Use of endoperoxy compounds alone or in combination with respiratory chain inhibitors in the preparation of antibacterial drugs, and antibacterial drugs

Abstract

The present application discloses a use of an endoperoxy compound in the preparation of an antimicrobial pathogenic bacteria drug. Under anaerobic conditions, in the culture medium of Mycobacterium tuberculosis, the representative artemisinin-based drug of the endoperoxy compound can partially and effectively inhibit the survival of Mycobacterium tuberculosis. The present application also discloses a use of an endoperoxy compound in combination with a respiratory chain inhibitor in the preparation of an antimicrobial pathogenic bacteria drug. Under normal and anaerobic conditions, in the culture medium of Mycobacterium tuberculosis, the endoperoxy compound and a variety of respiratory chain inhibitors have a synergistic effect, and the combined use has a more effective inhibitory effect on Mycobacterium tuberculosis. The endoperoxy compounds include artemisinin and its derivatives, as well as other endoperoxy compounds with a double oxygen bridge structure, which may have a similar mechanism of action, and the internal oxygen bridge is the key group for them to play a role. The present application aims to kill pathogens more quickly or effectively, improve the cure efficiency or shorten the course of treatment, overcome the problem of drug resistance, and reduce the amount of drugs to reduce side effects on patients by introducing endoperoxy compounds represented by artemisinin as drugs.

Description

Technical Field

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

[0002] Tuberculosis is one of the three major infectious diseases recognized by the World Health Organization (WHO) and is the leading infectious cause of death among adults worldwide. In 2020, about 1.5 million people died, most of whom were in low- and middle-income countries. Mycobacterium tuberculosis is the causative agent of tuberculosis. It is a type of slow-growing microaerophilic bacillus. At present, commonly used anti-tuberculosis drugs are composed of three major categories: antibiotics, synthetic drugs, and traditional Chinese medicine. Among them, more than 20 Western medicines are national basic treatment drugs. The first-line treatment drugs are mainly isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide, rifamycins, etc. In recent years, fluoroquinolones have also been used to treat tuberculosis and play an important role in combined treatment. Rifampicin-resistant tuberculosis and multidrug-resistant tuberculosis still cannot be quickly diagnosed and effective treatment plans cannot be adopted in time, including effective management of adverse reactions of second-line drugs. This leads to the continued spread of the epidemic, low cure rates, and the spread of drug-resistant tuberculosis. Patients with highly drug-resistant TB experience poorer treatment outcomes, including high mortality, especially in patients 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 good antimalarial drugs with the advantages of strong species specificity, rapid drug action, and few toxic side effects. The double oxygen bridge in the structure of artemisinin is the key pharmacophore. The opening or breaking of the double oxygen bridge, that is, the activation of artemisinin, is a prerequisite for its efficacy. Deoxyartemisinin obtained by replacing the double oxygen bridge with a single oxygen bridge will lose its antimalarial activity. In addition to antimalarial, artemisinin drugs also have the effects of killing other types of parasites and antivirals, and also have therapeutic effects on diabetes and the autoimmune disease lupus erythematosus.

[0004] Currently, the cure rate of tuberculosis treatment is low, the side effects of drugs are large, the treatment cycle is long, and the problem of drug resistance is becoming increasingly serious. During the treatment period, a large number of drug tablets need to be taken orally and long-term daily injections are required. Patients bear huge economic burdens and physical pain for a long time. Although the efficacy of artemisinin against tuberculosis bacteria has been reported, it has no clinical application significance because researchers are unclear about the mechanism of action of artemisinin, the drug is used in extremely high concentrations, and the effect is not ideal. Summary of the invention

[0005] 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.

[0006] In order to achieve the aforementioned invention objectives, the technical solutions adopted in this application include:

[0007] The invention discloses a use of an endoperoxy compound in preparing an antibacterial drug, wherein the endoperoxy 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; under anoxic conditions, the drug concentration of the endoperoxy compound is: the drug concentration is ≤100 μM.

