Application of tetrathiomolybdate in preparation of medicine for inhibiting inflammatory factor storm

Abstract

The invention provides application of tetrathiomolybdate in preparation of drugs for inhibiting inflammatory factor storm, and belongs to the technical field of drugs for treating inflammatory factor storm and respiratory system diseases. The general molecular formula of the tetrathiomolybdate is X < + > MoS < 42-Y < + >, and the positions of ionic bonds of four sulfur ions of the tetrathiomolybdate are interchangeable; x + and Y + are the same or different cations; the positive ions are inorganic positive ions and / or organic positive ions which can be dissociated in a solution to generate MoS < 2-> or can be subjected to enzymolysis in a biological system to generate MoS < 2->. The invention discloses unique advantages of a single drug scheme in inflammatory factor storm and respiratory system related diseases, especially bacterial acute respiratory system inflammation, for the first time, and tetrathiomolybdate is used as a single active component for preparing the drug for treating bacterial pneumonia and non-viral acute lung injury / acute respiratory distress syndrome.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for treating cytokine storms and bacterial acute respiratory inflammatory diseases, and in particular to the application of a tetrathiomolybdate in the preparation of drugs that inhibit cytokine storms. Background Technology

[0002] Acute respiratory inflammatory diseases (such as bacterial pneumonia, non-viral acute lung injury / acute respiratory distress syndrome) are common critical illnesses in clinical practice. Their core pathological mechanism involves the overactivation of the immune system, manifested as the cascade release of pro-inflammatory cytokines (such as TNF-α, IL-1β, and IL-6), massive neutrophil infiltration, and oxidative damage to lung tissue. These diseases progress rapidly, easily leading to alveolar-capillary barrier disruption, pulmonary edema, and multiple organ dysfunction, resulting in a high mortality rate. Current standard treatment mainly relies on broad-spectrum antibiotics combined with glucocorticoids to control infection and suppress the inflammatory response. However, long-term use of glucocorticoids can easily cause serious side effects such as immunosuppression, glucose metabolism disorders, and osteoporosis, and their efficacy is limited in some critically ill patients (such as those with drug-resistant bacterial infections or immunodeficiency), highlighting the inadequacy of current treatments.

[0003] Tetrathiomolybdate (TTM), a sulfur-containing metal complex, has been the subject of much previous research, primarily focusing on its copper-chelating ability in the treatment of Wilson's disease and its protective antioxidant properties in liver fibrosis models. These studies have confirmed TTM's therapeutic potential by modulating metal ion homeostasis and oxidative stress pathways, but its mechanism of action in respiratory diseases remains unclear. Notably, in existing technologies, TTM is mostly used for non-inflammatory diseases or in combination with other drugs. For example, patent CN 116546987 A, "Methods and Compositions for Treating Coronavirus, Influenza, and Acute Respiratory Distress Syndrome," discloses a combination regimen of TTM with a 5-lipoxygenase inhibitor (such as ethamazol) for viral respiratory diseases. However, this regimen targets viral pathogens, and combination therapy may increase the risk of drug interactions; it does not address the applicability of TTM as a monotherapy for bacterial acute respiratory inflammation.

[0004] Currently, while combination therapy can partially alleviate inflammation, it suffers from significant individual variability and cumulative side effects. Especially for bacterial inflammation, monotherapy offers potential advantages such as simplified administration, improved safety, and reduced clinical management costs. However, the role of TTM monotherapy in regulating lung tissue-specific inflammatory pathways (such as inhibiting NF-κB signaling and reducing neutrophil migration) remains unclear, and its anti-inflammatory effects and mechanisms in bacterial pneumonia models are still unexplored areas. Therefore, clarifying the feasibility and uniqueness of TTM as a monotherapy for bacterial acute respiratory inflammation not only fills existing technological gaps but also provides an important direction for the development of novel anti-inflammatory drugs. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides an application of tetrathiomolybdate in the preparation of drugs to inhibit cytokine storms. Compared with existing technologies, this invention reveals for the first time the unique advantages of monotherapy in bacterial acute respiratory inflammation, using tetrathiomolybdate as a single active ingredient in the preparation of drugs for treating bacterial pneumonia and non-viral acute lung injury / acute respiratory distress syndrome.

[0006] The technical solution of the present invention is as follows: The use of a tetrathiomolybdate in the preparation of a drug to inhibit cytokine storms, wherein the cytokine storm is associated with respiratory diseases. Further still, the respiratory disease is bacterial acute respiratory inflammation.

[0007] The general molecular formula of the tetrathiomolybdate is X. + MoS4 2- Y + Its structural formula is: ; The positions of the four sulfur ions in the tetrathiomolybdate are interchangeable; X + and Y + The cations may be the same or different; the cations are those that can dissociate in solution to produce MoS4. 2- MoS4 may be produced by enzymatic hydrolysis within a biological system. 2- Inorganic cations and / or organic cations.

