Use of amiloride in the preparation of a drug for preventing and treating infective endocarditis or inhibiting its pathogenic bacteria

Abstract

The present invention provides an application of amiloride in the preparation of a drug for preventing and treating infective endocarditis or inhibiting its pathogenic bacteria, belonging to the field of pharmaceutical technology. The results show that after treatment with amiloride, the vacuoles of macrophages disappear significantly, the release of LDH decreases significantly, and macrophage death is significantly reduced. At the same time, the results also show that injecting amiloride can significantly inhibit the pathogenic bacteria of endocarditis, thereby effectively treating infective endocarditis. Therefore, the present invention discloses for the first time that amiloride can prevent and treat infective endocarditis, and amiloride itself is a drug for clinical application with high safety. Therefore, the present invention provides a theoretical basis for new substances for treating infective endocarditis and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the use of amiloride in the preparation of drugs for preventing and treating infectious endocarditis or inhibiting its pathogenic bacteria. Background Art

[0002] Infectious endocarditis (IEC) is relatively common clinically. It is mainly caused by the invasion of pathogenic bacteria such as bacteria and fungi into the body, with Gram-positive bacteria being the main ones, seriously affecting the quality of life and life safety of patients. Research has found that Gram-positive strains in IEC pathogenic bacteria account for at least 45.0%, and the proportions of Gram-negative strains and fungal strains are mostly lower than 5.0%. If patients with IEC do not receive timely and effective treatment, it is not only easy to lead to the progression of the disease and increase the difficulty of treatment, but in severe cases, it may even endanger life. Therefore, it is extremely necessary to strengthen the treatment of IEC clinically.

[0003] Currently, the clinical treatment of IEC mainly uses antibacterial drugs such as vancomycin, and the clinical efficacy is relatively significant. However, long-term treatment with vancomycin can easily lead to drug resistance in pathogenic bacteria, resulting in adverse reactions such as dyspnea, hypotension, and wheezing, weakening the drug efficacy. Amiloride is an oral potassium-sparing diuretic, which is often used as an antihypertensive drug clinically. Existing research has found that as a multi-channel ion blocker, amiloride has great development prospects in central nervous system diseases, such as epilepsy, migraine, multiple sclerosis, cerebral ischemia-reperfusion injury, etc. However, there is no literature reporting that amiloride can treat infectious endocarditis mainly caused by pathogenic bacteria. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the use of amiloride in the preparation of drugs for preventing and treating infectious endocarditis or inhibiting its pathogenic bacteria.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides the use of amiloride in the preparation of drugs for preventing and treating infectious endocarditis.

[0007] Preferably, the amiloride inhibits the vacuolization death of cells induced by infectious endocarditis pathogenic bacteria.

[0008] Preferably, the amiloride inhibits the release of lactate dehydrogenase.

[0009] Preferably, the amiloride inhibits infectious endocarditis pathogenic bacteria.

[0010] The present invention also provides the use of amiloride in the preparation of drugs for inhibiting infectious endocarditis pathogenic bacteria.

[0011] Preferably, the pathogen causing infective endocarditis includes Berger's bacteria.

[0012] The present invention also provides an application of amiloride in the preparation of a drug for inhibiting cell vacuolization death.

[0013] Preferably, the drug comprises an active ingredient amiloride and a pharmaceutically acceptable excipient.

[0014] Preferably, the concentration of amiloride is 0.2 - 1 μM.

[0015] Preferably, the dosage form of the drug is a granule, pill, tablet, injection, capsule or powder.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides an application of amiloride in the preparation of a drug for preventing and treating infective endocarditis or inhibiting its pathogen. The results show that after macrophages are treated with amiloride, the vacuoles of macrophages significantly disappear, the release of LDH significantly decreases, and macrophage death is significantly reduced. At the same time, the results also show that injecting amiloride can significantly inhibit the pathogen causing endocarditis, thereby effectively treating infective endocarditis. Therefore, the present invention discloses for the first time that amiloride can prevent and treat infective endocarditis, and amiloride itself is a drug for clinical application with high safety. Therefore, the present invention provides a theoretical basis for new substances for treating infective endocarditis and has broad application prospects. Description of the Drawings

[0018] Figure 1 Shows the effects of different treatment groups on the vacuolization of BMDM cells;

[0019] Figure 2 Shows the effects of different treatment groups on the pathogen causing endocarditis. Detailed Embodiments

[0020] Based on the fact that in the prior art, antibacterial drugs such as vancomycin are usually used to treat infective endocarditis, resulting in drug resistance and various adverse reactions, therefore, the present invention provides an application of amiloride in the preparation of a drug for preventing and treating infective endocarditis.

[0021] The amiloride of the present invention is preferably amiloride hydrochloride, with the chemical formula C6H8ClN7O·HCl, and the chemical structural formula is as follows:

[0022]

[0023] The present invention utilizes amiloride to inhibit the vacuolization death of cells induced by pathogens of infective endocarditis, the release of lactate dehydrogenase, and the pathogens of infective endocarditis. In the examples of the present invention, the pathogens of infective endocarditis are isolated from patients with infective endocarditis and identified as Bergeyella cardium (see Pan H, Li W, Sun E, et al. Characterization and whole genome sequencing of a novel strain of Bergeyella cardium related to infective endocarditis [J]. BMC Microbiology, 2020, 20.). The pathogens of infective endocarditis include Bergeyella.

[0024] Based on this, the present invention also provides an application of amiloride in the preparation of a drug for inhibiting cell vacuolization death.

