Use of endoperoxide alone or in combination with respiratory chain inhibitor in preparing Anti-bacterial drug and Anti-bacterial drug and use thereof in treating tuberculosis

Abstract

Provided is use of an endoperoxide in preparing an anti-microbial drug. In an anoxic condition, the endoperoxide can partially and effectively inhibit the survival of Mycobacterium tuberculosis. Provided is use of an endoperoxide in combination with a respiratory chain inhibitor in preparing an anti-microbial drug. The endoperoxide includes artemisinin and derivatives thereof, and other endoperoxides with a two-oxygen bridge structure. The endoperoxide has synergistic effects with a variety of respiratory chain inhibitors, and the combination has a more effective inhibition effect on Mycobacterium tuberculosis.

Description

Use of endoperoxyl compounds alone or in combination with respiratory chain inhibitors in the preparation of antibacterial drugs, antibacterial drugs, and use in the treatment of tuberculosis Technical Field

[0001] The present application relates to the use of an endoperoxide compound in the preparation of an antimicrobial pathogen drug, and belongs to the field of biochemical technology. Background Art

[0002] Tuberculosis (TB), one of the three major infectious diseases recognized by the World Health Organization (WHO), is the leading infectious cause of death among adults worldwide, accounting for approximately 1.5 million deaths in 2020, the majority in low- and middle-income countries. Mycobacterium tuberculosis, the causative agent of TB, is a slow-growing, microaerophilic bacillus. Currently, commonly used anti-TB drugs consist of three main categories: antibiotics, synthetic drugs, and traditional Chinese medicines. Over 20 Western medicines are designated as essential treatments in China, with first-line treatments primarily consisting of isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide, and rifamycins. In recent years, fluoroquinolones have also been used in TB treatment, playing an important role in combination therapy. However, rapid diagnosis and timely implementation of effective treatment options, including effective management of adverse reactions to second-line drugs, remain challenging for both rifampicin-resistant TB and multidrug-resistant TB. This has led to continued epidemic growth, low cure rates, and the spread of drug-resistant TB. Patients with highly drug-resistant TB experience poorer treatment outcomes, including high mortality, particularly among those co-infected with HIV, even when they are receiving antiretroviral drugs. Therefore, there is an urgent need for newer, shorter, and more effective (non-injectable) treatments.

[0003] Artemisinin and its derivatives are effective antimalarial drugs, boasting strong species specificity, rapid drug action, and minimal toxic side effects. The dioxygen bridge in the artemisinin structure is a key pharmacophore. The opening or breaking of this dioxygen bridge, or activation of artemisinin, is a prerequisite for its efficacy. Replacing this dioxygen bridge with a single oxygen bridge results in deoxyartemisinin, which loses its antimalarial activity. In addition to antimalarial properties, artemisinin-based drugs also have antiparasitic and antiviral properties, and are effective in treating diabetes and the autoimmune disease lupus erythematosus.

[0004] Current tuberculosis treatments have low cure rates, significant side effects, long treatment cycles, and a growing problem of drug resistance. Treatment requires large oral doses of tablets and long-term daily injections, placing a significant financial burden and physical pain on patients. While there are reports of artemisinin's efficacy against tuberculosis, its mechanism of action remains unclear, requiring extremely high concentrations and achieving suboptimal results, making it clinically ineffective.

[0005] Summary of the Invention

[0006] The first object of the present application is to provide a use of an endoperoxide compound in the preparation of an antibacterial drug, so as to overcome the problems of drug resistance and severe drug side effects in the prior art of tuberculosis treatment.

[0007] To achieve the aforementioned invention objectives, the technical solutions adopted in this application include:

[0008] The invention relates to the use of an endoperoxyl compound in the preparation of an antibacterial drug, wherein the endoperoxyl compound comprises the following structural units: 1,2-dioxane, 1,2,4-trioxane, 1,2,4,5-tetraoxane, 1,2-dioxolane or 1,2,4-trioxolane; the antibacterial drug is a drug for inhibiting Mycobacterium tuberculosis; and under hypoxic conditions, the administration concentration of the endoperoxyl compound is: the administration concentration is: ≤100 μM.

[0009] In this application, a sealed anoxic culture tube filled with Middlebrook 7H9 broth was used as a culture model for Mycobacterium tuberculosis. Mycobacterium tuberculosis was inoculated into the culture tube to an OD600 of 0.01 and then cultured until anoxic conditions were reached. Methylene blue was used as an indicator. The dosing concentration of the endoperoxide compound was calculated from the in vitro dosing concentration. When the dosing concentration was ≤100 μM, the endoperoxide compound had a strong inhibitory effect on Mycobacterium tuberculosis. Low dosing concentrations can help reduce drug side effects.