[0008] In the present application, a sealed anoxic culture tube filled with Middlebrook 7H9 broth medium is used as a culture model of Mycobacterium tuberculosis, Mycobacterium tuberculosis is inoculated in the culture tube to OD600=0.01, and then cultured to anoxic conditions. Methylene blue is used as an indicator. The drug concentration of the endoperoxide compound here is converted from the in vitro drug concentration. Under the condition of drug concentration ≤100 μM, the endoperoxide compound has a good inhibitory effect on Mycobacterium tuberculosis, and the low drug concentration is conducive to reducing drug side effects.

[0009] Further, the 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.

[0010] Among them, artemisinin and its derivatives are good antimalarial drugs with the advantages of strong species specificity, rapid drug action, and low toxicity and side effects. The dioxygen bridge in the structure of artemisinin is the key pharmacophore. The opening or breaking of the dioxygen bridge, that is, the activation of artemisinin, is the prerequisite for its efficacy. Deoxyartemisinin obtained by replacing the dioxygen bridge with a single oxygen bridge will lose its antimalarial activity. In addition to antimalarial, artemisinin drugs also have the effects of killing other types of parasites and antivirals, and have therapeutic effects on diabetes and the autoimmune disease lupus erythematosus. Artefenomel (OZ439) is an orally active, synthetic antimalarial compound containing the artemisinin pharmacophore, which has a mechanism of action similar to that of artemisinin. HEM is an extract of the Chinese medicine Senecio radiata, which is an extract mixture containing multiple eremophilanolid sesquiterpenes. Ascaridol is an extract of the Chinese medicine Santoinian. Majapolene A is an extract of the concave-capped algae. Yingzhaosu A is Yingzhaosu A, also known as Yingzhaosu A, which is a sesquiterpene derivative with a peroxide group isolated from the herb Yingzhao root. Aceranol acetate is an extract of the traditional Chinese medicine Acer truncatum.

[0011] Furthermore, the drug concentration of the endoperoxide compound is: 12.5 μM≤drug concentration≤50 μM.

[0012] Further reducing the drug concentration, experiments have confirmed that the drug 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.

[0013] The second object of the present application is to provide a use of an endoperoxide compound in combination with a respiratory chain inhibitor 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.

[0014] In order to achieve the aforementioned invention objectives, the technical solutions adopted in this application include:

[0015] 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.

[0016] In the present application, a sealed culture tube filled with Middlebrook 7H9 broth medium is used as a culture model of 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.

[0017] Further, the 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.

[0018] Furthermore, the Mycobacterium tuberculosis respiratory chain inhibitor is one or more of bedaquiline, Q203, Aurachin D or lansoprazole.

[0019] 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 drug concentration of artemisinin is: drug concentration ≤100 μM; the drug concentration of bedaquiline is: drug concentration ≤4 μM; the drug concentration of Q203 is: drug concentration <10 nM; the drug concentration of Aurachin D is: drug concentration ≤20 μM; the drug concentration of lansoprazole is: drug concentration ≤20 μM.

[0020] Thus, under normal oxygen conditions, the combination of artemisinin and bedaquiline, Q203, lansoprazole or Aurachin D can reduce the survival rate of mycobacteria compared with the use of the corresponding respiratory chain inhibitor alone. It can be seen that under normal circumstances, the combination of artemisinin and respiratory chain inhibitors can improve the inhibitory effect of respiratory chain inhibitors on Mycobacterium tuberculosis. Under the above drug concentration conditions, the total drug concentration of artemisinin combined with bedaquiline, Q203, lansoprazole or Aurachin D is low, which is conducive to reducing drug side effects.

[0021] 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 drug concentration of artemisinin is: drug concentration ≤100 μM; the drug concentration of Q203 is: drug concentration ≤1 nM.