[0025] In the present invention, all the above-mentioned drugs involve the active ingredient amiloride and pharmaceutically acceptable excipients. The mass of the active ingredient accounts for 1-99% of the drug, and the concentration of amiloride is preferably 0.2-1 μM. The excipients in the present invention include one or more of antioxidants, excipients, stabilizers, and solvents, such as starch, lactose, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, microcrystalline silica, gum arabic, gelatin solution, sodium carboxymethyl cellulose, sodium dodecyl sulfate, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, magnesium stearate, sodium sulfite, ascorbic acid, talc, silica, etc. The dosage form of the drug is granule, pill, tablet, injection, capsule, or powder. The drug dosage of the present invention can be determined by factors such as the type of disease to be treated, the severity of the disease, the administration route, the age, gender, and health status of the patient. For example, the dosage of the drug of the present invention can be 0.01 μg-1000 mg / day per patient.

[0026] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they cannot be understood as limiting the protection scope of the present invention.

[0027] In the following examples, the pathogens of endocarditis involved in the examples are Bergeyella cardium HPQL.

[0028] Example 1

[0029] 1. Effect of hydrochloric amiloride on BMDM cells infected with pathogens of endocarditis

[0030] (1) Preparation of primary mouse BMDM cells: Adult C57 / BL mice at 4 - 6 weeks old were euthanized. The femurs and tibias were isolated under sterile conditions and placed in PBS buffer. The muscles, cartilage, and epiphyses on the bone surface were scraped off with a scalpel. After cutting both ends of the bone to expose the bone marrow cavity, 1 mL syringe was used to aspirate DMEM - F12 medium to rinse the bone marrow cavity, and the cells were collected. After the cells were evenly pipetted, they were counted and the cell density was adjusted to 1 - 2 Million / mL. 20 ng / mL of M - CSF growth factor was added to the DMEM - F12 complete medium, and the cells were seeded in a cell culture plate and cultured in a CO2 incubator at 5% CO2 and 37 °C. After static culture for 5 days, the adherent cells were harvested for corresponding experiments.

[0031] (2) The endocarditis pathogen was activated on Columbia blood agar plate at 37 °C and 5% CO2 until typical colonies of bacteria grew.

[0032] (3) Grouping: Control group, endocarditis pathogen infection group, and amiloride hydrochloride test group. In the control group, the adherent BMDM cells were only treated with DMEM - F12 + FBS. In the endocarditis pathogen infection group, the adherent BMDM cells were first treated with DMEM - F12 + FBS for 6 hours, and then infected with 400 MOI of endocarditis pathogen for 24 hours. In the amiloride hydrochloride test group, the adherent BMDM cells were treated with amiloride hydrochloride at a working concentration of 0.5 μM for 6 hours, and then infected with 400 MOI of endocarditis pathogen for 24 hours. Among them, the 0.5 μM amiloride hydrochloride was prepared by dissolving it in water to a storage solution with a concentration of 5 mM, and then diluted to a working concentration of 0.5 μM with DMEM - F12 complete medium.

[0033] The BMDM cells in the control group, endocarditis pathogen infection group, and amiloride hydrochloride test group were photographed (the results are shown in Figure 1 a), and at the same time, the supernatants of the cultured cells in each group were collected for the detection of lactate dehydrogenase (LDH) (the results are shown in Figure 1 b).

[0034] As shown by Figure 1 , compared with the cells in the control group, the cells in the endocarditis pathogen infection group showed obvious vacuolization, while the number of vacuoles in the amiloride hydrochloride test group was significantly reduced (see Figure 1 a). Compared with the cells in the control group, the release of LDH in the endocarditis pathogen infection group was significantly increased, while the release of LDH in the amiloride hydrochloride test group was significantly reduced (see Figure 1In b) thereof. Therefore, in the present invention, mouse BMDM cells are treated with amiloride hydrochloride, which can significantly inhibit the vacuolization state of the cells and reduce cell death.

[0035] 2. Effect of amiloride hydrochloride on endocarditis pathogens

[0036] Use 4×10 8 CFU dose of endocarditis pathogens to infect mice treated with amiloride hydrochloride by nasal drip. After 24 hours, take the lung tissue of the mice to count the bacterial load in the lungs and observe the bacterial load in the lungs of the mice.

[0037] The experiment is divided into two groups: (1) Endocarditis pathogen group, that is, infect mice with 4×10 8 CFU dose of endocarditis pathogens by nasal drip for 24 hours; (2) Amiloride hydrochloride group, that is, infect mice with 4×10 8 CFU dose of endocarditis pathogens by nasal drip and simultaneously inject 100 μL of amiloride hydrochloride 100 mg / mL via the tail vein. After 12 hours, inject 100 μL of amiloride hydrochloride 100 mg / mL via the tail vein again.

[0038] Figure 2 The results show that the bacterial load in the lung tissue of the mice in the amiloride hydrochloride group is significantly lower than that in the endocarditis pathogen group. Therefore, amiloride hydrochloride can significantly inhibit the infection of endocarditis pathogens.

[0039] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of amiloride in the preparation of a drug for inhibiting pathogens of infective endocarditis, characterized in that, The pathogen of infective endocarditis is Berger's bacterium.

2. The application according to claim 1, wherein The drug includes an active ingredient amiloride and a pharmaceutically acceptable excipient.

3. The application according to claim 1, wherein The concentration of amiloride is 0.2 to 1 μM.

4. The application according to claim 2, wherein the dosage form of the drug is a granule, a pill, a tablet, an injection, a capsule or a powder.

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