[0010] Further, endoperoxy compounds include artemisinin, dihydroartemisinin, artemether, arteether, artesunate, HEM, ascaridol, majapolene A, Norterpene Peroxides, yinzaosu A, artefenomel (OZ439), aceranol acetate, and deoxycholic acid derivatives containing internal oxygen bridges.

[0011] Artemisinin and its derivatives are effective antimalarial drugs, boasting strong species specificity, rapid drug action, and minimal toxic side effects. The dioxygen bridge in the artemisinin structure is a key pharmacophore. The opening or cleavage of the dioxygen bridge, i.e., the activation of artemisinin, is a prerequisite for its efficacy. Deoxyartemisinin, obtained by replacing the dioxygen bridge with a single oxygen bridge, loses its antimalarial activity. In addition to antimalarial properties, artemisinin-based drugs also have antiparasitic and antiviral properties, and are also effective in treating diabetes and the autoimmune disease lupus erythematosus. Artefenomel (OZ439) is an orally active, synthetic antimalarial compound containing the artemisinin pharmacophore, with a mechanism of action similar to that of artemisinin. HEM, an extract from the Chinese medicinal herb Senecio radiata, is a mixture of various eremophilanolid sesquiterpenes. Ascaridol, an extract from the Chinese medicinal herb Sandao Nian, is also available. Majapolene A, an extract from the algae Concavocapsula, is also available. Yingzhaosu A is a sesquiterpene derivative with a peroxide group isolated from the root of the herb Yingzhaosu. Aceranol acetate is an extract from the traditional Chinese medicine Acer truncatum.

[0012] Furthermore, the administration concentration of the endoperoxide compound is: 12.5 μM≤administration concentration≤50 μM.

[0013] Further reducing the dosage concentration, experiments have confirmed that the dosage concentration of endoperoxide in the range of 12.5μM-50μM has a good inhibitory effect on Mycobacterium tuberculosis, which is beneficial to further reduce the side effects of the drug.

[0014] The second object of the present application is to provide a method for preparing an antibacterial drug by combining an endoperoxy compound with a respiratory chain inhibitor, so as to overcome the problems of drug resistance and severe drug side effects in the prior art of tuberculosis treatment.

[0015] To achieve the aforementioned invention objectives, the technical solutions adopted in this application include:

[0016] The endoperoxygen compound includes the following structural units: 1,2-dioxane, 1,2,4-trioxane, 1,2,4,5-tetraoxane, -1,2-dioxolane or 1,2,4-trimethoxolane; the antibacterial drug is a drug that inhibits Mycobacterium tuberculosis; and the respiratory chain inhibitor is a Mycobacterium tuberculosis respiratory chain inhibitor.

[0017] In the present application, a sealed culture tube filled with Middlebrook 7H9 broth medium is used as a culture model for Mycobacterium tuberculosis. In the experiment of the culture model, the combination of endoperoxides and respiratory chain inhibitors can enhance the inhibitory effect of the respiratory chain inhibitors on Mycobacterium tuberculosis.

[0018] Further, endoperoxy compounds include artemisinin, dihydroartemisinin, artemether, arteether, artesunate, HEM, ascaridol, majapolene A, Norterpene Peroxides, yinzaosu A, artefenomel (OZ439), aceranol acetate, and deoxycholic acid derivatives containing internal oxygen bridges.

[0019] Furthermore, the Mycobacterium tuberculosis respiratory chain inhibitor is one or more of bedaquiline, Q203, Aurachin D or lansoprazole.

[0020] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, Aurachin D or lansoprazole; under normal oxygen conditions, the administration concentration of the artemisinin is: administration concentration ≤100 μM; the administration concentration of bedaquiline is: administration concentration ≤4 μM; the administration concentration of Q203 is: administration concentration <10 nM; the administration concentration of Aurachin D is: administration concentration ≤20 μM; the administration concentration of lansoprazole is: administration concentration ≤20u M.

[0021] Thus, under normal oxygen conditions, the combination of artemisinin with bedaquiline, Q203, lansoprazole, or aurachin D reduced the survival rate of mycobacteria compared to the corresponding respiratory chain inhibitor alone. This suggests that under normal circumstances, the combination of artemisinin and respiratory chain inhibitors can enhance the inhibitory effect of respiratory chain inhibitors against Mycobacterium tuberculosis. Under the aforementioned dosing concentration conditions, the combined use of artemisinin with bedaquiline, Q203, lansoprazole, or aurachin D results in a low total dose, which helps reduce drug side effects.

[0022] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, AurachinD or lansoprazole; under normal oxygen conditions, the administration concentration of artemisinin is: administration concentration ≤100 μM; the administration concentration of Q203 is: administration concentration ≤1 nM.

