Combined synergistic pharmaceutical composition for resisting mycobacterial infection

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of a combined synergistic pharmaceutical composition in resisting mycobacterial infection. The pimozide (PMZ) provided by the invention can be used as an active component and applied to the medicines for combined anti-mycobacterium tuberculosis, a new application of the PMZ is developed, the PMZ is combined with the anti-tuberculosis medicines (Bedaquiline BDQ, clofazimine CFZ and the like) to improve the anti-tuberculosis activity, the PMZ can be used as a synergist, the treatment course is expected to be shortened, the drug resistance is reduced, and a new treatment strategy is provided. The MIC (Minimum Inhibitory Concentration) of the mycobacterium tuberculosis sensitive bacterium H37Rv to the BDQ is reduced from 0.06 g / ml to 0.0038 g / ml (the synergy is 16 times) by combining the PMZ with the concentration of 0.5 g / ml with the BDQ; 0.5 g / ml of pimozide is combined with BDQ, so that the MIC (minimal inhibitory concentration) of the Rv0678 mutant strain on the BDQ is reduced from 1g / ml to 0.0313 g / ml (synergistic effect is 32 times) and is lower than that of a wild strain H37Rv MIC (0.06 g / ml), the drug resistance of the BDQ is reversed, and the activity of resisting mycobacterium infection is further enhanced. And the mycobacterium tuberculosis can be completely killed after the combination of 1.5 mu g / ml of PMZ and 0.03 mu g / ml of BDQ for 14 days.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to application of a combined synergistic drug composition in resisting mycobacterial infection. Background Art

[0002] Mycobacterial infection is a disease caused by bacteria of the genus Mycobacterium ( Mycobacterium ) bacteria, including Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) and nontuberculous mycobacteria (NTM). Mycobacterium infection can affect multiple organ systems, including the lungs, skin, lymph nodes, bones, etc. Mycobacterium tuberculosis is the main pathogen that causes tuberculosis and is mainly transmitted through the respiratory tract. After infection, patients may develop latent infection or active tuberculosis. Latent infection may turn into active disease under certain conditions (such as immunodeficiency, diabetes, etc.) (Lu Lunshan. Diagnosis and prevention and treatment measures of Mycobacterium tuberculosis infection [J]. World Latest Medical Information Abstracts, 2016.).

[0003] Tuberculosis is caused by Mycobacterium tuberculosis ( Mycobacterium tuberculosis) is a chronic infectious disease caused by tuberculosis, which is mainly transmitted through the air, especially when patients cough, sneeze or spit, releasing droplets containing the bacteria (SeunNurudeen Akorede, Ayodotun Edward Ajayi et al. "Knowledge of Tuberculosisamong Tuberculosis Patients Attending Federal Medical Centre, Owo, OndoState, Nigeria." Unnes Journal of Public Health(2021).). Tuberculosis can affect multiple organs, but lung infection is the most common, called pulmonary tuberculosis. Tuberculosis remains a serious public health problem worldwide. The main method of treating tuberculosis is the use of anti-tuberculosis drugs, including isoniazid, rifampicin, ethambutol and pyrazinamide (Anny Thuraidah, Rima Agnes Widya Astuti et al. "Anemia dan Lama KonsumsiObat Anti Tuberculosis." Medical laboratory technology(2017).). Treatment is divided into an intensive phase and a consolidation phase. The intensive phase is treated twice a day to reduce the number of bacteria in the body, while the consolidation phase lasts longer but at a lower dose. However, due to the drug resistance of Mycobacterium tuberculosis, treatment becomes complicated and difficult (Xu Yin, Meng Xianmin, Zhang Yongxin. Current status of drug-resistant tuberculosis and research progress of anti-tuberculosis drugs [J]. Shanghai Medicine, 2013.). Drug-resistant tuberculosis (especially multidrug-resistant tuberculosis, MDR-TB) has become a major challenge to global public health (Shen Xin, Song Shenchao, Lei Shiguang. Current status of drug-resistant tuberculosis and prevention and control measures [J]. Occupation and Health, 2017.). The treatment of drug-resistant tuberculosis faces many challenges. Although chemotherapy is still the main treatment, its effect is not ideal for patients with multidrug-resistant and extensively drug-resistant tuberculosis (Zhou Wenqiang, Chu Naihui. Progress in the treatment of drug-resistant tuberculosis [J]. Chinese Journal of Clinicians, 2020.). The situation of drug-resistant tuberculosis is serious. There are limited drug options for treating drug-resistant tuberculosis, long treatment courses, and a low cure rate of about 63%.

