Anti-mycobacterial infection pharmaceutical composition

Abstract

The present invention belongs to the technical field of biopharmaceuticals and specifically relates to use of a pharmaceutical composition in combating mycobacterial infection. The use of zuclopenthixol in combination with bedaquiline exhibits significantly enhanced anti-mycobacterial infection activity. Compared to the use of bedaquiline alone, the use of 2 μg / ml zuclopenthixol in combination with bedaquiline results in varying reductions in colony counts of the MmpL5-MmpS5 strain when the concentration of bedaquiline ranges from 1 / 16 MIC to 2 MIC. As an enhancer of bedaquiline, zuclopenthixol can enhance the anti-mycobacterial infection activity of bedaquiline. The minimum inhibitory concentration of zuclopenthixol against the Rv0678 mutant strain in vitro is 16 μg / ml, and the minimum inhibitory concentration of BDQ against the Rv0678 mutant strain in vitro is 1 μg / mL. The use of 1 μg / ml zuclopenthixol in combination with BDQ reduces the MIC of BDQ against the Rv0678 mutant strain from 1 μg / ml to 0.03 μg / ml (a 32-fold increase in efficacy), which is lower than the MIC against the wild strain H37Rv (0.06 μg / ml), reversing resistance to BDQ and further enhancing the anti-mycobacterial infection activity.

Description

A pharmaceutical composition for resisting mycobacterium infection TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of a pharmaceutical composition in resisting mycobacterium infection. BACKGROUND

[0002] Mycobacterium includes the Mycobacterium tuberculosis complex (including M. tuberculosis, M. bovis, M. africanum, M. microti, M. caprae, M. pinnipedii, M. suricattae and M. mungi), non-tuberculous Mycobacteria (NTM) and M. leprae.

[0003] Tuberculosis caused by Mycobacterium tuberculosis (MTB) is a chronic respiratory infectious disease that seriously endangers human health. Drug resistance of tuberculosis is serious, and the selection of drugs for treating drug-resistant tuberculosis is limited, the treatment course is long, and the cure rate is low, about 63%. In 2019 and 2022, bedaquiline and clofazimine were listed as Group A and Group B drugs in the core drugs for treating multidrug-resistant tuberculosis by WHO, but bedaquiline-resistant strains were soon identified. The research team has found clinical drug-resistant strains of the new drug bedaquiline, especially in tuberculosis patients who have not been exposed to and used the drug, and isolated bedaquiline and clofazimine cross-resistant strains (Rv0678 gene mutation) (Xu J, et al. Antimicrob Agents Chemother, 2017). Although atpE gene mutation encoding ATP synthase and non-target PepQ gene mutation are associated with bedaquiline resistance, the most reported in clinical practice is the Rv0678 gene mutation of Mycobacterium tuberculosis, which can cause the activity of the anti-mycobacterium infection chemotherapy regimen containing bedaquiline to decrease, the treatment cycle to prolong and even the treatment to fail. It was found that the Rv0678 mutant could also cause resistance to the B group drug of the core drug for treating multidrug-resistant tuberculosis, clofazimine, and the new anti-tuberculosis drug PBTZ169 in clinical phase II. It was found that the Rv0678 mutant strain still had partial cross-resistance to the new generation of bedaquiline derivatives (TBAJ-587) and clofazimine derivatives with stronger activity (Xu J, et al. Antimicrob Agents Chemother, 2021; Xu J, et al. Antimicrob Agents Chemother, 2019).

[0004] The Rv0678 gene is a transcriptional repressor of the MmpL5-MmpS5 efflux system, and its mutation can cause overexpression of MmpL5 and MmpS5. Transcriptional and proteomic studies have also found that the mutation of Rv0678 causes the expression of MmpL5 and MmpS5 to be up-regulated by more than 2 times in bedaquiline-resistant Mycobacterium tuberculosis (Xu J, et al. Antimicrob Agents Chemother, 2023). One of the methods to combat drug resistance caused by drug efflux is to develop drug efflux inhibitors for use in combination with antibiotics. In the study of the role and function of MmpL5-MmpS5, it was found that in both M. smegmatis and M. tuberculosis, the co-expression of MmpL5 and MmpS5 forms an efflux pump, leading to bedaquiline resistance, while MmpL5 alone does not play a role (Li Dongshuo; Wang Bin; Lu Yu; Xu Jian. Expression and functional study of Mycobacterium tuberculosis membrane proteins MmpS5-MmpL5. Chinese Journal of Antituberculosis 2022; Xu J, et al. Antimicrob Agents Chemother, 2023). This study lays the foundation for the interaction of MmpL5-MmpS5 as a new target.