[0023] In this way, the dosage concentration of Q203 is further reduced. Experiments have confirmed that the dosage concentration of Q203 is no higher than 1nM and combined with artemisinin, which has a good inhibitory effect on Mycobacterium tuberculosis, which is conducive to further reducing the dosage and thus reducing drug side effects.

[0024] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, Aurachin D or lansoprazole; under hypoxic conditions, the administration concentration of artemisinin is: administration concentration ≤50 μM; the administration concentration of bedaquiline is: administration concentration <10 μM; the administration concentration of Q203 is: administration concentration <5 nM; the administration concentration of Aurachin D is: administration concentration <20 μM; the administration concentration of lansoprazole is: administration concentration <20 μM.

[0025] Thus, under hypoxic conditions, the combination of artemisinin with bedaquiline, Q203, lansoprazole, or aurachin D reduced the survival rate of mycobacteria compared to the corresponding respiratory chain inhibitor alone. This suggests that the combination of artemisinin and respiratory chain inhibitors can enhance the inhibitory effect of respiratory chain inhibitors against Mycobacterium tuberculosis under hypoxic conditions. Under the aforementioned dosing concentration conditions, the combined use of artemisinin with bedaquiline, Q203, lansoprazole, or aurachin D resulted in a low total dose, which helps reduce drug side effects.

[0026] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, Aurachin D or lansoprazole; under hypoxic conditions, the administration concentration of artemisinin is: administration concentration ≤50 μM; the administration concentration of bedaquiline is: administration concentration ≤2 μM; the administration concentration of Q203 is: administration concentration ≤0.5 nM.

[0027] In this way, the dosage concentrations of bedaquiline and Q203 are further reduced. Experiments have confirmed that when the dosage concentration of bedaquiline is not higher than 2 μM, it has a good inhibitory effect on Mycobacterium tuberculosis when combined with artemisinin. When the dosage concentration of Q203 is not higher than 0.5 nM, it has a good inhibitory effect on Mycobacterium tuberculosis when combined with artemisinin. This is conducive to further reducing the dosage and thus reducing drug side effects.

[0028] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is two of bedaquiline, Q203, AurachinD, or lansoprazole; under normal oxygen conditions, the administration concentrations of artemisinin and the respiratory chain inhibitor are:

[0029] The dosing concentration of artemisinin is ≤50 μM, the dosing concentration of Aurachin D is: dosing concentration <10 μM, and the dosing concentration of Q203 is: dosing concentration <5 nM;

[0030] or,

[0031] The administration concentration of artemisinin is ≤50 μM, the administration concentration of Aurachin D is: administration concentration <10 μM, and the administration concentration of lansoprazole is: administration concentration <10 μM.

[0032] These experimental results demonstrate that, under normal conditions, the combination of artemisinin with Q203 and aurachin D, or the combination of artemisinin with lansoprazole and aurachin D, can reduce the survival rate of mycobacteria compared to the use of either respiratory chain inhibitor alone. This suggests that, under normal circumstances, the combination of artemisinin with multiple respiratory chain inhibitors can enhance the inhibitory effect of these inhibitors against Mycobacterium tuberculosis. Under the aforementioned dosing concentration conditions, the combined use of artemisinin with two respiratory chain inhibitors results in a low total dose, which helps reduce drug side effects.

[0033] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is two of bedaquiline, Q203, AurachinD, or lansoprazole; under normal oxygen conditions, the administration concentrations of artemisinin and the respiratory chain inhibitor are:

[0034] The dosing concentration of artemisinin is ≤25 μM, the dosing concentration of Aurachin D is: dosing concentration ≤2.5 μM, and the dosing concentration of Q203 is: dosing concentration ≤4 nM;

[0035] or,

[0036] The administration concentration of artemisinin is ≤25 μM, the administration concentration of Aurachin D is: administration concentration ≤2.5 μM, and the administration concentration of lansoprazole is: administration concentration ≤2 μM.

[0037] In this way, the dosage concentrations of Q203, AurachinD and lansoprazole were further reduced. Experiments have confirmed that under normal conditions, when the dosage concentration of Aurachin D is no higher than 2.5μM and the dosage concentration of Q203 is no higher than 4nM, they can be combined with artemisinin to have a good inhibitory effect on Mycobacterium tuberculosis; when the dosage concentration of AurachinD is no higher than 2.5μM and the dosage concentration of lansoprazole is no higher than 2μM, they can be combined with artemisinin to have a good inhibitory effect on Mycobacterium tuberculosis, which is conducive to further reducing the dosage and thus reducing drug side effects.

[0038] The third object of the present application is to provide a drug for treating tuberculosis, comprising an effective amount of an endoperoxy compound and a Mycobacterium tuberculosis respiratory chain inhibitor and a pharmaceutically acceptable carrier; the Mycobacterium tuberculosis respiratory chain inhibitor is one or more of bedaquiline, Q203, Aurachin D or lansoprazole; the endoperoxy compound includes artemisinin, dihydroartemisinin, artemether, arteether, artesunate, HEM, ascaridol, majapolene A, Norterpene Peroxides, yinzaosu A, artefenomel (OZ439), aceranol acetate, and a deoxycholic acid derivative containing an internal oxygen bridge.