[0004] In recent years, some new anti-tuberculosis drugs have been developed. For example, in 2019 and 2022, WHO listed bedaquiline (BDQ) and clofazimine (CFZ) as Group A and Group B drugs in the core drugs for the treatment of multidrug-resistant tuberculosis, respectively. However, clinical drug-resistant strains of the new drug BDQ have been found, especially cross-resistant strains of BDQ and CFZ have been isolated from tuberculosis patients who have not been exposed to and have not used the drug ( Rv0678Gene mutations) (Xu J, et al. Antimicrob Agents Chemother, 2017; 61(6): e00239-17). The Rv0678 gene encodes a transcriptional regulator that regulates the expression of the MmpS5-MmpL5 efflux pump. When the Rv0678 gene mutates, it leads to upregulation of the efflux pump, thereby increasing the minimum inhibitory concentration (MIC) of BDQ and CFZ, and thus producing cross-resistance (K. Kaniga, N. Lounis et al. “Impact of Rv0678 mutations on patients with drug-resistant TB treated with bedaquiline.” TheInternational Journal of Tuberculosis and Lung Disease(2022).). Specifically, mutations in the Rv0678 gene reduce the susceptibility of BDQ to Mycobacterium tuberculosis, and this mutation also leads to reduced sensitivity to CFZ. This cross-resistance has been confirmed in some clinical isolates. For example, in the resistant strains reported in Pakistan, the Rv0678 gene mutant strains showed high MIC values ​​for both BDQ and CFZ (A. Ghodousi, A. Rizvi et al. "Acquisition of Cross-Resistance to Bedaquiline and Clofazimine following Treatment for Tuberculosis in Pakistan." Antimicrobial Agents and Chemotherapy (2019).). In addition, the study also found that some Mycobacterium tuberculosis strains carried Rv0678 mutations even in the absence of previous treatment with CFZ or BDQ, indicating that cross-resistance may be a naturally occurring resistance mechanism (Cristina Villellas, N. Coeck et al. "Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline." Journal of Antimicrobial Chemotherapy (2016).).

[0005] The treatment of drug-resistant tuberculosis requires longer time and more expensive drugs, which places a huge burden on the medical system and patients' families. Therefore, there is an urgent need to develop new drugs. The research and development cycle of anti-tuberculosis drugs is long and difficult. Developing anti-tuberculosis drug enhancers to enhance the anti-tuberculosis activity of existing drugs and reverse their resistance is a very important treatment strategy. Antimicrobial drug enhancers themselves are not antibiotics, but when used in combination with antibiotics, they can reverse bacterial resistance and restore the sensitivity of resistant bacteria to antibiotics, while slowing down the development of bacterial resistance. They are an effective and sustainable strategy to combat resistant bacteria.

[0006] Pimozide (PMZ) belongs to the dibenzopiperidine class of compounds (C. Lorenzo, J. Koo. "Pimozide in dermatologic practice: a comprehensive review.." American journal of clinical dermatology (2004).), which was launched in 1970. It is an antipsychotic drug used to treat acute and chronic schizophrenia, Tourette syndrome, etc. It is a specific central dopamine receptor antagonist. The chemical structure of Pimozide is 1-(1-[4,4-bis(4-fluorophenyl) phenyl]-4-piperidinyl)benzo[1,2-a:5,4-e']dibenzopiperidine; there is no report on its combined anti-tuberculosis drug synergistic anti-mycobacterial activity. Summary of the invention

[0007] In the present invention, it is found that the combination of pimozide and bedaquiline can enhance the anti-mycobacterial infection activity of bedaquiline and / or clofazimine, and the drug combined with pimozide and bedaquiline and / or clofazimine has the effect of anti-mycobacterial infection. Based on this, the present invention is completed.

