decreased number of bone marrow eosinophil progenitor cells compared to the model group. Eosinophils are the terminal effector cells of allergic asthma and play an important role in the occurrence and development of asthma. A decrease in eosinophils reflects reduced inflammation and is beneficial for asthma treatment. Bone marrow eosinophil progenitor cells are an important source of mature eosinophils; a decrease in bone marrow eosinophil progenitor cells ultimately leads to a decrease in mature eosinophils.

[0097] Results of IL-4, IL-5 and IL-13 levels in mouse bronchoalveolar lavage fluid

[0098] Cytokine detection ( Figure 4-6 The results showed that compared with the blank control group, the concentrations of Th2 cytokines IL-4, IL-5, and IL-13 in the BALF of the model group mice were significantly increased. Compared with the asthma group, the concentrations of Th2 cytokines IL-4, IL-5, and IL-13 in the BALF of the TM group mice were significantly decreased.

[0099] Pathological morphological changes in lung tissue

[0100] HE staining ( Figure 7 The results showed that, compared with the blank control group, the lung tissue of the asthmatic mice showed peribronchial inflammatory cell infiltration, tracheal wall thickening, alveolar wall thickening, and increased lung parenchyma. After administration of tetrathiomolybdate, the lung pathology significantly improved.

[0101] PAS staining ( Figure 8 The results showed that the asthma group exhibited significant mucus secretion in the airways. Compared to the model group, the group treated with tetrathiomolybdate showed reduced mucus secretion.

[0102] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of tetrathiomolybdate in the preparation of a medicament for the prevention and / or treatment of asthma in a subject.

2. The use according to claim 1, characterized in that, The asthma mentioned is allergic asthma.

3. The use according to claim 1 or 2, characterized in that, The drug is intended for use selected from the group consisting of: (a) Inhibit eosinophilic infiltration in the lungs of the subjects; (b) Reduce the total number of white blood cells in the lungs of the subject; (c) Inhibit the lung Th2 cytokine levels of the subjects; (d) Reduce mucus production in the lung goblet cells of the subject; or / and (e) Reduced collagen deposition in the lungs of the subjects.

4. The use according to claim 1 or 2, characterized in that, The drug is used to suppress the type II immune response in the lung tissue of patients with allergic asthma.

5. The use according to any one of claims 1-4, characterized in that, The subjects had characteristics selected from the following group: (i) The total white blood cell count in the bronchoalveolar lavage fluid of the subjects was high; (ii) The lung tissue of the subjects had elevated levels of eosinophils; (iii) The subjects had elevated levels of eosinophilic progenitor cells in their bone marrow; (iv) The bronchoalveolar lavage fluid of the subjects had elevated levels of Th2 cytokines; (v) The subjects had elevated mucus secretion from goblet cells in their lungs; or / and (vi) The subjects had elevated levels of collagen deposition in their lungs.

6. The use according to claim 3, characterized in that, The Th2 cytokines include IL-4, IL-5, and IL-13.

7. The use according to claim 1, characterized in that, The tetrathiomolybdate is selected from: alkali metal salts, alkaline earth metal salts, aluminum salts and ammonium salts of tetrathiomolybdate, and salts that can be prepared from physiologically acceptable organic bases.

8. The use according to claim 1, characterized in that, The drug is an oral preparation.

9. The use according to claim 8, characterized in that, The dosage form of the oral preparation is selected from the following group: tablets, capsules, granules, pills, powders, solutions, emulsions, and suspensions.

10. The use according to claim 8, characterized in that, The unit dose of the oral formulation is 0.1 to 200 mg / kg, preferably 0.2 to 100 mg / kg, more preferably 0.4 to 20 mg / kg, and even more preferably 0.4 to 4 mg / kg, based on the weight of the subject.

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