[0039] The fourth object of this application is to provide an antibacterial drug for the treatment of tuberculosis.

[0040] This application aims to introduce endoperoxide compounds, represented by artemisinin, as drugs to more quickly or effectively kill pathogens, particularly Mycobacterium tuberculosis. This has potential applications in the treatment of tuberculosis, improving cure rates or shortening treatment courses, overcoming drug resistance, and reducing drug dosage and side effects on patients. Compared with existing technologies, this application can achieve the following technical benefits:

[0041] 1) Different mechanisms of action help overcome drug resistance;

[0042] 2) It can reduce the dosage of drugs and reduce the side effects of drugs;

[0043] 3) Compared with existing technologies, it is safer and has fewer toxic and side effects;

[0044] 4) It helps shorten the course of treatment, reduce treatment costs, and alleviate the burden on patients and the medical system. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 is a schematic diagram of the combined action of artemisinin and a respiratory chain inhibitor to inhibit Mycobacterium tuberculosis in an embodiment of the present application.

[0046] FIG2 is an analysis of the inhibition of Mycobacterium tuberculosis by using artemisinin and bedaquiline alone under normal and hypoxic conditions in Example 1, Control Example 1 and Supporting Example 1 of the present application.

[0047] FIG3 is an analysis of the inhibition of Mycobacterium tuberculosis by using artemisinin in combination with bedaquiline, Q203, Aurachin D or lansoprazole under normal conditions in Example 2 of the present application.

[0048] Figure 4 is an analysis of the inhibition of Mycobacterium tuberculosis by using different concentrations of artemisinin in combination with fixed concentrations of Q203 and bedaquiline under normal and hypoxic conditions in Example 3 of the present application.

[0049] FIG5 is an analysis of the inhibition of Mycobacterium tuberculosis by using artemisinin and Aurachin D in combination with Q203 or lansoprazole, respectively, under normal conditions in the examples of the present application.

[0050] In the figure, normal and aerobic refer to normal oxygen conditions, ART-Artemisinin-Artemisinin, Bedaquiline-Bedaquiline, Lansoprazole-Lansoprazole. DETAILED DESCRIPTION

[0051] The present application is further described below with reference to the embodiments, but is not limited thereto.

[0052] Unless otherwise specified, the materials and reagents used in the following examples are commercially available. In this application, "normal conditions" refers to normal oxygen conditions; "anoxic conditions" refers to the color change of the methylene blue indicator in a sealed anoxic culture tube, typically indicating an oxygen content below 5%.

[0053] Terminology Notes:

[0054] HEM: Senecio oleifera extract, a blend of extracts from various eremophilanolid sesquiterpenes.

[0055] Ascaridol: Ascaris chinensis extract, CAS: 512-85-6

[0056] majapolene A: majapolene A, NSC-692207, CHEMBL450193, [4-(4-bromo-3,3-dimethylcyclohexyl)-2,3-dioxabicyclo[2.2.2]oct-5-en-1-yl]methanol

[0057] YingzhaosuA: Yingzhaosu A, also known as Yingzhaosu A; CAS: 73301-54-9

[0058] Artefenomel(OZ439):CAS:1029939-86-3

[0059] aceranol acetate:CAS:1221260-33-8

[0060] OD600: OD600 refers to the absorbance of a solution at a wavelength of 600nm. The absorbance is proportional to the concentration of the absorbing substance in the solution and can be used to characterize bacterial cell density.

[0061] OADC: (=oleic acid, albumin, dextrose and catalase), a mixture of oleic acid, albumin, dextrose and catalase;

[0062] Middlebrook 7H9 broth medium: purchased from Beijing Coolbo Technology Co., Ltd., product number: MKC301; How to prepare: prepare according to the product instructions.

[0063] Q203: CAS No. 1334719-95-7;

[0064] Aurachin D: CAS No. 108354-13-8.

[0065] Example 1: This example illustrates the effect of artemisinin against Mycobacterium tuberculosis through in vitro experiments, comprising the following steps:

[0066] S1: dissolving artemisinin powder in dimethyl sulfoxide to prepare artemisinin solution;

[0067] S2: Inoculate Mycobacterium tuberculosis to an OD600 of 0.01 in a sealed anoxic culture tube containing Middlebrook 7H9 broth and continue incubating until anoxic conditions are reached. Methylene blue, an indicator, changes color when bacterial growth depletes oxygen in the culture tube, indicating the presence of anoxic conditions.