[0008] In a first aspect, the present invention provides a combined synergistic pharmaceutical composition for resisting mycobacterial infection, wherein the combined synergistic pharmaceutical composition comprises pimozide in combination with another drug for resisting mycobacterial infection, wherein the other drug for resisting mycobacterial infection is bedaquiline and / or clofazimine, and the pharmaceutical composition has at least one of the following effects: a) inhibiting mycobacterial activity; b) protection against mycobacterial infections; c) Prevention and / or treatment of diseases caused by mycobacteria.

[0009] Furthermore, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria and / or Mycobacterium leprae.

[0010] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0011] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0012] Furthermore, the diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0013] Furthermore, the drug-resistant tuberculosis includes but is not limited to mono-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0014] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

[0015] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0016] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0017] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0018] Furthermore, the non-tuberculous mycobacteria include but are not limited to Mycobacterium avium ( M. avium ), Mycobacterium intracellulare ( Intracellular M. ), Mycobacterium kansasii ( M. kansasii ), Mycobacterium fortuitum ( M. fortuitous ), Mycobacterium abscessus ( M. abscess ), Mycobacterium ulcerans ( Ulcerative muscle ) and / or Mycobacterium marinum ( Marine M. )wait.

[0019] Furthermore, the disease caused by nontuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphatic disease, disseminated NTM disease and / or other NTM diseases.

[0020] Furthermore, the pharmaceutical composition may also contain other active ingredients against mycobacterial infection.

[0021] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0022] Furthermore, the pharmaceutical composition can be prepared into various forms such as tablets, powders, granules, capsules, oral liquids, injection preparations or aerosols; the above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0023] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0024] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption enhancers and / or lubricants.

[0025] Furthermore, 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.

[0026] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0027] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0028] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0029] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0030] Furthermore, 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.

[0031] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0032] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0033] Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0034] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0035] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0036] Furthermore, the pharmaceutical composition can be introduced into the body by physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0037] Furthermore, the pimozide also includes pharmaceutically acceptable salts or esters.

[0038] In a second aspect, the present invention provides a use of pimozide as a synergist, wherein the function of the synergist is that pimozide can increase the anti-mycobacterial infection effect of bedaquiline and / or clofazimine, that is, when pimozide is used in combination with bedaquiline and / or clofazimine, pimozide can enhance the anti-mycobacterial infection activity of bedaquiline and / or clofazimine.

[0039] Furthermore, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria and / or Mycobacterium leprae.

[0040] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0041] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0042] Furthermore, the diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0043] Furthermore, the drug-resistant tuberculosis includes but is not limited to mono-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0044] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

[0045] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0046] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0047] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0048] Furthermore, the non-tuberculous mycobacteria include but are not limited to Mycobacterium avium ( M. avium ), Mycobacterium intracellulare ( Intracellular M. ), Mycobacterium kansasii ( M. kansasii )、Mycobacterium fortuitum ( M. fortuitous ), Mycobacterium abscessus ( M. abscess ), Mycobacterium ulcerans ( Ulcerative muscle ) and / or Mycobacterium marinum ( Marine M. )wait.

[0049] Furthermore, the disease caused by nontuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphatic disease, disseminated NTM disease and / or other NTM diseases.

[0050] Furthermore, the pimozide also includes pharmaceutically acceptable salts or esters.

[0051] In a third aspect, the present invention provides a use of pimozide in combination with bedaquiline and / or clofazimine in the preparation of an anti-mycobacterial infection pharmaceutical composition, wherein the pimozide in combination with bedaquiline and / or clofazimine exerts an anti-mycobacterial infection effect, and the pimozide can enhance the anti-mycobacterial infection activity of bedaquiline and / or clofazimine.

[0052] Furthermore, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria and / or Mycobacterium leprae.