[0005] Non-tuberculous mycobacteria (NTM) was once named as atypical mycobacteria, atypical acid-fast bacilli, non-classified mycobacteria, unclassified mycobacteria, anonymous mycobacteria, wild mycobacteria, opportunistic mycobacteria, paratuberculosis bacillus, and pseudotuberculosis bacillus. NTM disease refers to the infection of NTM in human body and the resulting lesions of related tissues and organs (Daley CL, Iaccarino JM, Lange C, et al. Treatment of nontuberculous mycobacterial pulmonary disease: an official ATS / ERS / ESCMID / IDSA clinical practice guideline [J]. Eur Respir J, 2020, 56(1): 200053.). NTM disease is a systemic disease, mainly affecting lung tissue, but all organ systems in the body can be affected, including systemic poisoning symptoms and local damage (Tang Shenjie, Gao Wen. Clinical Tuberculosis [M]. 2nd ed. Beijing: People's Medical Publishing House, 2019: 1026-1046.). Without the results of sterile species identification, NTM disease can be misdiagnosed as tuberculosis and bronchiectasis for a long time. NTM disease has different clinical manifestations due to different infection species, affected tissues and organs. In recent years, NTM disease has shown a rapid increasing trend and has become one of the important public health problems threatening human health (Furuuchi K, Morimoto K, Yoshiyama T, et al. Interrelational changes in the epidemiology and clinical features of nontuberculous mycobacterial pulmonary disease and tuberculosis in a referral hospital in Japan [J]. Respir Med, 2019, 152: 74-80.).

[0006] In the field of diagnosis, GeneXpert MTB / RIF has become the main method for rapid diagnosis of tuberculosis and rapid detection of rifampicin resistance. In the field of treatment, the multidrug-resistant / rifampicin-resistant tuberculosis (MDR / RR-TB) all-oral short-course treatment regimen has become the recommended treatment regimen in guidelines. However, the proportion of drug-resistant diseases is still rising, and there is a lack of rapid diagnostic methods, long treatment course, and treatment regimen containing injections, which cannot meet the rapid diagnosis of the disease and affect the patient's compliance to treatment, so there is an urgent need for new treatment methods.

[0007] Zuclopenthixol is a thioxanthene derivative, which acts by blocking dopamine receptors, and has significant antipsychotic effect and sedative effect. Zuclopenthixol was discovered in 1962 and began to be used for antipsychotic, and has been marketed in more than 30 countries in Europe, Latin America and Canada. At present, there is no report on its activity of resisting Mycobacterium tuberculosis and synergistic anti-mycobacterium infection. With the increasingly serious problem of drug resistance of tuberculosis, it is urgent to develop new treatment strategies, enhance the activity of bedaquiline against mycobacterium infection, and reduce the occurrence of drug resistance of new drugs.

[0008] SUMMARY

[0009] In the present application, it is found that zuclopenthixol combined with bedaquiline can enhance the activity of bedaquiline against mycobacterium infection, and the drug combined with zuclopenthixol and bedaquiline has the effect of resisting mycobacterium infection. Based on this, the present application is completed.

[0010] In a first aspect, the present application provides a pharmaceutical composition for resisting mycobacterium infection, which comprises zuclopenthixol and bedaquiline, and the pharmaceutical composition has at least one of the following effects:

[0011] a) inhibiting the activity of mycobacterium;

[0012] b) resisting mycobacterium infection;

[0013] c) preventing and / or treating diseases caused by mycobacterium.

[0014] Further, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculosis mycobacterium and / or leprosy mycobacterium.

[0015] Further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis clinical isolates, Mycobacterium tuberculosis standard strains and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0016] Further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection.

[0017] Further, the diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis and the like.

[0018] Further, the drug-resistant tuberculosis includes but is not limited to single-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis.

[0019] Further, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, trachea-bronchus tuberculosis, tuberculous pleurisy, and bacterium-negative pulmonary tuberculosis.

[0020] Further, the pulmonary tuberculosis includes, but is not limited to, lymphatic tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0021] Further, the Mycobacterium tuberculosis includes multi-drug resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0022] Further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, and Mycobacterium microti.

[0023] Further, the non-tuberculosis Mycobacterium includes, but is not limited to, Mycobacterium avium (M. avium), Mycobacterium intracellulare (M. intracellulare), Mycobacterium kansasii (M. kansasii), Mycobacterium fortuitum (M. fortuitum), Mycobacterium abscessus (M. abscessus), Mycobacterium ulcerans (M. ulcerans), and / or Mycobacterium marinum (M. marinum), etc.

[0024] Further, the disease caused by the non-tuberculosis Mycobacterium is selected from one or more of NTM pulmonary disease, NTM lymphatic disease, disseminated NTM disease, and / or other NTM disease.

[0025] Further, the pharmaceutical composition can further contain other active ingredients against Mycobacterium infection.