[0068] S3: First, prepare a 200 mM dimethyl sulfoxide stock solution of artemisinin. When using, add different amounts of artemisinin stock solution to the experimental group as needed, and then dilute with dimethyl sulfoxide to make the concentrations of artemisinin in the culture medium 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, respectively. Continue incubating at 37°C for 6 days. Only dimethyl sulfoxide solvent is added to the control group.

[0069] Comparative Example 1:

[0070] The implementation method is basically the same as that of Example 1, except that: (1) the test is carried out under normal (aerobic) conditions;

[0071] (2) In step S2, the density of Mycobacterium tuberculosis is OD600=0.003.

[0072] Supporting example 1:

[0073] The effectiveness of the experimental process is demonstrated by Supporting Example 1. At the same time, bedaquiline, a clinically used anti-tuberculosis drug, is used to treat Mycobacterium tuberculosis in the same manner as in Example 1 (the concentrations of drug treatment are 0.25 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM, respectively). It can be observed that there is a significant inhibitory effect under both normal conditions and hypoxic conditions.

[0074] From Example 1, Control Example 1, Supporting Example 1, and Figure 2, it can be seen that under normal conditions, when different concentrations of artemisinin were incubated with the same number of Mycobacterium tuberculosis (OD600 = 0.003), after culturing at 37°C for 6 days, the survival rate of Mycobacterium tuberculosis was not significantly different from that of the control group; under hypoxic conditions, when different concentrations of artemisinin were incubated with the same number of Mycobacterium tuberculosis (OD600 = 0.01), after culturing at 37°C for 6 days, the survival rate of Mycobacterium tuberculosis was significantly reduced compared with the control group, and 25 μM artemisinin was able to significantly inhibit the survival rate of Mycobacterium tuberculosis.

[0075] Example 2:

[0076] The implementation method is basically the same as that of Example 1, except that: (1) the experiment is carried out under normal (aerobic) conditions; (2) the combination and concentration of the treatment drugs are different. The specific drug combination and concentration are shown in Table 1.

[0077] Table 1 Drug concentrations of each group tested in Example 2

[0078] The experimental results are shown in Figure 3. These results demonstrate that, under normal conditions, artemisinin combined with bedaquiline, Q203, lansoprazole, or aurachin D reduced the survival rate of mycobacteria compared to the corresponding respiratory chain inhibitor alone. This suggests that, under normal conditions, the combination of artemisinin and respiratory chain inhibitors can enhance the inhibitory effect of respiratory chain inhibitors against Mycobacterium tuberculosis. Artemisinin was administered at concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0079] Specifically, as shown in Figure 3, combined with the experimental data analysis is as follows:

[0080] In Experiments 1-18, under normal conditions, when bedaquiline was used alone, the survival rate of Mycobacterium tuberculosis gradually decreased with increasing bedaquiline concentrations (0.03125 μM, 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM). When artemisinin was used in combination with bedaquiline, the addition of artemisinin significantly enhanced the inhibitory effect of bedaquiline on Mycobacterium tuberculosis at every concentration tested.

[0081] In Experiments 19-36, under normal conditions, when Q203 was used alone, the survival rate of Mycobacterium tuberculosis gradually decreased with increasing Q203 concentrations (0.03 nM, 0.0625 nM, 0.125 nM, 0.25 nM, 0.5 nM, and 1 nM). When artemisinin was used in combination with Q203, the addition of artemisinin significantly enhanced the inhibitory effect of Q203 against Mycobacterium tuberculosis at each concentration tested. Additionally, experiments were conducted at Q203 concentrations of 2 nM, 4 nM, and 8 nM (not shown in the chart). The results demonstrated that under normal conditions, the addition of artemisinin significantly enhanced the inhibitory effect of Q203 against Mycobacterium tuberculosis, with survival rates of Mycobacterium tuberculosis remaining below 10%.

[0082] In Trials 37-54, under normal conditions, when lansoprazole was used alone, the survival rate of Mycobacterium tuberculosis generally decreased with increasing lansoprazole concentrations (0.625μM, 1.25μM, 2.5μM, 5μM, 10μM, and 20μM). When artemisinin was used in combination with lansoprazole, the addition of artemisinin significantly enhanced the inhibitory effect of bedaquiline against Mycobacterium tuberculosis at every concentration tested.

[0083] In Experiments 55-72, under normal conditions, when Aurachin D was used alone, the survival rate of Mycobacterium tuberculosis did not change significantly compared to the control group as the Aurachin D concentration increased (0.625μM, 1.25μM, 2.5μM, 5μM, 10μM, and 20μM). When artemisinin was used in combination with the respiratory chain inhibitor Aurachin D, at artemisinin concentrations of 0-50μM and Aurachin D concentrations of 0.625-5μM, artemisinin did not enhance the inhibitory effect of Aurachin D on Mycobacterium tuberculosis. However, when Aurachin D concentrations were 10-20μM and artemisinin concentrations were 100-200μM, the addition of artemisinin significantly enhanced the inhibitory effect of Aurachin D on Mycobacterium tuberculosis.