[0053] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0054] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0055] Furthermore, the diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0056] Furthermore, the drug-resistant tuberculosis includes but is not limited to mono-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0057] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

[0058] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0059] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0060] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0061] Furthermore, the non-tuberculous mycobacteria include but are not limited to Mycobacterium avium (M. avium ), Mycobacterium intracellulare ( Intracellular M. ), Mycobacterium kansasii ( M. kansasii )、Mycobacterium fortuitum ( M. fortuitous ), Mycobacterium abscessus ( M. abscess ), Mycobacterium ulcerans ( Ulcerative muscle ) and / or Mycobacterium marinum ( Marine M. )wait.

[0062] Furthermore, the disease caused by nontuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphatic disease, disseminated NTM disease and / or other NTM diseases.

[0063] Furthermore, the pharmaceutical composition may also contain other active ingredients against mycobacterial infection.

[0064] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0065] Furthermore, the pharmaceutical composition can be prepared into various forms such as tablets, powders, granules, capsules, oral liquids, injection preparations or aerosols; the above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0066] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0067] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption enhancers and / or lubricants.

[0068] Furthermore, 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.

[0069] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0070] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0071] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0072] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0073] Furthermore, 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.

[0074] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0075] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0076] Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0077] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0078] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0079] Furthermore, the pharmaceutical composition can be introduced into the body by physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0080] Furthermore, the pimozide also includes pharmaceutically acceptable salts or esters.

[0081] Beneficial Effects The PMZ described in the present invention can be used as an active ingredient in a drug combined with anti-tuberculosis Mycobacterium tuberculosis, which opens up a new use for PMZ. It can be used in combination with anti-tuberculosis drugs (bedaquiline, clofazimine, etc.) to enhance the anti-tuberculosis activity. PMZ can be used as a synergist, which is expected to shorten the course of treatment and reduce drug resistance. It will be a new treatment strategy. 0.5 µg / ml of pimozide combined with BDQ reduced the MIC of Mycobacterium tuberculosis sensitive bacteria H37Rv to BDQ from 0.06µg / ml to 0.0038µg / ml (16 times the synergy); 0.5 µg / ml of pimozide combined with BDQ Rv0678 The mutant strain's MIC for BDQ decreased from 1µg / ml to 0.0313µg / ml (32-fold increase), which was lower than the wild-type H37Rv MIC (0.06µg / ml), reversing BDQ resistance and further enhancing anti-mycobacterial activity. 1.5μg / ml PMZ combined with 0.03μg / ml BDQ for 14 days was able to completely kill Mycobacterium tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The anti-tuberculosis activity of different concentrations of PMZ combined with BDQ in macrophages infected with Mycobacterium tuberculosis H37Rv.

[0083] Figure 2 This is the time-kill curve of PMZ combined with BDQ against Mycobacterium tuberculosis. DETAILED DESCRIPTION

[0084] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0085] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0086] the term Bedaquiline (BDQ): Its chemical name is 1-(6-bromo-2-methoxyquinolin-3-yl)-4-dimethylamino-1-phenyl-2-(1-naphthyl)-2-butanol, and its trade name is Sirturo. It is a new type of diarylquinoline antimycobacterial drug. It inhibits the activity of the proton pump of the ATP synthase of Mycobacterium tuberculosis to affect the ATP synthesis of Mycobacterium tuberculosis, thereby exerting antibacterial and bactericidal effects. It is clinically used to treat multidrug-resistant pulmonary tuberculosis (MDR-PTB) in adults.

[0087] The synergist in the present invention refers to pimozide. When pimozide and bedaquiline are used in combination, the anti-mycobacterial infection activity of bedaquiline can be enhanced. The anti-tuberculosis ability of the pharmaceutical composition after pimozide and bedaquiline are combined is enhanced compared with the use of bedaquiline alone.