[0022] In this way, the drug concentration of Q203 is further reduced. Experiments have confirmed that when the drug concentration of Q203 is not higher than 1nM, it has a good inhibitory effect on Mycobacterium tuberculosis when combined with artemisinin, which is conducive to further reducing the dosage and thus reducing the side effects of the drug.

[0023] 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 drug concentration of artemisinin is: drug concentration ≤50μM; the drug concentration of bedaquiline is: drug concentration <10μM; the drug concentration of Q203 is: drug concentration <5nM; the drug concentration of Aurachin D is: drug concentration <20μM; the drug concentration of lansoprazole is: drug concentration <20μM.

[0024] Thus, under hypoxic conditions, the combination of artemisinin and bedaquiline, Q203, lansoprazole or Aurachin D can reduce the survival rate of mycobacteria compared with the use of the corresponding respiratory chain inhibitor alone. It can be seen that the combination of artemisinin and respiratory chain inhibitors under hypoxia can improve the inhibitory effect of respiratory chain inhibitors on Mycobacterium tuberculosis. Under the above drug concentration conditions, the total drug concentration of artemisinin combined with bedaquiline, Q203, lansoprazole or Aurachin D is low, which is conducive to reducing drug side effects.

[0025] 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 drug concentration of artemisinin is: drug concentration ≤50μM; the drug concentration of bedaquiline is: drug concentration ≤2μM; the drug concentration of Q203 is: drug concentration ≤0.5nM.

[0026] In this way, the drug concentrations of bedaquiline and Q203 are further reduced. Experiments have confirmed that when the drug concentration of bedaquiline is not higher than 2 μM, it has a good inhibitory effect on Mycobacterium tuberculosis when combined with artemisinin. When the drug concentration of Q203 is not higher than 0.5 nM, it has a good inhibitory effect on Mycobacterium tuberculosis when combined with artemisinin, which is conducive to further reducing the dosage and thus reducing drug side effects.

[0027] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is two of bedaquiline, Q203, Aurachin D or lansoprazole; under normal oxygen conditions, the drug concentrations of artemisinin and the respiratory chain inhibitor are:

[0028] The drug concentration of artemisinin is ≤50 μM, the drug concentration of Aurachin D is: drug concentration <10 μM, and the drug concentration of Q203 is: drug concentration <5 nM;

[0029] or,

[0030] The drug concentration of artemisinin is ≤50 μM, the drug concentration of Aurachin D is: drug concentration <10 μM, and the drug concentration of lansoprazole is: drug concentration <10 μM.

[0031] Thus, the test results show that under normal conditions, the combination of artemisinin, Q203 and Aurachin D, or the combination of artemisinin, lansoprazole and Aurachin D, can reduce the survival rate of mycobacteria compared with the use of two corresponding respiratory chain inhibitors alone. It can be seen that under normal conditions, the combination of artemisinin and multiple respiratory chain inhibitors can improve the inhibitory effect of respiratory chain inhibitors on Mycobacterium tuberculosis. Under the above drug concentration conditions, the total drug concentration of artemisinin combined with two respiratory chain inhibitors is low, which is conducive to reducing drug side effects.

[0032] Furthermore, the endoperoxygen compound is artemisinin, and the Mycobacterium tuberculosis respiratory chain inhibitor is two of bedaquiline, Q203, Aurachin D or lansoprazole; under normal oxygen conditions, the drug concentrations of artemisinin and the respiratory chain inhibitor are:

[0033] The drug concentration of artemisinin is ≤25 μM, the drug concentration of Aurachin D is: drug concentration ≤2.5 μM, and the drug concentration of Q203 is: drug concentration ≤4 nM;

[0034] or,

[0035] The drug concentration of artemisinin is ≤25 μM, the drug concentration of Aurachin D is: drug concentration ≤2.5 μM, and the drug concentration of lansoprazole is: drug concentration ≤2 μM.