[0084] Example 3:

[0085] The methods used in Example 1 and Control Example 1 were essentially the same, except that artemisinin and Q203, or artemisinin and bedaquiline, were used to treat Mycobacterium tuberculosis under normal and hypoxic conditions, respectively. The drug combinations and concentrations are shown in Table 2. (Under normal conditions, the Mycobacterium tuberculosis concentration was OD600 = 0.003; under hypoxic conditions, the Mycobacterium tuberculosis concentration was OD600 = 0.01.)

[0086] Table 2 Drug concentrations of each group tested in Example 3

[0087] The test results are shown in Figure 4. The test results show that under normal conditions, the combination of artemisinin and bedaquiline / Q203 can reduce the survival rate of Mycobacterium tuberculosis compared with the use of artemisinin alone or bedaquiline / Q203 alone.

[0088] Under normal conditions, when artemisinin is used alone, the survival rate of Mycobacterium tuberculosis decreases with the increase of artemisinin concentration (12.5μM, 25μM, 50μM, 100μM). When the artemisinin concentration is 100μM, the survival rate of Mycobacterium tuberculosis is 88.62%.

[0089] Under normal conditions, the survival rate of Mycobacterium tuberculosis was 60.22% when treated alone with 4 μM bedaquiline. When artemisinin was used in combination with 4 μM bedaquiline, the survival rate decreased with increasing artemisinin concentrations. At a 50 μM artemisinin concentration, the survival rate dropped to 14.65%. When the artemisinin concentration was increased to 100 μM, the survival rate further decreased to 7.08%. This suggests that under normal conditions, the combined use of artemisinin and bedaquiline can enhance the inhibitory effect of both artemisinin and bedaquiline against Mycobacterium tuberculosis.

[0090] Under normal conditions, the survival rate of Mycobacterium tuberculosis was 51.03% when treated with 1 nM Q203 alone. When artemisinin was combined with 1 nM Q203, the survival rate decreased with increasing artemisinin concentrations. At a concentration of 50 μM, the survival rate dropped to 7.42%. Further increases in the artemisinin concentration to 100 μM had no significant effect on the survival rate. This suggests that under normal conditions, the combined use of artemisinin and Q203 can enhance the inhibitory effects of both artemisinin and Q203 against Mycobacterium tuberculosis.

[0091] Under hypoxic conditions, a lower dosage concentration is required to achieve a better inhibitory effect on Mycobacterium tuberculosis than under normal conditions. Specifically, when artemisinin is used alone, the survival rate of Mycobacterium tuberculosis decreases with increasing artemisinin dosage concentrations (6.25μM, 12.5μM, 25μM, 50μM). When the artemisinin dosage concentration is 25μM, the survival rate of Mycobacterium tuberculosis is 49.48%. When the artemisinin dosage concentration is increased to 50μM, the survival rate further decreases to 37.69%. It can be seen that artemisinin can effectively inhibit Mycobacterium tuberculosis under hypoxic conditions.

[0092] Under hypoxic conditions, the survival rate of Mycobacterium tuberculosis was 56.76% when treated with 2μM bedaquiline alone. When artemisinin was combined with 2μM bedaquiline, the survival rate decreased with increasing artemisinin concentrations. At a 25μM artemisinin concentration, the survival rate dropped to 16.09%. When the artemisinin concentration was increased to 50μM, the survival rate further decreased to 5.97%. This suggests that the combined use of artemisinin and bedaquiline under hypoxic conditions can enhance the inhibitory effect of artemisinin and bedaquiline against Mycobacterium tuberculosis.

[0093] Under hypoxic conditions, the survival rate of Mycobacterium tuberculosis was 52.64% when treated with 0.5nM Q203 alone. When artemisinin was combined with 0.5nM Q203, the survival rate decreased with increasing artemisinin concentrations. At a 25μM artemisinin concentration, the survival rate dropped to 12.09%. When the artemisinin concentration was increased to 50μM, the survival rate further decreased to 5.19%. This suggests that the combined use of artemisinin and Q203 under hypoxic conditions can enhance the inhibitory effects of both artemisinin and Q203 against Mycobacterium tuberculosis.

[0094] In addition, experiments were conducted using bedaquiline at concentrations of 4 μM, 6 μM, and 8 μM (not shown in the chart), and artemisinin at a concentration of 50 μM. The results demonstrated that under hypoxic conditions, the addition of artemisinin significantly enhanced the inhibitory effect of bedaquiline on Mycobacterium tuberculosis, with the survival rate of Mycobacterium tuberculosis remaining below 10%.