[0008] The inventors' experiments show that the active unit of this tetrathiomolybdate is mainly tetrathiomolybdate ion (MoS4). 2- Therefore, the range of cations that can be selected is relatively wide, and there is a lot of room for selection and adjustment of later products.

[0009] In the above applications, preferably, the inorganic cation includes Na. + K + or NH4 +At least one of them.

[0010] Preferably, the organic cation includes at least one of amine ions or choline ions.

[0011] More preferably, the tetrathiomolybdate includes at least one of (NH4)2MoS4, Na2MoS4, or K2MoS4.

[0012] Furthermore, the drug can be administered orally, by inhalation, intravenous injection, intramuscular injection, or inhalation via airway.

[0013] Furthermore, the dosage forms of the drug include oral formulations, inhaled formulations, intravenous formulations, intramuscular injection formulations, or airway infusion formulations.

[0014] Furthermore, the dosage forms of the oral preparations include capsules, granules, and tablets; the dosage forms of the inhaled preparations include sprays, aerosols, dry powder inhalers, or combinations thereof.

[0015] Furthermore, the drug also includes a pharmaceutically acceptable carrier, the carrier comprising at least one of a propellant, a dispersant, a surfactant, a liquid carrier, and / or a solid carrier.

[0016] Furthermore, the subjects to which the above-mentioned drugs are applied include humans or non-human mammals, preferably humans, mice, or rats.

[0017] The beneficial technical effects of this invention are as follows: 1. This invention utilizes tetrathiomolybdate to prepare a drug for treating bacterial acute respiratory inflammation, achieving the therapeutic effect. The drug of this invention contains only tetrathiomolybdate as the active ingredient and does not contain other active ingredients such as 5-lipoxygenase inhibitors (e.g., ethamazol, ziljub). 2. Compared with the prior art, the present invention confirms that the drug can improve lung function and pulmonary edema in cases of acute lung injury, reduce serum TNF-α, IL-1β, and IL-6 levels, reduce total protein concentration and TNF-α, IL-1β, and IL-6 levels in bronchoalveolar lavage fluid, and reduce myeloperoxidase (MPO) activity, TNF-α, IL-1β, and IL-6 levels and mRNA expression in lung tissue, thereby achieving the purpose of treating bacterial acute respiratory inflammation without hepatotoxicity. Attached Figure Description

[0018] Figure 1 This is a comparison chart of lung function in mice; Figure 2 This is a comparison chart of rectal temperatures in mice; Figure 3 This is a comparison chart of mouse lung edema measurement (wet and dry weight method); Figure 4 This is a comparison image of H&E staining in mouse lung tissue; Figure 5 The ELISA method is used to detect the levels of TNF-α, IL-1β, and IL-6 inflammatory factors in mouse serum. Figure 6 The study aimed to detect the total protein concentration in mouse bronchoalveolar lavage fluid and to detect the levels of TNF-α, IL-1β, and IL-6 inflammatory factors in mouse bronchoalveolar lavage fluid using ELISA. Figure 7 The methods used were ELISA to detect the levels of MPO, TNF-α, IL-1β, and IL-6 in mouse lung tissue and qRT-PCR to detect the mRNA expression levels of TNF-α, IL-1β, and IL-6 in mouse lung tissue. Figure 8 This is a comparison chart of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels to assess drug-induced hepatotoxicity. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] Example: Tetrathiomolybdate improves lipopolysaccharide (LPS)-induced acute lung injury model in mice.

[0022] 1. Materials and Methods: 1.1 Animal grouping: SPF-grade male C57BL / 6 mice (23-26g, 8 weeks old) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and randomly divided into four groups: normal group, tetrathiomolybdate group (using (NH4)2MoS4), LPS treatment group and tetrathiomolybdate LPS treatment group, with 6 mice in each group.

[0023] 1.2 Drug pretreatment: Tetrathiomolybdate solution of 10 mg / kg / d was accurately calculated and prepared according to the mice’s drinking water volume. The daily drinking water of mice in the tetrathiomolybdate group and the tetrathiomolybdate LPS treatment group was replaced with tetrathiomolybdate solution 72 hours before LPS treatment.

[0024] 1.3 Acute lung injury modeling: 72 hours after administration of tetrathiomolybdate, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The mice were kept upright with their heads facing upwards. The mice's tongues were carefully removed with forceps to expose the airway opening. LPS (5 mg / kg) was instilled into the airway using a tubing to complete the acute lung injury modeling.