[0088] MIC (minimum inhibitory concentration): Minimum inhibitory concentration is an indicator for measuring the antibacterial activity of antibacterial drugs. It refers to the lowest drug concentration that can inhibit the growth of pathogens in the culture medium after 7 to 10 days of in vitro culture of Mycobacterium tuberculosis. In vitro pharmacodynamic interactions refer to the evaluation of the interaction and effective dose of the research drugs, so as to determine the feasibility of further development as a drug for the treatment of pulmonary tuberculosis. The commonly used quantitative method is the chessboard dilution method. Taking the interaction between two drugs as an example, the MIC of the two research drugs against Mycobacterium tuberculosis is first determined respectively. According to the MIC value, the highest concentration is set as 2×MIC of the single drug, and then 2-fold dilutions are performed one by one (in the vertical and horizontal columns of the square array respectively). Each tube (well) contains a mixture of different concentrations of the two drugs. Generally, 6 to 8 dilutions are designed. The initial inoculum is 5×105CFU / mL, incubated at 37℃ for 7 days, and the results are observed and the fractional inhibitory concentration index (FICI) is calculated. In this application, the FICI calculation formula is as follows: FICI = MIC A United / MIC A Single + MIC B United / MIC B alone Among them, MICA alone and MICB alone represent the MIC of drug A and drug B alone against Mycobacterium tuberculosis, and MICA combined and MICB combined represent the lowest concentrations of drug A and drug B that prevent the color from changing from blue to pink after the combined application of drug A and drug B. The in vitro activity judgment criteria of the two-drug combination: FICI ≤ 0.5, synergistic effect; FICI 0.5-4, irrelevant effect; FICI> 4, antagonistic effect.

[0089] Time-Kill Curves can dynamically observe the interaction between drugs (compounds) and Mycobacterium tuberculosis, and can compare the effects of different concentrations of compounds on the growth of Mycobacterium tuberculosis at different action times, providing more basis for evaluating the activity of compounds against mycobacterial infection. The experimental cycle of the time-kill curve is usually 14 days. Mycobacterium tuberculosis is cultured continuously in a drug-containing culture medium for 14 days. During this period, part of the bacterial solution is taken every 3-4 days to culture on a solid culture medium, and the colony count is performed after 3-4 weeks of culture. The time-kill curve is drawn with the time point of taking the bacterial solution as the horizontal axis (x-axis) and the Log10 of the colony count result as the vertical axis (y-axis). Time-kill curves are also used to evaluate the interaction between drugs in a drug combination in vitro.

[0090] Microplate Alamar Blue Assay (MAB A) is a fast, simple and low-cost chromatographic method for determining the minimum inhibitory concentration (MIC) of microorganisms. This method uses Alamar Blue dye as an indicator to assess bacterial growth by measuring color changes. In the study of drug resistance of Mycobacterium tuberculosis, MABA is widely used to evaluate the inhibitory effects of different drugs on tuberculosis.

[0091] Example Example 1 Screening of Pimozide pass Rv0678 Mutant strain (MmpL5-MmpS5 overexpression) of Mycobacterium tuberculosis ( Rv0678 The mutant strain (BDQMIC=1 µg / ml) was used as a functional screening model to screen compounds that could reduce the MIC of bedaquiline from the drug library.

[0092] From the screening of 4000 compound libraries, Pimozide was found to have the best activity against mycobacterial infection by enhancing bedaquiline, and its structural formula is as follows: Example 2 Determination of the Minimum Inhibitory Concentration and Combined Synergistic Activity of Pimozide by MABA Method 2.1 Test methods In this part, the microplate Alamar Blue assay (MAB A) was used to determine the MICs of PMZ and fixed PMZ combined with BDQ against Mycobacterium tuberculosis.

[0093] Mycobacterium tuberculosis was inoculated into 7H9 medium supplemented with 10% OADC enrichment solution, 0.2% glycerol and 0.05% Tween 80, and cultured in a constant temperature incubator until the logarithmic growth phase.

[0094] Take the Mycobacterium tuberculosis bacterial solution and culture medium in the logarithmic growth phase and place them in the well plate. Measure the OD value of each well at a wavelength of 570nm, where OD (strain) = OD (bacterial solution) - OD (7H9 culture medium). OD = 0.1 is equivalent to 1×10 7 CFU / mL, dilute each bacterial solution so that the final concentration of each strain is set to 1×10 5 CFU / mL.

[0095] Add culture medium and drug stock solution, dilute the drugs in the first column of microwells to the tenth column by two-fold dilution method, and the final concentration of bacterial solution in each microwell is 1×10 5 CFU / mL. On the 7th day of culture, Tween-80 and resazurin indicator were added to each microwell and incubation continued. The color change was observed the next day, and the fluorescence value of each well was measured at 530nm and 590nm excitation wavelengths, and the color of each well was recorded. Blue indicated no growth of the strain, and red indicated bacterial growth; MIC indicated the lowest drug concentration at which the blue color changed to red.