[0036] In this way, the drug concentrations of Q203, Aurachin D and lansoprazole are further reduced. Experiments have confirmed that under normal conditions, when the drug concentration of Aurachin D is not higher than 2.5μM and the drug concentration of Q203 is not higher than 4nM, they can be combined with artemisinin to have a good inhibitory effect on Mycobacterium tuberculosis; when the drug concentration of Aurachin D is not higher than 2.5μM and the drug concentration of lansoprazole is not 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 the side effects of the drugs.

[0037] The third object of the present application is to provide an antibacterial drug, comprising an effective amount of an endoperoxide 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 endoperoxide compound includes artemisinin, dihydroartemisinin, artemether, arteether, artesunate, HEM, ascaridol, majapolene A, Norterpene Peroxides, yinzaosuA, artefenomel (OZ439), aceranol acetate, and deoxycholic acid derivatives containing internal oxygen bridges.

[0038] The present application aims to kill pathogens, especially Mycobacterium tuberculosis, more quickly or effectively by introducing endoperoxide compounds represented by artemisinin as drugs, which has potential applications in the treatment of tuberculosis, improves the cure efficiency or shortens the course of treatment, overcomes the problem of drug resistance, and reduces the amount of drugs to reduce side effects on patients. Compared with the prior art, the present application can bring the following technical effects:

[0039] 1) Different mechanisms of action help overcome drug resistance;

[0040] 2) It can reduce the dosage of drugs and reduce the side effects of drugs;

[0041] 3) Compared with existing technologies, it is safer and has fewer toxic and side effects;

[0042] 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

[0043] Figure 1 This is a schematic diagram of the combined action of artemisinin and a respiratory chain inhibitor to inhibit Mycobacterium tuberculosis in the examples of the present application.

[0044] Figure 2 This 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.

[0045] Figure 3 This is an analysis of the inhibition of Mycobacterium tuberculosis using artemisinin in combination with bedaquiline, Q203, Aurachin D or lansoprazole under normal conditions in Example 2 of the present application.

[0046] Figure 4 This is an analysis of the inhibition of Mycobacterium tuberculosis by using different drug concentrations of artemisinin in combination with fixed drug concentrations of Q203 and bedaquiline under normal and hypoxic conditions in Example 3 of the present application.

[0047] Figure 5 This is an analysis of the inhibition of Mycobacterium tuberculosis using artemisinin and Aurachin D in combination with Q203 or lansoprazole, respectively, under normal conditions in the examples of the present application.

[0048] In the figure, normal and aerobic refer to normal oxygen conditions, ART-Artemisinin, Bedaquiline-Bedaquiline, Lansoprazole-Lansoprazole. DETAILED DESCRIPTION

[0049] The present application is further described below with reference to embodiments, but is not limited thereto.

[0050] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. In this application, normal conditions refer to normal oxygen conditions; hypoxic conditions refer to the color change of the methylene blue indicator in a sealed hypoxic culture tube, usually when the oxygen content is less than 5%.

[0051] Terminology Note:

[0052] HEM: Senecio oleifera extract, a blend of extracts from various eremophilanolid sesquiterpenes.

[0053] Ascaridol: Ascaridol extract, CAS: 512-85-6

[0054] majapolene A: extract of concave algae, NSC-692207, CHEMBL450193, [4-(4-bromo-3,3-dimethylcyclohexyl)-2,3-dioxabicyclo[2.2.2]oct-5-en-1-yl]methanol

[0055] Yingzhaosu A: Yingzhaosu A, also known as Yingzhaosu A; CAS: 73301-54-9

[0056] Artefenomel(OZ439):CAS:1029939-86-3

[0057] aceranol acetate:CAS:1221260-33-8

[0058] 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 the density of bacterial cells.

[0059] OADC: (=oleic acid, albumin, dextrose and catalase), i.e. a mixture of oleic acid, albumin, dextrose and catalase;

[0060] Middlebrook 7H9 broth medium: purchased from Beijing Coolbo Technology Co., Ltd., product number: MKC301; How to prepare: prepare according to the product manual.