[0026] Further, one or more pharmaceutically acceptable carriers can be added to the pharmaceutical composition.

[0027] Further, the pharmaceutical composition can be prepared in the form of injection, tablet, powder, granule, capsule, oral liquid, injection preparation, or aerosol, etc.; and the above-mentioned various dosage forms of the pharmaceutical can be prepared according to the conventional method in the field of pharmacy.

[0028] Further, the preparation can be one or more of ordinary preparation, sustained-release preparation, controlled-release preparation, and / or various microparticle drug delivery systems.

[0029] Further, the tablet can widely use various carriers known in the art, including one or more of diluent and absorbent, humectant and binder, disintegrating agent, disintegrating inhibitor, absorption promoter, and / or lubricant.

[0030] Further, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and / or aluminum silicate.

[0031] Further, the wetting agent and the binder include, but are not limited to, one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, sugar paste, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and / or polyvinylpyrrolidone.

[0032] Further, the disintegrant includes, but is not limited to, one or more of dried starch, alginate, agar powder, fucoidin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, and / or ethyl cellulose.

[0033] Further, the disintegration inhibitor includes, but is not limited to, one or more of sucrose, glycerin tristearate, cocoa butter, and / or hydrogenated oil, etc.

[0034] Further, the absorption promoter includes, but is not limited to, one or more of quaternary ammonium salt and / or sodium dodecyl sulfate.

[0035] Further, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0036] Further, the tablet can be further manufactured into a coated tablet, including a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a double-layer tablet, and a multi-layer tablet.

[0037] Further, the injection preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder, and / or a suspension.

[0038] Further, the injection preparation can use all diluents commonly used in the art, including, but not limited to, one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and / or polyoxyethylene sorbitol fatty acid ester.

[0039] Further, the injection preparation can be added with an appropriate amount of one or more of sodium chloride, glucose, glycerin, a conventional cosolvent, a buffer, and / or a pH adjuster in order to prepare an isotonic injection.

[0040] Further, the various preparations can also be added with a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetener, or other materials, if necessary.

[0041] Further, the pharmaceutical composition can be introduced into the body, such as muscle, intradermally, subcutaneously, or intravenously, by a physical or chemical-mediated method.

[0042] Further, the impurity of the chloquelone also includes a pharmaceutically acceptable salt or ester.

[0043] Further, the pharmaceutically acceptable salt includes, but is not limited to, acetate, hydrochloride and other pharmaceutically acceptable salt.

[0044] Further, the pharmaceutically acceptable ester includes, but is not limited to, decanoate.

[0045] In a second aspect, the present application provides a use of a zalcitabine as a synergist of bedaquiline against mycobacterial infection, wherein the zalcitabine enhances the activity of bedaquiline against mycobacterial infection when the zalcitabine is used in combination with bedaquiline.

[0046] Further, the mycobacterium is selected from the group consisting of Mycobacterium tuberculosis, non-tuberculosis mycobacterium and / or Mycobacterium leprae.

[0047] Further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis clinical isolates, Mycobacterium tuberculosis standard strains and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0048] Further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection.

[0049] Further, the disease caused by Mycobacterium tuberculosis includes, but is not limited to, drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis and the like.

[0050] Further, the drug-resistant tuberculosis includes, but is not limited to, single drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis.

[0051] Further, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, bacterium-negative pulmonary tuberculosis and the like.

[0052] Further, the extrapulmonary tuberculosis includes, but is not limited to, lymph node tuberculosis, intestinal tuberculosis, kidney tuberculosis, bone and joint tuberculosis and the like.

[0053] Further, the Mycobacterium tuberculosis includes multi-drug resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0054] Further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti and Mycobacterium microti.

[0055] Further, the non-tuberculous mycobacteria include, but are not limited to, M. avium, M. intracellulare, M. kansasii, M. fortuitum, M. abscessus, M. ulcerans, and / or M. marinum, etc.

[0056] Further, the disease caused by the non-tuberculous mycobacteria is selected from one or more of NTM pulmonary disease, NTM lymphatic disease, disseminated NTM disease, and / or other NTM disease.

[0057] Further, the pirlimycin also includes a pharmaceutically acceptable salt or ester.

[0058] Still further, the pharmaceutically acceptable salt includes, but is not limited to, acetate, hydrochloride, and other medicinal salts.

[0059] Still further, the pharmaceutically acceptable ester includes, but is not limited to, decanoate.

[0060] In a third aspect, the present application provides a use of pirlimycin and bedaquiline in combination in the preparation of a pharmaceutical composition for resisting mycobacterial infection, wherein the pirlimycin and bedaquiline in combination have an effect of resisting mycobacterial infection, and the pirlimycin can enhance the activity of bedaquiline in resisting mycobacterial infection.

[0061] Further, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria, and / or Mycobacterium leprae.