[0096] The combined activity was determined by the MIC assay method, except that PMZ at a final concentration of 2μg / ml, 1μg / ml, and 0.5μg / ml was added to the culture medium, and the subsequent operations were performed using this culture medium. The first 6 wells were used as negative control wells, and the diluted bacterial solution was added to the last 6 wells as positive control wells. Then, 198μL of the culture medium containing PMZ was added to the first column of microwells in the BH row of the 96-well plate, and 100μl of the culture medium containing PMZ was added to the remaining microwells. 2μl of BDQ stock solution was added to the first column of microwells in the BH row, and after mixing the drug solution and the culture medium containing PMZ, the operation was repeated to the last column of microwells by the two-fold dilution method. In this way, the MIC of bedaquiline under the combined action of a fixed concentration of PMZ and the multiple of reduction were determined.

[0097] 2.2 Test results As shown in Table 1, pimozide has an in vitro activity against Mycobacterium tuberculosis H37Rv and Rv0678 The minimum inhibitory concentration of the mutant strains was 3.13 μg / ml. Rv0678The minimum inhibitory concentration of the mutant strain was 1 µg / mL (as shown in Table 1). The combination of 0.5 µg / ml pimozide and BDQ reduced the MIC of Mycobacterium tuberculosis sensitive strain H37Rv to BDQ from 0.06 µg / ml to 0.0038 µg / ml (16-fold increase in efficacy). Rv0678 The mutant strain's MIC for BDQ decreased from 1µg / ml to 0.0313µg / ml (32-fold increase), which was lower than the wild-type H37Rv MIC (0.06µg / ml), reversing BDQ resistance and further enhancing anti-mycobacterial activity. As the concentration of pimozide increased, the MIC of BDQ decreased more significantly, showing a dose-effect relationship at 0.5-2µg / ml.

[0098] Example 3 Determination of the combined activity of pimozide and different anti-tuberculosis drugs against different strains by chessboard method 3.1 Test methods The checkerboard assay is a method based on MIC for determining the interaction between two drugs.

[0099] The chessboard method was used to determine the FIC values ​​of PMZ and BDQ, CFZ, INH, RFP, LVX, and PBTZ169 combinations against the standard strain H37Rv.

[0100] Based on the MIC of each drug when applied alone, a total of 7 concentration gradients were set up with a concentration range of 2×MIC-1 / 32×MIC. The drugs were distributed in a chessboard manner, as shown in Table 2. B2-H9 is 2×MIC-1 / 32×MIC of drug A (PMZ), A3-H9 is 2×MIC-1 / 32×MIC of drug B (BDQ, CFZ, INH, RFP, LVX, PBTZ169). Drug B was diluted twice horizontally and drug B was diluted twice vertically. The B10-H10 and B11-H11 columns were negative control wells and positive control wells, respectively. The MIC of each drug was determined by observing the color change, and the drug interaction relationship was determined by FICI. After 7 days of culture, Alamar Blue indicator was added. After 24 hours, the color changes of drug A and drug B were observed to determine their respective MIC wells. The combined effect of the two drugs was determined by calculating the FICI value according to the calculation formula: FICI=MIC A United / MIC A Single + MIC B United / MIC B alone Among them, MICA alone and MICB alone represent the MIC of drug A and drug B alone against Mycobacterium tuberculosis, and MICA combined and MICB combined represent the lowest concentrations of drug A and drug B that prevent the color from changing from blue to pink after the combined application of drug A and drug B. The in vitro activity judgment criteria of the two-drug combination: FICI ≤ 0.5, synergistic effect; FICI 0.5-4, irrelevant effect; FICI> 4, antagonistic effect.

[0101] Note: X is drug A, i.e., pimozide: PMZ; X1-X7 are 7 concentration gradients of PMZ: 2×MIC-1 / 32×MIC; Y is drug B, which are bedaquiline: BDQ; isoniazid: INH; rifampicin RFP; levofloxacin: LVX and PBTZ169; Y1-Y7 are 7 concentration gradients of drug B: 2×MIC-1 / 32×MIC.