[0061] Q203: CAS No. 1334719-95-7;

[0062] Aurachin D: CAS No. 108354-13-8.

[0063] Example 1: This example illustrates the effect of artemisinin in fighting Mycobacterium tuberculosis through in vitro experiments, comprising the following steps:

[0064] S1: dissolving artemisinin powder in dimethyl sulfoxide to prepare artemisinin solution;

[0065] S2: Inoculate Mycobacterium tuberculosis in a sealed anoxic culture tube filled with Middlebrook 7H9 broth medium to OD600 = 0.01, and then continue to culture until anoxic conditions. The methylene blue indicator added to the culture medium will deplete the oxygen in the culture tube when the bacteria grow to a certain extent, and the indicator will change color, indicating that anoxic conditions have been reached.

[0066] S3: First, prepare a 200mM artemisinin dimethyl sulfoxide stock solution. 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 6.25μM, 12.5μM, 25μM, 50μM, and 100μM, respectively. Continue to incubate at 37°C for 6 days. Only add the solvent dimethyl sulfoxide to the control group.

[0067] Comparative Example 1:

[0068] The implementation method is basically the same as that of Example 1, except that: (1) the test is carried out under normal (aerobic) conditions;

[0069] (2) In step S2, the density of Mycobacterium tuberculosis is OD600=0.003.

[0070] Supporting example 1:

[0071] The effectiveness of the experimental process is demonstrated by Supporting Example 1. At the same time, the clinically used anti-tuberculosis drug bedaquiline was used to treat Mycobacterium tuberculosis in the same manner as in Example 1 (the drug treatment concentrations were 0.25 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM, respectively). It was observed that there was a significant inhibitory effect under both normal conditions and hypoxic conditions.

[0072] Example 1 combined with Comparative Example 1, Supporting Example 1 and Figure 2It can be seen that: under normal conditions, different concentrations of artemisinin were incubated with the same number (OD600=0.003) of Mycobacterium tuberculosis. After 6 days of culture at 37°C, there was no significant difference in the survival rate of Mycobacterium tuberculosis compared with the control group; under hypoxic conditions, different concentrations of artemisinin were incubated with the same number (OD600=0.01) of Mycobacterium tuberculosis. After 6 days of culture at 37°C, the survival rate of Mycobacterium tuberculosis was significantly reduced compared with the control group. 25 μM artemisinin can significantly inhibit the survival rate of Mycobacterium tuberculosis.

[0073] Embodiment 2:

[0074] 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.

[0075] Table 1 Drug concentrations of each group of tests in Example 2

[0076]

[0077]

[0078] The test results are as follows Figure 3 As shown in the figure, the experimental results show that under normal conditions, the combination of artemisinin and bedaquiline, Q203, lansoprazole or Aurachin D can reduce the survival rate of mycobacteria compared with the corresponding respiratory chain inhibitor alone. It can be seen that under normal conditions, the combination of artemisinin and respiratory chain inhibitors can improve the inhibitory effect of respiratory chain inhibitors on Mycobacterium tuberculosis. The drug concentrations of artemisinin are 6.25μM, 12.5μM, 25μM, 50μM, 100μM, and 200μM.

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

[0080] In experiments 1-18, under normal conditions, bedaquiline was used alone. As the concentration of bedaquiline increased (0.03125 μM, 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM), the survival rate of Mycobacterium tuberculosis gradually decreased. When artemisinin was used in combination with bedaquiline, at each drug concentration in the experiment, the addition of artemisinin could significantly improve the inhibitory effect of bedaquiline on Mycobacterium tuberculosis.