[0095] In addition, experiments were conducted using Q203 at concentrations of 1 nM, 2 nM, and 4 nM (not shown in the chart), along with artemisinin at a concentration of 50 μM. The results demonstrated that under hypoxic conditions, the addition of artemisinin significantly enhanced the inhibitory effect of Q203 on Mycobacterium tuberculosis, with survival rates remaining below 10%.

[0096] Example 4:

[0097] The implementation method is basically the same as that of Example 1, except that: (1) the experiment is carried out under normal (aerobic) conditions; (2) the combination and concentration of the treatment drugs are different. The specific drug combination and concentration are shown in Table 3.

[0098] Table 3 Drug concentrations of each group tested in Example 4

[0099] The experimental results are shown in Figure 5. These results demonstrate that, under normal conditions, the combination of artemisinin with Q203 and Aurachin D, or the combination of artemisinin with lansoprazole and Aurachin D, can reduce the survival rate of mycobacteria compared to the use of either respiratory chain inhibitor alone. This suggests that, under normal circumstances, the combination of artemisinin with multiple respiratory chain inhibitors can enhance the inhibitory effect of respiratory chain inhibitors against Mycobacterium tuberculosis.

[0100] In addition, experiments were conducted using Q203 at concentrations of 1 nM, 2 nM, and 4 nM, Aurachin D at concentrations of 5 μM and 6.25 μM, and artemisinin at a concentration of 50 μM (not shown in the chart). The results showed that under normal conditions, the combination of artemisinin, Q203, and Aurachin D reduced the survival rate of mycobacteria by less than 10%, compared to the use of the two corresponding respiratory chain inhibitors alone.

[0101] In addition, experiments were conducted using lansoprazole at concentrations of 4 μM, 6 μM, and 8 μM, Aurachin D at concentrations of 5 μM and 6.25 μM, and artemisinin at a concentration of 50 μM (not shown in the chart). The results showed that under normal conditions, the combination of artemisinin, lansoprazole, and Aurachin D reduced the survival rate of mycobacteria by less than 10%, compared to the use of the two corresponding respiratory chain inhibitors alone.

[0102] As shown in Figure 1, the possible mechanism by which the drugs involved in this article inhibit Mycobacterium tuberculosis is that Q203 and lansoprazole inhibit Mycobacterium tuberculosis cytochrome oxidase bcc-aa3, Aurachin D inhibits cytochrome oxidase bd, bedaquiline inhibits ATP synthase, and artemisinin is activated by electrons generated by the respiratory chain to destroy the cell membrane potential. When the downstream electron transport chain is inhibited by inhibitors, the respiratory chain will transfer more electrons to activate artemisinin, thereby enhancing the cell-killing effect of artemisinin.

[0103] Although the present application has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions, and / or additions may be made and that substantial equivalents may be substituted for elements of the described embodiments without departing from the spirit and scope of the present application. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present application without departing from the scope of the present application. Therefore, it is not intended that the present application be limited to the particular embodiments disclosed for carrying out the present application, but rather that the present application will encompass all embodiments falling within the scope of the appended claims.

Claims

1. Use of endoperoxides in the preparation of antibacterial drugs, Characterized in that: The endoperoxides include the following structural units: 1,2-dioxane, 1,2,4-trioxane, 1,2,4,5-tetraoxane, 1,2-dioxolane or 1,2,4-trioxolane; the antibacterial drug is a drug that inhibits Mycobacterium tuberculosis; under hypoxic conditions, the administration concentration of the endoperoxide is: administration concentration ≤ 100 μM.

2. Use of the endoperoxide according to claim 1 in the preparation of antibacterial drugs, Characterized in that: The endoperoxides include artemisinin, dihydroartemisinin, artemether, arteether, artesunate, HEM, ascaridol, majapolene A, Norterpene Peroxides, yinzaosu A, artefenomel (OZ439), aceranol acetate, deoxycholic acid derivatives containing an endo-oxygen bridge.

3. Use of the endoperoxide according to claim 2 in the preparation of antibacterial drugs, Characterized in that: The administration concentration of the endoperoxide is: 12.5 μM ≤ administration concentration ≤ 50 μM.

4. Use of the combination of endoperoxides and respiratory chain inhibitors in the preparation of antibacterial drugs, Characterized in that: The endoperoxides include the following structural units: 1,2-dioxane, 1,2,4-trioxane, 1,2,4,5-tetraoxane, -1,2-dioxolane or 1,2,4-trimethoxolane; the antibacterial drug is a drug that inhibits Mycobacterium tuberculosis; the respiratory chain inhibitor is a Mycobacterium tuberculosis respiratory chain inhibitor.