[0062] Further, the Mycobacterium tuberculosis includes a clinical isolate of Mycobacterium tuberculosis, a standard strain of Mycobacterium tuberculosis, and / or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

[0063] Further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0064] Further, the disease caused by the Mycobacterium tuberculosis includes, but is not limited to, drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, and extrapulmonary tuberculosis.

[0065] Still further, the drug-resistant tuberculosis includes, but is not limited to, single-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, extensively drug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0066] Further, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, and bacteriologically negative pulmonary tuberculosis.

[0067] Further, the pulmonary tuberculosis includes, but is not limited to, lymphatic tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0068] Further, the Mycobacterium tuberculosis includes multi-drug resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0069] Further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, and Mycobacterium microti.

[0070] Further, the non-tuberculosis Mycobacterium includes, but is not limited to, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium ulcerans, and / or Mycobacterium marinum, etc.

[0071] Further, the disease caused by the non-tuberculosis Mycobacterium is selected from one or more of NTM pulmonary disease, NTM lymphatic disease, disseminated NTM disease, and / or other NTM disease.

[0072] Further, the pharmaceutical composition can further contain other active ingredients against Mycobacterium infection.

[0073] Further, one or more pharmaceutically acceptable carriers can be added to the pharmaceutical composition.

[0074] Further, the pharmaceutical composition can be prepared in the form of injection, tablet, powder, granule, capsule, oral solution, injection preparation, or aerosol, etc.; and the above-mentioned various dosage forms of the pharmaceutical composition can be prepared according to the conventional method in the field of pharmacy.

[0075] Further, the preparation can be one or more of ordinary preparation, sustained-release preparation, controlled-release preparation, and / or various microparticle drug delivery systems.

[0076] Further, the tablet can widely use various carriers known in the art, including one or more of diluent and absorbent, humectant and binder, disintegrant, disintegration inhibitor, absorption promoter, and / or lubricant.

[0077] Further, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and / or aluminum silicate.

[0078] Further, the wetting agent and the binder include, but are not limited to, one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, sugar paste, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and / or polyvinylpyrrolidone.

[0079] Further, the disintegrant includes, but is not limited to, one or more of dried starch, alginate, agar powder, fucoidin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, and / or ethyl cellulose.

[0080] Further, the disintegration inhibitor includes, but is not limited to, one or more of sucrose, glycerin tristearate, cocoa butter, and / or hydrogenated oil, etc.

[0081] Further, the absorption promoter includes, but is not limited to, one or more of quaternary ammonium salt and / or sodium dodecyl sulfate.

[0082] Further, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0083] Further, the tablet can be further manufactured into a coated tablet, including a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a double-layer tablet, and a multi-layer tablet.

[0084] Further, the injection preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder, and / or a suspension.

[0085] Further, the injection preparation can use all diluents commonly used in the art, including, but not limited to, one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and / or polyoxyethylene sorbitol fatty acid ester.

[0086] Further, the injection preparation can be added with an appropriate amount of one or more of sodium chloride, glucose, glycerin, a conventional cosolvent, a buffer, and / or a pH adjuster in order to prepare an isotonic injection.

[0087] Further, the various preparations can also be added with a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetener, or other materials, if necessary.

[0088] Further, the pharmaceutical composition can be introduced into the body by physical or chemical-mediated methods, such as intramuscularly, intradermally, subcutaneously, or intravenously.

[0089] Further, the impurity of the pharmaceutical composition can be removed by a conventional method.

[0090] Further, the pharmaceutically acceptable salt includes, but is not limited to, acetate, hydrochloride and other pharmaceutically acceptable salts.

[0091] Further, the pharmaceutically acceptable ester includes, but is not limited to, decanoate. Beneficial effects

[0092] In the present application, the combination of clofoctol and bedaquiline shows obvious enhanced activity against mycobacterial infection:

[0093] Compared with bedaquiline alone, 2 μg / ml of clofoctol combined with bedaquiline reduces the colony number of MmpL5-MmpS5 strain from 1 / 16 MIC to 2 MIC of bedaquiline to varying degrees.

[0094] Clofoctol as a synergist of bedaquiline can enhance the activity of bedaquiline against mycobacterial infection:

[0095] The minimum inhibitory concentration of clofoctol in vitro against Rv0678 mutant strain is 16 μg / ml, and the minimum inhibitory concentration of BDQ in vitro against Rv0678 mutant strain is 1 μg / ml. 1 μg / ml of clofoctol combined with BDQ reduces the MIC of Rv0678 mutant strain to BDQ from 1 μg / ml to 0.03 μg / ml (32-fold synergistic effect), which is lower than the MIC (0.06 μg / ml) of wild strain H37Rv, reverses the drug resistance of BDQ and further enhances the activity against mycobacterial infection. BRIEF DESCRIPTION OF DRAWINGS

[0096] Figure 1 Time-kill curve of clofoctol (ZUC) combined with bedaquiline (BDQ) on Mycobacterium tuberculosis H37Rv.