[0081] Test 19-36, under normal conditions, Q203 was used alone. As the concentration of Q203 increased (0.03nM, 0.0625nM, 0.125nM, 0.25nM, 0.5nM, 1nM), the survival rate of Mycobacterium tuberculosis gradually decreased. When artemisinin was used in combination with Q203, at each drug concentration in the test, the addition of artemisinin could significantly improve the inhibitory effect of Q203 on Mycobacterium tuberculosis. In addition, tests were also conducted with Q203 drug concentrations of 2nM, 4nM, and 8nM (not shown in the chart). The test results showed that under normal conditions, the addition of artemisinin could significantly improve the inhibitory effect of Q203 on Mycobacterium tuberculosis, and the survival rate of Mycobacterium tuberculosis was below 10%.

[0082] In experiments 37-54, under normal conditions, when lansoprazole was used alone, the survival rate of Mycobacterium tuberculosis basically showed a downward trend as the concentration of lansoprazole increased (0.625μM, 1.25μM, 2.5μM, 5μM, 10μM, 20μM). When artemisinin was used in combination with lansoprazole, at each drug concentration in the experiment, the addition of artemisinin could significantly improve the inhibitory effect of bedaquiline on Mycobacterium tuberculosis.

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

[0084] Embodiment 3:

[0085] The implementation method is basically the same as that of Example 1 and Control Example 1, and artemisinin and Q203, artemisinin and bedaquiline are used to treat Mycobacterium tuberculosis under normal and hypoxic conditions, respectively; the combination and concentration of the treatment drugs are shown in Table 2. (Under normal conditions, the concentration of Mycobacterium tuberculosis is OD600 = 0.003; under hypoxic conditions, the concentration of Mycobacterium tuberculosis is OD600 = 0.01)

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

[0087]

[0088]

[0089] The test results are as follows Figure 4 As shown, the experimental results show that under normal conditions, the combination of artemisinin and bedaquiline / Q203 can reduce the survival rate of Mycobacterium tuberculosis compared with artemisinin alone or bedaquiline / Q203 alone.

[0090] Under normal conditions, when artemisinin is used alone, the survival rate of Mycobacterium tuberculosis decreases with the increase of artemisinin drug concentration (12.5μM, 25μM, 50μM, 100μM). When the drug concentration of artemisinin is 100μM, the survival rate of Mycobacterium tuberculosis is 88.62%.

[0091] Under normal conditions, the survival rate of Mycobacterium tuberculosis was 60.22% when 4μM bedaquiline was used alone. When artemisinin was used in combination with 4μM bedaquiline, the survival rate of Mycobacterium tuberculosis decreased with the increase of artemisinin drug concentration. When the drug concentration of artemisinin was 50μM, the survival rate of Mycobacterium tuberculosis could be reduced to 14.65%. When the drug concentration of artemisinin was increased to 100μM, the survival rate of Mycobacterium tuberculosis further decreased to 7.08%. It can be seen that under normal conditions, the combination of artemisinin and bedaquiline can improve the inhibitory effect of artemisinin and bedaquiline on Mycobacterium tuberculosis.

[0092] Under normal conditions, the survival rate of Mycobacterium tuberculosis was 51.03% when 1nM Q203 was used alone. When artemisinin was used in combination with 1nM Q203, the survival rate of Mycobacterium tuberculosis decreased with the increase of artemisinin drug concentration. When the drug concentration of artemisinin was 50μM, the survival rate of Mycobacterium tuberculosis could be reduced to 7.42%. The drug concentration of artemisinin continued to increase to 100μM, which had no significant effect on the survival rate of Mycobacterium tuberculosis. It can be seen that under normal conditions, the combination of artemisinin and Q203 can improve the inhibitory effect of artemisinin and Q203 on Mycobacterium tuberculosis.

[0093] Under hypoxic conditions, less drug concentration is needed to have 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 the increase of artemisinin drug concentration (6.25μM, 12.5μM, 25μM, 50μM). When the drug concentration of artemisinin is 25μM, the survival rate of Mycobacterium tuberculosis is 49.48%. When the drug concentration of artemisinin is increased to 50μM, the survival rate is further reduced to 37.69%. It can be seen that artemisinin can effectively inhibit Mycobacterium tuberculosis under hypoxic conditions.