[0025] 1.4 Mouse Lung Function Testing: Mice were anesthetized 24 hours after intratracheal instillation of LPS, followed by intraperitoneal injection of 1% sodium pentobarbital. A tracheal incision was made in the neck, the trachea was exposed and dissected, and half of the trachea was cut open for endotracheal intubation. Lung function was assessed using an experimental animal lung function testing system (model: PFT, manufacturer: DSI Buxco, country of origin: USA). Mice were placed in individual animal volume tracking chambers, and respiratory parameters were continuously recorded for 10 minutes.

[0026] 1.5 Animal Sample Collection and Index Detection: Mice were administered LPS intratracheally 24 hours later, and their rectal temperature was measured. After deep CO2 anesthesia, blood was collected from the eyes via enucleation into coagulation-promoting blood collection tubes containing separating gel. The tubes were placed at 4 ℃ for 2 hours, then centrifuged at 3000 g for 10 min at 4 ℃. The supernatant serum was collected and aliquoted into eight-tube sets, stored at -80 ℃, avoiding repeated freeze-thaw cycles. ELISA was performed to detect serum levels of TNF-α, IL-1β, and IL-6 inflammatory factors.

[0027] After anesthesia and fixation, the trachea was exposed and dissected through a neck incision. Half of the trachea was cut open, and endotracheal intubation was performed. 0.5 mL of sterile saline was drawn with a 1 mL syringe and injected into the trachea through the endotracheal tube. This was repeated three times, and the fluid was collected in a centrifuge tube. The above operation was repeated twice, and the fluid was collected in a centrifuge tube. The total protein concentration of the collected bronchoalveolar lavage fluid was determined, and the levels of TNF-α, IL-1β, and IL-6 inflammatory factors in the bronchoalveolar lavage fluid were detected by ELISA.

[0028] Mouse lung tissue was collected, and the wet weight was measured using an electronic scale. The lungs were then dried in an oven (65 °C) for 72 hours and weighed again to determine the dry weight. The wet weight to dry weight ratio was calculated to assess pulmonary edema. A portion of the lung tissue was preserved in 4% paraformaldehyde, and paraffin sections were prepared. H&E staining was then performed to observe the pathological structural changes in the mouse lung tissue. The remaining lung tissue was used for ELISA to detect the levels of MPO, TNF-α, IL-1β, and IL-6 inflammatory factors in the lung tissue. qRT-PCR was performed to detect the mRNA expression levels of TNF-α, IL-1β, and IL-6 in the lung tissue.

[0029] 1.6 Drug Hepatotoxicity Assessment: SPF-grade male C57BL / 6 mice (23-26g, 8 weeks old) were selected and divided into two groups: a control group and a tetrathiomolybdate group, with 6 mice in each group. A tetrathiomolybdate solution of 10 mg / kg / day was precisely prepared based on the mice's water intake. Mice in the tetrathiomolybdate group had their daily drinking water replaced with the tetrathiomolybdate solution, while the control group did not. After 96 hours, the mice were deeply anesthetized with CO2, and blood was collected from their eyes by enucleation. The blood was collected in a coagulation-promoting blood collection tube containing separating gel. After the blood collection tube was placed in a 4 ℃ refrigerator for 2 hours, it was centrifuged at 3000 g for 10 min at 4 ℃. The supernatant serum was used for ELISA to detect ALT and AST levels.

[0030] 2. Results and Analysis Figure 1 The figure shows a comparison of lung function in mice. In the figure, Con represents the normal control group, Vehicle represents the solvent control group, ALI represents the LPS-induced acute lung injury group, and TM represents the tetrathiomolybdate treatment group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and the same applies below. As can be seen from the figure, 24 hours after LPS treatment, mice pre-treated with tetrathiomolybdate showed significantly lower lung resistance and expiratory time, and significantly higher maximum vital capacity, lung compliance, and minute ventilation compared to the untreated group, indicating that tetrathiomolybdate can improve lung function in mice.

[0031] Figure 2 The study showed a comparison of rectal temperatures in mice. After 24 hours of LPS treatment, mice pre-treated with tetrathiomolybdate had significantly higher rectal temperatures than the untreated group, indicating that tetrathiomolybdate can protect the body temperature of mice.

[0032] Figure 3 The study demonstrated lung edema in mice. After 24 hours of LPS treatment, mice pre-treated with tetrathiomolybdate showed significantly lower levels of lung edema compared to the untreated group, indicating that tetrathiomolybdate can alleviate lung edema.

[0033] Figure 4 The study demonstrated the pathological structural changes in mouse lung tissue. After 24 hours of LPS treatment, mice pre-treated with tetrathiomolybdate showed improved lung tissue pathological structure compared to the untreated group, specifically reduced edema, repair of alveolar damage, relief of congestion, reduced interstitial hyperplasia, and reduced inflammatory cell infiltration, indicating that tetrathiomolybdate can alleviate pathological structural changes in the lungs.