[0097] Figure 2 Time-kill curve of clofoctol (ZUC) combined with bedaquiline (BDQ) on Mycobacterium tuberculosis MmpL5-mmpS5 overexpression strain.

[0098] Figure 3 Time-kill curve of different concentrations of bedaquiline (BDQ) on MmpL5-MmpS5 overexpression strain.

[0099] Figure 4 Time-kill curve of different concentrations of clofoctol (ZUC) combined with bedaquiline (BDQ) on MmpL5-MmpS5 overexpression strain. DETAILED DESCRIPTION

[0100] The specific embodiments of the present application are further described below. It should be noted that the description of these embodiments is intended for the purpose of aiding in the understanding of the present application, and is not intended to limit the present application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0101] The experimental methods in the following examples are all conventional methods, and the experimental materials used in the following examples are all commercially available, unless otherwise specified.

[0102] The term

[0103] Thiothixene: Thiothixene is a thioxanthene derivative, which is a cis-isomer of chlorpromazine. It acts by blocking dopamine receptors, and has significant antipsychotic effect and specific sedative effect, and is especially suitable for patients with schizophrenia. Thiothixene has low acute toxicity and chronic toxicity, and no toxicity is found at therapeutic dose; no special toxicity needs to be paid attention to when used by women of childbearing age; no mutagenicity and carcinogenicity, etc. It is especially suitable for elderly patients.

[0104] Bedaquiline (BDQ): The chemical name of bedaquiline is 1-(6-bromo-2-methoxyquinoline-3-yl)-4-dimethylamino-1-phenyl-2-(1-naphthyl)-2-butanol, and the trade name is Sirturo. It is a new type of diarylquinoline anti-mycobacterial drug, which can affect the ATP synthesis of Mycobacterium tuberculosis by inhibiting the activity of Mycobacterium tuberculosis ATP synthase proton pump, thereby playing an antibacterial and bactericidal effect. It is clinically used for the treatment of adult multidrug-resistant pulmonary tuberculosis (MDR-PTB).

[0105] The synergist in the present application refers to thiothixene. When thiothixene and bedaquiline are used together, the activity of bedaquiline against mycobacterial infection can be enhanced. The drug composition after the combination of thiothixene and bedaquiline has enhanced anti-tuberculosis ability compared with the use of bedaquiline alone.

[0106] MIC (minimum inhibitory concentration): MIC is an index for measuring the antibacterial activity of an antibacterial drug, and refers to the minimum drug concentration that can inhibit the growth of a pathogenic bacterium in a culture medium after 7 to 10 days of in vitro culture of Mycobacterium tuberculosis. Pharmacodynamic interactions in vitro refer to the evaluation of the interactions and effective dose of a study drug, thereby determining the feasibility of further development of the drug as a treatment regimen for pulmonary tuberculosis. The commonly used quantitative method is the checkerboard dilution method. Taking the interaction of two drugs as an example, first, the MIC of each of the two study drugs for Mycobacterium tuberculosis is determined, and according to the MIC value, the highest concentration is set to 2xMIC of the single drug, and then 2-fold dilution is performed one by one (in the vertical column and horizontal column of the square, respectively), and each tube (well) contains a mixture of different concentrations of the two drugs. Generally, 6 to 8 dilution degrees are designed. The initial inoculum is 5x105CFU / mL, and incubation is performed at 37°C for 7 days, and the results are observed and the fractional inhibitory concentration index (FICI) is calculated. The FICI index calculation formula is (MIC of drug A when used in combination) / (MIC of drug A when used alone)+(MIC of drug B when used in combination) / (MIC of drug B when used alone). According to the FICI value, it is judged to be synergistic (≤0.5), additive (0.5-1), irrelevant (1-2), and antagonistic (>2). FICI is one of the pharmacodynamic (PD) parameters of antibacterial drugs, and is an index for combined drug sensitivity of two antibacterial drugs (when two antibacterial drugs are used at the same time, synergistic, additive, irrelevant, and four situations can occur).

[0107] Time-kill curves can dynamically observe the interaction between a drug (compound) and Mycobacterium tuberculosis, and can compare the effects of different concentrations of compounds on the growth of Mycobacterium tuberculosis at different action times, thereby providing more basis for evaluating the activity of the compound against Mycobacterium tuberculosis infection. The experimental period of time-kill curves is usually 14 days, and Mycobacterium tuberculosis is continuously cultured in a drug-containing culture medium for 14 days, and during this period, part of the bacterial solution is taken every 3-4 days for solid culture, and after 3-4 weeks of culture, colony counting is performed. The time-kill curve is plotted with the time point of taking the bacterial solution as the horizontal coordinate (x-axis) and the Log10 of the colony count result as the vertical coordinate (y-axis). Time-kill curves are also used to evaluate the interaction between each drug in a drug combination in vitro.