[0094] Under hypoxic conditions, the survival rate of Mycobacterium tuberculosis was 56.76% when 2μM bedaquiline was used alone. When artemisinin was used in combination with 2μM bedaquiline, the survival rate of Mycobacterium tuberculosis decreased with the increase of artemisinin drug concentration. When the drug concentration of artemisinin was 25μM, the survival rate of Mycobacterium tuberculosis could be reduced to 16.09%. When the drug concentration of artemisinin was increased to 50μM, the survival rate of Mycobacterium tuberculosis further decreased to 5.97%. It can be seen that the combination of artemisinin and bedaquiline under hypoxia can improve the inhibitory effect of artemisinin and bedaquiline on Mycobacterium tuberculosis.

[0095] Under hypoxic conditions, the survival rate of Mycobacterium tuberculosis was 52.64% when 0.5nM Q203 was used alone. When artemisinin was used in combination with 0.5nM Q203, the survival rate of Mycobacterium tuberculosis decreased with the increase of artemisinin drug concentration. When the drug concentration of artemisinin was 25μM, the survival rate of Mycobacterium tuberculosis could be reduced to 12.09. When the drug concentration of artemisinin was increased to 50μM, the survival rate of Mycobacterium tuberculosis further decreased to 5.19%. It can be seen that the combination of artemisinin and Q203 under hypoxic conditions can improve the inhibitory effect of artemisinin and Q203 on Mycobacterium tuberculosis.

[0096] In addition, experiments were conducted with bedaquiline drug concentrations of 4 μM, 6 μM, and 8 μM (not shown in the chart), and artemisinin drug concentration of 50 μM. The test results show that under hypoxic conditions, the addition of artemisinin can significantly improve the inhibitory effect of bedaquiline on Mycobacterium tuberculosis, and the survival rate of Mycobacterium tuberculosis is below 10%.

[0097] In addition, experiments were conducted with Q203 drug concentrations of 1 nM, 2 nM, and 4 nM (not shown in the chart), and artemisinin drug concentration of 50 μ M. The test results showed that under hypoxic conditions, the addition of artemisinin can significantly improve the inhibitory effect of Q203 on Mycobacterium tuberculosis, and the survival rate of Mycobacterium tuberculosis is below 10%.

[0098] Embodiment 4:

[0099] 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.

[0100] Table 3 Drug concentrations of each group of tests in Example 4

[0101]

[0102] The test results are as follows Figure 5As shown in the results, the test results show that under normal conditions, the combination of artemisinin, Q203 and Aurachin D, or the combination of artemisinin, lansoprazole and Aurachin D, can reduce the survival rate of mycobacteria compared with the use of two corresponding respiratory chain inhibitors alone. It can be seen that under normal conditions, the combination of artemisinin and multiple respiratory chain inhibitors can improve the inhibitory effect of respiratory chain inhibitors on Mycobacterium tuberculosis.

[0103] In addition, experiments were conducted with Q203 drug concentrations of 1nM, 2nM, and 4nM, Aurachin D drug concentrations of 5μM and 6.25μM, and artemisinin drug concentration of 50μM (not shown in the chart). The test results showed that under normal conditions, the combination of artemisinin, Q203, and Aurachin D can reduce the survival rate of mycobacteria, compared with the use of two corresponding respiratory chain inhibitors alone, and the survival rate is within 10%.

[0104] In addition, experiments were conducted with lansoprazole at drug concentrations of 4 μM, 6 μM, and 8 μM, Aurachin D at drug concentrations of 5 μM and 6.25 μM, and artemisinin at a drug concentration of 50 μM (not shown in the chart). The experimental results showed that under normal conditions, the combination of artemisinin, lansoprazole, and Aurachin D can reduce the survival rate of mycobacteria, compared with the use of two corresponding respiratory chain inhibitors alone, and the survival rate is within 10%.