5. Use of the combination of the endoperoxide and the respiratory chain inhibitor according to claim 4 in the preparation of antibacterial drugs, Characterized in that: The endoperoxides include artemisinin, dihydroartemisinin, artemether, arteether, artesunate, HEM, ascaridol, majapolene A, Norterpene Peroxides, yinzaosu A, artefenomel (OZ439), aceranol acetate, deoxycholic acid derivatives containing an endo-oxygen bridge.

6. Use of the combination of the endoperoxide and the respiratory chain inhibitor according to claim 4 in the preparation of antibacterial drugs, Characterized in that, The Mycobacterium tuberculosis respiratory chain inhibitor is one or more of bedaquiline, Q203, Aurachin D or lansoprazole.

7. Use of the combination of the endoperoxide and the respiratory chain inhibitor according to claim 6 in the preparation of antibacterial drugs, Characterized in that: The endoperoxide is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, Aurachin D or lansoprazole; under normal oxygen conditions, the administration concentration of artemisinin is: administration concentration ≤ 100 μM; the administration concentration of bedaquiline is: administration concentration ≤ 4 μM; the administration concentration of Q203 is: administration concentration < 10 nM; the administration concentration of Aurachin D is: administration concentration ≤ 20 μM; the administration concentration of lansoprazole is: administration concentration ≤ 20 μM.

8. Use of the combination of the endoperoxide and the respiratory chain inhibitor according to claim 7 in the preparation of an antibacterial drug, characterized in that: The endoperoxide is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, Aurachin D or lansoprazole; under normal oxygen conditions, the administration concentration of artemisinin is: administration concentration ≤ 100 μM; the administration concentration of Q203 is: administration concentration ≤ 1 nM.

9. Use of the combination of the endoperoxide and the respiratory chain inhibitor according to claim 6 in the preparation of an antibacterial drug, characterized in that: The endoperoxide is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, Aurachin D or lansoprazole; under hypoxic conditions, the administration concentration of artemisinin is: administration concentration ≤ 50 μM; the administration concentration of bedaquiline is: administration concentration < 10 μM; the administration concentration of Q203 is: administration concentration < 5 nM; the administration concentration of Aurachin D is: administration concentration < 20 μM; the administration concentration of lansoprazole is: administration concentration < 20 μM.

10. Use of the combination of the endoperoxide and the respiratory chain inhibitor according to claim 9 in the preparation of an antibacterial drug, characterized in that: The endoperoxide is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is one of bedaquiline, Q203, Aurachin D or lansoprazole; under hypoxic conditions, the administration concentration of artemisinin is: administration concentration ≤ 50 μM; the administration concentration of bedaquiline is: administration concentration ≤ 2 μM; the administration concentration of Q203 is: administration concentration ≤ 0.5 nM.

11. Use of the combination of the endoperoxide and the respiratory chain inhibitor according to claim 6 in the preparation of an antibacterial drug, characterized in that: The endoperoxide is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is two of bedaquiline, Q203, Aurachin D or lansoprazole; under normal oxygen conditions, the administration concentrations of artemisinin and the respiratory chain inhibitor are: the administration concentration of artemisinin ≤ 50 μM, and the administration concentration of Aurachin D is: administration concentration < 10 μM, and the administration concentration of Q203 is: administration concentration < 5 nM; or, The administration concentration of artemisinin ≤ 50 μM, and the administration concentration of Aurachin D is: the administration concentration < 10 μM, and the lansoprazole is: the administration concentration < 10 μM.

12. Use of the endoperoxide in combination with a respiratory chain inhibitor according to claim 11 in the preparation of an antibacterial drug, characterized in that: the endoperoxide is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is two of bedaquiline, Q203, Aurachin D or lansoprazole; under normal oxygen conditions, the administration concentrations of artemisinin and the respiratory chain inhibitor are: the administration concentration of artemisinin ≤ 25 μM, and the administration concentration of Aurachin D is: the administration concentration ≤ 2.5 μM, and the administration concentration of Q203 is: the administration concentration ≤ 4 nM; or, the administration concentration of artemisinin ≤ 25 μM, and the administration concentration of Aurachin D is: the administration concentration ≤ 2.5 μM, and the lansoprazole is: the administration concentration ≤ 2 μM.

13. A drug for treating tuberculosis, characterized in that: it contains an effective amount of an endoperoxide and a Mycobacterium tuberculosis respiratory chain inhibitor and a pharmaceutically acceptable carrier; the Mycobacterium tuberculosis respiratory chain inhibitor is one or more of bedaquiline, Q203, Aurachin D or lansoprazole; the endoperoxide includes artemisinin, dihydroartemisinin, artemether, arteether, artesunate, HEM, ascaridol, majapolene A, Norterpene Peroxides, yinzaosu A, artefenomel (OZ439), aceranol acetate, deoxycholic acid derivatives containing an endo-oxygen bridge.

14. Use of the antibacterial drug according to claim 10 in the treatment of tuberculosis.

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