[0108] Verapamil: A derivative of strychnine, commonly used as its hydrochloride salt, which is a white powder; odorless. Soluble in methanol, ethanol or chloroform, soluble in water. Melting point 140-145°C. It was introduced as a coronary vasodilator in 1962. Verapamil, also known as isoptin, pacerone, is a calcium channel blocker. In recent years, it is used for the treatment of hypertension, angina pectoris, arrhythmia, cerebrovascular disease, digital vasospasm, abdominal pain, esophageal achalasia, migraine, pulmonary hypertension and prevention of premature birth.

[0109] Examples

[0110] Example 1 Screening of Perlapine

[0111] Mycobacterium tuberculosis (Rv0678 mutant: BDQ MIC = 1 μg / ml) of Rv0678 mutant (MmpL5-MmpS5 overexpression) was used as a functional screening model to screen compounds from a drug library that can reduce the MIC of bedaquiline.

[0112] The best active perlapine for synergizing bedaquiline against mycobacterial infection was found by screening a library of 4000 compounds, and its structure is as follows:

[0113] Example 2 Determination of minimum inhibitory concentration and synergistic activity of perlapine by MABA method

[0114] 2.1 Test content

[0115] This part uses microplate Alamar Blue assay (MABA) to determine the minimum inhibitory concentration.

[0116] Add culture medium and drug stock solution to the microwells:

[0117] Weigh the drug powder, add liposoluble drugs to DMSO to dissolve, so that the concentration of the drug stock solution is 2 mg / ml. Take the logarithmic growth phase of Mycobacterium tuberculosis and culture it in the microwells. Measure the OD value of each well at 570 nm wavelength, wherein OD (strain) = OD (bacterial solution) - OD (7H9 culture medium). OD = 0.1 corresponds to 1 x 10 7 CFU / mL, dilute each bacterial solution so that the final concentration of each strain is set to 1 x 10 5 CFU / mL.

[0118] Dilute the drug in the first column of microwells to the tenth column by double dilution method, and the final concentration of each microwell bacterial solution is 1 x 10 5CFU / mL. Cultures were incubated for 7 days, then the micro-wells were incubated after adding Tween-80 solution and 20 Alamar blue indicator. The next day, color changes were observed and the fluorescence values of each well were measured at excitation wavelengths of 530 nm and 590 nm, respectively. The color of each well was recorded, with blue indicating no growth of the strain and red indicating growth of the strain; the MIC was the lowest drug concentration that changed from blue to red.

[0119] The method for measuring the joint activity was the same as the MIC, except that 1 μg / mL of beclomycin or 10 μg / mL of verapamil was added to the medium, and the subsequent operations were performed using medium containing 1 μg / mL of beclomycin or 10 μg / mL of verapamil. The first 6 wells were used as negative control wells, and the last 6 wells were used as positive control wells after adding the diluted bacterial solution. Then, 198 μL of medium containing 1 μg / mL of beclomycin or 10 μg / mL of verapamil was added to the first column of micro-wells in rows B-H of the 96-well plate, and 100 μL of medium containing 1 μg / mL of beclomycin or 10 μg / mL of verapamil was added to the remaining micro-wells. 2 μL of BDQ stock solution was added to the first column of micro-wells in rows B-H, and the drug solution and medium containing 1 μg / mL of beclomycin or 10 μg / mL of verapamil were mixed and then the operation was repeated in the last column of micro-wells using a two-fold dilution method. In this way, the MIC of bedaquiline under the joint action of a fixed concentration of beclomycin or verapamil was determined, as well as the fold reduction.

[0120] 2.2 Test Results

[0121] The minimum inhibitory concentration of beclomycin for the Rv0678 mutant strain in vitro was 16 μg / mL, and the minimum inhibitory concentration of BDQ for the Rv0678 mutant strain in vitro was 1 μg / mL (as shown in Table 1). The combination of 1 μg / mL of beclomycin and BDQ reduced the MIC of BDQ for the Rv0678 mutant strain from 1 μg / mL to 0.03 μg / mL (32-fold synergistic effect), which was lower than the MIC of the wild strain H37Rv (0.06 μg / mL), reversed the resistance of BDQ, and further enhanced the activity against Mycobacterium infection. The efflux pump inhibitor verapamil at a concentration of 10 μg / mL had no effect on the MIC of BDQ in the Rv0678 mutant strain (as shown in Table 1).