[0034] Figure 5This study demonstrates the detection of TNF-α, IL-1β, and IL-6 inflammatory factors in mouse serum using ELISA. Twenty-four hours after LPS treatment, mice pre-treated with tetrathiomolybdate showed significantly lower levels of TNF-α, IL-1β, and IL-6 inflammatory factors in their serum compared to the untreated group, indicating that tetrathiomolybdate can improve the degree of circulating inflammation.

[0035] Figure 6 This study demonstrates the total protein concentration in mouse bronchoalveolar lavage fluid (BALFL) and the levels of TNF-α, IL-1β, and IL-6 inflammatory factors in BALFL detected by ELISA. After 24 hours of LPS treatment, mice pre-treated with tetrathiomolybdate showed significantly lower total protein concentrations in BALFL compared to the untreated group. Simultaneously, the levels of TNF-α, IL-1β, and IL-6 inflammatory factors were also significantly reduced, indicating that tetrathiomolybdate can improve lung tissue structure, repair the alveolar-capillary barrier, inhibit inflammatory factor exudation, and reduce lung inflammation levels.

[0036] Figure 7 This study demonstrates the detection of MPO, TNF-α, IL-1β, and IL-6 levels in mouse lung tissue using ELISA and the detection of TNF-α, IL-1β, and IL-6 mRNA expression levels in mouse lung tissue using qRT-PCR. Mice pre-treated with tetrathiomolybdate showed significantly lower MPO activity in their lung tissue compared to the untreated group, suggesting that tetrathiomolybdate can reduce neutrophil infiltration. Furthermore, mice pre-treated with tetrathiomolybdate showed significantly lower levels of TNF-α, IL-1β, and IL-6 in their lung tissue and lower mRNA expression levels compared to the untreated group, indicating that tetrathiomolybdate can alleviate lung inflammation and improve lung function.

[0037] Figure 8 The study demonstrated the detection of serum ALT and AST activities in mice using ELISA. Mice pre-administered with tetrathiomolybdate showed no significant difference in ALT and AST activities compared to the unadministered group, indicating that tetrathiomolybdate has no hepatotoxicity.

[0038] Replacing tetrathiomolybdate (NH4)2MoS4 in the above experiment with dicholine tetrathiomolybdate yielded similar results to (NH4)2MoS4.

[0039] In summary, the experimental data show that tetrathiomolybdate pretreatment can significantly improve LPS-induced bacterial acute respiratory inflammation. This is achieved by improving lung function in mice, maintaining stable rectal temperature, reducing pulmonary edema, improving lung tissue pathology, inhibiting TNF-α, IL-1β, and IL-6 levels in serum and bronchoalveolar lavage fluid, and simultaneously reducing MPO activity in lung tissue and downregulating TNF-α, IL-1β, IL-6 levels and mRNA expression levels in lung tissue, without hepatotoxicity. This demonstrates that it comprehensively alleviates lung pathological damage through a triple mechanism of multi-target inhibition of inflammatory factor release, repair of alveolar barrier integrity, and blocking neutrophil migration, providing clear experimental evidence for the treatment of acute respiratory inflammation.

[0040] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, and for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the present invention is not limited to the specific details without departing from the general concept defined by the claims and their equivalents.

Claims

1. The application of a tetrathiomolybdate in the preparation of drugs that inhibit cytokine storms.

2. The application according to claim 1, characterized in that, The cytokine storm is associated with respiratory diseases.

3. The application according to claim 2, characterized in that, The respiratory disease is bacterial acute respiratory inflammation.

4. The application according to claim 1, wherein the general molecular formula of the tetrathiomolybdate is X. + MoS4 2- Y + Its structural formula is: ； The positions of the four sulfur ions in the tetrathiomolybdate are interchangeable; X + and Y + The cations may be the same or different; the cations are those that can dissociate in solution to produce MoS4. 2- MoS4 may be produced by enzymatic hydrolysis within a biological system. 2- Inorganic cations and / or organic cations.

5. The application according to claim 1, characterized in that, The inorganic cations include Na + K + or NH4 + At least one of the following; the organic cation includes at least one of amine ions or choline ions.

6. The application according to claim 1, characterized in that, The tetrathiomolybdate includes at least one of (NH4)2MoS4, Na2MoS4, or K2MoS4.

7. The application according to claim 1, characterized in that, The drug can be administered orally, by inhalation, intravenous injection, intramuscular injection, or by airway infusion.

8. The application according to claim 1, characterized in that, The dosage forms of the drug include oral formulations, inhaled formulations, intravenous injection formulations, intramuscular injection formulations, or airway infusion formulations.

9. The application according to claim 8, characterized in that, The dosage forms of the oral preparations include capsules, granules, and tablets; the dosage forms of the inhaled preparations include sprays, aerosols, dry powder inhalers, or combinations thereof.

10. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier, which includes at least one of a propellant, a dispersant, a surfactant, a liquid carrier, and / or a solid carrier.

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