[0105] like Figure 1 As shown, the possible mechanism of the drugs involved in this article inhibiting 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 produced by the respiratory chain to destroy the cell membrane potential. When the downstream of the electron transport chain is inhibited by the inhibitor, the respiratory chain will transfer more electrons to activate artemisinin, thereby enhancing the effect of artemisinin in killing cells.

[0106] Although the present application has been described with reference to illustrative embodiments, it will be appreciated by those skilled in the art that various other changes, omissions, and / or additions may be made and that elements of the described embodiments may be substituted with substantial equivalents 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 herein to limit the present application to the disclosed specific embodiments for carrying out the present application, but rather it is intended that the present application will include all embodiments within the scope of the appended claims.

Claims

1. Use of an endoperoxygen compound in combination with a respiratory chain inhibitor in the preparation of an antibacterial drug, characterized in that: The endoperoxygen compound is artemisinin; the antibacterial drug is a drug that inhibits Mycobacterium tuberculosis; the respiratory chain inhibitor is a Mycobacterium tuberculosis respiratory chain inhibitor; The Mycobacterium tuberculosis respiratory chain inhibitor is: Bedaquiline, under normal oxygen conditions, the drug concentration of artemisinin is: 12.5 μM ≤ drug concentration ≤ 100 μM; the drug concentration of bedaquiline is: drug concentration ≤ 4 μM; Or, Q203, then under normal oxygen conditions, the drug concentration of artemisinin is: 12.5 μM ≤ drug concentration ≤ 100 μM; the drug concentration of Q203 is: drug concentration < 10 nM; Or, Aurachin D, then under normal oxygen conditions, the drug concentration of artemisinin is: 100 μM or 200 μM; the drug concentration of Aurachin D is: drug concentration ≤ 20 μM; Or, lansoprazole, then under normal oxygen conditions, the drug concentration of artemisinin is: drug concentration ≤ 100 μM; the drug concentration of lansoprazole is: drug concentration ≤ 20 μM; Alternatively, the Mycobacterium tuberculosis respiratory chain inhibitor is any one of a combination of Aurachin D and Q203, or a combination of Aurachin D and lansoprazole; under normal oxygen conditions, the drug concentrations of artemisinin and the respiratory chain inhibitor are: The drug concentration of artemisinin is ≤50 μM, the drug concentration of Aurachin D is: drug concentration <10 μM, and the drug concentration of Q203 is: drug concentration <5 nM; or, The drug concentration of artemisinin is ≤50 μM, the drug concentration of Aurachin D is: drug concentration <10 μM, and the drug concentration of lansoprazole is: drug concentration <10 μM.

2. A drug for inhibiting Mycobacterium tuberculosis, characterized in that: The invention comprises an effective amount of an endoperoxy compound and a respiratory chain inhibitor of Mycobacterium tuberculosis and a pharmaceutically acceptable carrier; the respiratory chain inhibitor of Mycobacterium tuberculosis is Q203, Aurachin D, Q203 and Aurachin D, or Aurachin D and lansoprazole; the endoperoxy compound is artemisinin; Under normal oxygen conditions, the Mycobacterium tuberculosis respiratory chain inhibitor is: Q203, the drug concentration of artemisinin is: 12.5 μM ≤ drug concentration ≤ 100 μM; the drug concentration of Q203 is: drug concentration < 10 nM; Or, Aurachin D, the drug concentration of artemisinin is: 100 μM or 200 μM; the drug concentration of Aurachin D is: drug concentration ≤ 20 μM; or Q203 and Aurachin D, the drug concentration of artemisinin is ≤50 μM, and the drug concentration of Aurachin D is: drug concentration <10 μM, and the drug concentration of Q203 is: drug concentration <5 nM; or Aurachin D and lansoprazole, the drug concentration of artemisinin is ≤50 μM, and the drug concentration of Aurachin D is: drug concentration <10 μM, and the drug concentration of lansoprazole is: drug concentration <10 μM.

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