[0122] Table 1 Synergistic activity of beclomycin on bedaquiline against the Rv0678 mutant strain

[0123] Note: MIC of BDQ for the wild strain H37Rv (0.06 μg / mL)

[0124] Example 3: Measurement of the joint activity of beclomycin and different anti-tuberculosis drugs against different strains by the checkerboard method

[0125] 3.1 Test content

[0126] The in vitro activity of zuclopenthixol combined with different anti-tuberculosis drugs and the activity of Mycobacterium tuberculosis with different sensitivity to bedaquiline were determined by chessboard method.

[0127] Based on the MIC of each drug alone, 7 concentration gradients were set, with a concentration range of 4xMIC-1 / 16xMIC. The drugs were distributed in a chessboard manner, as shown in Table 2. In 96-well plates X1-X7 were 4xMIC-1 / 16xMIC of A drug (zuclopenthixol: ZUC), Y1-Y7 were 4xMIC-1 / 16xMIC of B drug (bedaquiline: BDQ; isoniazid: INH; PA-824; moxifloxacin: MXF and linezolid: LZD). A9-H9 and A10-H10 columns were negative control wells and positive control wells, respectively (as shown in Table 2). The A drug was diluted by two-fold vertically, and the B drug was diluted by two-fold horizontally.

[0128] After 7 days of culture, Alamar Blue indicator was added. After 24 h, the color change of A drug alone and B drug alone was observed to determine their respective MIC wells. The combined effect of the two drugs was determined by calculating the FICI value according to the formula:

[0129] FICI = MICA combined / MICA alone + MICB combined / MICB alone

[0130] Wherein, MICA alone and MICB alone represent the MIC of A drug and B drug alone on Mycobacterium tuberculosis, respectively, and MICA combined and MICB combined represent the lowest concentrations of A drug and B drug after combined application to prevent the color from changing from blue to pink.

[0131] Table 2 Two-dimensional chessboard design

[0132] Note: X is A drug, i.e. zuclopenthixol: ZUC; X1-X7 are 7 concentration gradients of zuclopenthixol: 4xMIC-1 / 16xMIC.

[0133] Y is B drug, which is bedaquiline: BDQ; isoniazid: INH; PA-824; moxifloxacin: MXF and linezolid: LZD, respectively; Y1-Y7 are 7 concentration gradients of B drug: 4xMIC-1 / 16xMIC.

[0134] 3.2 Test results

[0135] The checkerboard method experiment showed that the in vitro synergistic anti-mycobacterial infection activity of beclometasone and BDQ for Rv0678 mutant strain was the best, and the FICI thereof was 0.156. In addition to the synergistic anti-tuberculosis effect of beclometasone and BDQ, beclometasone had no interaction with isoniazid (INH), PA-824, moxifloxacin (MXF), linezolid (LZD) and other anti-tuberculosis drugs (as shown in Table 3).

[0136] Table 3 Activity of beclometasone and anti-tuberculosis drugs in combination for Rv0678 mutant strain

[0137] Note: The MIC of BDQ alone is 1 μg / mL, the combined MIC is 0.03125 μg / mL, and the concentration of beclometasone is 2 μg / mL; the MIC of INH alone is 0.04 μg / mL, the combined MIC is 0.00125 μg / mL, and the concentration of beclometasone is 16 μg / mL; the MIC of PA-824 alone is 0.16 μg / mL, the combined MIC is 16 μg / mL, and the concentration of beclometasone is 16 μg / mL; the MIC of MFX alone is 1 μg / mL, the combined MIC is 0.03 μg / mL, and the concentration of beclometasone is 16 μg / mL; the MIC of LZD alone is 0.29 μg / mL, the combined MIC is 0.009 μg / mL, and the concentration of beclometasone is 16 μg / mL.

[0138] Further, the activity of beclometasone combined with bedaquiline in different bedaquiline-resistant strains was determined, and the synergistic activity was studied on the constructed genetically engineered MmpL5-MmpS5 overexpression strain, the sensitive strain H37Rv, different Rv0678 mutant strains, and the atpE mutant strain of bedaquiline target gene.

[0139] The checkerboard method for determining interaction found that beclometasone and bedaquiline had synergistic activity in all the strains determined, but the synergistic activity in Rv0678 mutant strains and MmpL5-MmpS5 overexpression strains was better than that in H37Rv strains and atpE mutant strains (as shown in Table 4).

[0140] Table 4 Activity of beclometasone combined with bedaquiline in different strains against mycobacterial infection

[0141] Note: H37Rv standard strain, BDQ single MIC is 0.06 μg / mL, combined MIC is 0.015 μg / mL, the concentration of clofazimine is 2 μg / mL; MmpL5-MmpS5 overexpression strain, BDQ single MIC is 0.5 μg / mL, combined MIC is 0.03125 μg / mL, the concentration of clofazimine is 2 μg / mL; Rv0678 mutant strain 2, BDQ single MIC is 1 μg / mL, combined MIC is 0.0625 μg / mL, the concentration of clofazimine is 2 μg / mL; atpE mutant strain, BDQ single MIC is 1.25 μg / mL, combined MIC is 0.3125 μg / mL, the concentration of clofazimine is 8 μg / mL.

[0142] Example 4 Evaluation of the antibacterial activity of the combination of clofazimine and bedaquiline by time-kill curve method

[0143] 4.1 Test content

[0144] In order to investigate the dynamic bactericidal activity of clofazimine combined with bedaquiline, a combined action bactericidal curve determination study was carried out.

[0145] The evaluation of the combination of antibacterial drugs includes single drug wells and two-drug combination wells, and ZUC 10 μg / mL and BDQ 1 / 2xMIC are selected for time-kill test evaluation.

[0146] Prepare drug and drug combination solutions of the corresponding concentrations, add H37Rv or Rv0678 mutant strain bacterial solution, so that the concentration of each bacterial solution reaches 2x10 5 CFU / mL, and culture. On the day of culture (D0) and on the 3rd, 7th, 10th and 14th days of culture, the bacterial solution stock or diluted bacterial solution was taken from the 24-well plate and inoculated on solid culture medium, spread evenly, and after 4 weeks of culture, colony counting was carried out. The culture time points of Mycobacterium tuberculosis were taken as the abscissa, and the logarithmic values of the number of colonies at different culture time points were taken as the ordinate, and the time-kill curve graphs of clofazimine and bedaquiline combination were drawn respectively.

[0147] 4.2 Test results

[0148] 10 μg / ml of clofazimine combined with 1 / 2MIC of bedaquiline can exert antibacterial activity against Mycobacterium infection on H37Rv and MmpL5-MmpS5 overexpression strains, while 10 μg / ml of clofazimine or 1 / 2MIC of bedaquiline alone have no bactericidal activity (as shown in Figure 1).

[0149] The bactericidal effect of clofazimine combined with bedaquiline on Rv0678 mutant strain is more obvious, and 10 days reach sterilization, while for H37Rv it takes 14 days to reach sterilization (as shown in Figure 2).

[0150] To further reduce the concentration of the effect of ball clozapine, combined with different concentrations of bedaquiline MmpL5-MmpS5 strain, compared with bedaquiline alone (as shown in figure 3), 2 μg / ml of ball clozapine combined with bedaquiline, from 1 / 16 MIC to 2 MIC of MmpL5-MmpS5 strain from bedaquiline, different degrees of reduction of colony number, showing obvious activity of enhancing the resistance to mycobacterial infection (as shown in figure 4).

Claims

1. A pharmaceutical composition for combating mycobacterial infection, said pharmaceutical composition comprising beclomethasone and bedaquiline, said pharmaceutical composition having at least one of the following effects: a) inhibiting mycobacterial activity; b) combating mycobacterial infection; c) preventing and / or treating mycobacterium-induced diseases.

2. Use of beclomethasone in combination with bedaquiline for the preparation of a pharmaceutical composition for combating mycobacterial infection, said beclomethasone and bedaquiline combination exerting an effect of combating mycobacterial infection, said beclomethasone being capable of enhancing the activity of bedaquiline against mycobacterial infection.

3. The pharmaceutical composition according to claim 1 or 2 can be prepared in the form of injection, tablet, powder, granule, capsule, oral solution, injection preparation or aerosol, etc.; the pharmaceutical composition in the above-mentioned various forms can be prepared according to the conventional method in the field of pharmacy.

4. The pharmaceutical composition according to claim 3 can also be added with coloring agent, preservative, perfume, flavoring agent, sweetening agent or other materials, if necessary.

5. The pharmaceutical composition according to any one of claims 1 to 3 can further contain other active ingredients against mycobacterial infection.

6. The pharmaceutical composition according to any one of claims 1 to 3 can further contain one or more pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to any one of claims 1 to 3 can be introduced into the body by physical or chemical mediated methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

8. Use of beclomethasone as a potentiator of the effect of bedaquiline against mycobacterial infection, said beclomethasone being capable of enhancing the activity of bedaquiline against mycobacterial infection when beclomethasone is used in combination with bedaquiline.

9. The mycobacterium according to any one of claims 1 to 7 is selected from the group consisting of Mycobacterium tuberculosis, non-tuberculous mycobacteria and / or Mycobacterium leprae. The pharmaceutically acceptable salts include, but are not limited to, acetate, hydrochloride and other pharmaceutically acceptable salts; the pharmaceutically acceptable esters include, but are not limited to, decanoate. ​ ​ 10. The bead biochaninol of any one of claims 1-7 further comprising a pharmaceutically acceptable salt or ester, wherein, ​

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