[0102] 3.2 Test results As shown in Table 3, the chessboard assay showed that pimozide and BDQ had the best in vitro synergistic anti-mycobacterial activity against H37Rv, with FICI = 0.375. In addition to the synergistic anti-tuberculosis effect with BDQ, pimozide also had a synergistic effect with CFZ (FICI = 0.5), and had no interaction with anti-tuberculosis drugs such as INH, RFP, levofloxacin (LVX), and PBTZ-169. Note: When the MIC of BDQ alone is 0.06μg / mL and the MIC of the combination is 0.0075μg / mL, the concentration of PMZ is 0.75μg / mL; when the MIC of CFZ alone is 0.125μg / mL and the MIC of the combination is 0.03125μg / mL, the concentration of PMZ is 0.75μg / mL; when the MIC of INH alone is 0.0625μg / mL and the MIC of the combination is 0.03125μg / mL, the concentration of PMZ is 1.5μg / mL; when the MIC of RFP alone is 0.03125μg / mL and the MIC of the combination is 0.0156μg / mL, the concentration of PMZ is 0.75μg / mL; when the MIC of LVX alone is 0.5μg / mL and the MIC of the combination is 0.25μg / mL When the MIC of PBTZ169 alone was 0.0008 μg / mL and the MIC of the combination was 0.0002 μg / mL, the concentration of PMZ was 3 μg / mL.

[0103] As shown in Table 4, the activity of PMZ combined with BDQ / CFZ in different clinically isolated Mycobacterium tuberculosis strains was further determined. Strains 17851 and 13385 were sensitive to BDQ and CFZ, while strains 11492 and 12657 were resistant to BDQ and CFZ.

[0104] The chessboard assay showed that pimozide and BDQ / CFZ had an in vitro synergistic effect on clinical isolates of Mycobacterium tuberculosis, whether BDQ-sensitive strains (17851, 13385) or BDQ-resistant strains (11492, 12657). The synergistic anti-tuberculosis effect of pimozide and BDQ was better than that of CFZ, and its FICI was small.

[0105] Example 4 Determination of the anti-tuberculosis activity of PMZ combined with BDQ in macrophages 4.1 Test methods Mycobacterium tuberculosis is an intracellular parasite, and macrophages are its parasitic site.

[0106] This application uses mouse mononuclear macrophage J774A.1 infected with Mycobacterium tuberculosis H37Rv as a research model.

[0107] J774A.1 cells were cultured in DMEM complete medium containing 10% FBS. The cells were digested and a cell suspension was obtained, and the medium was diluted to a final concentration of 4×10 5 The diluted J774A.1 cells were added to the cell culture plate at a volume of 1 ml / well and incubated. H37Rv in the logarithmic growth phase were added and the final concentration of the bacterial solution was set to 2×10 6 CFU / mL, and continue incubation. Discard the culture medium and wash away the H37Rv that has not been infected into J774 A.1 cells with PBS.

[0108] Add 2 ml of drug-containing culture medium, PMZ 1, 1.5 µg / ml, BDQ 0.3, 0.6 µg / ml and the combination of the two, set up 2 drug parallel control groups, and set up 6 negative control groups, and incubate. Add 0.1% SDS at a volume of 0.2 mL / well to lyse the cells, and replenish DMEM complete culture medium at a volume of 0.8 mL / well. Dilute the bacterial solution by 10-fold dilution method, aspirate 100 µL of the diluted bacterial solution on 7H10 culture medium, spread it evenly, and count CFU after 4 weeks of culture. The bactericidal activity of the two-drug combination on macrophages was determined by paired t-test. If the P value of the reduced CFU count of the two-drug combination compared with the single drug is <0.05, the combination activity is considered to be enhanced.

[0109] 4.2 Test results PMZ at 1.5 µg / ml also had no intracellular anti-TB activity in macrophages infected with Mycobacterium tuberculosis H37Rv. PMZ at 1 µg / ml and 1.5 µg / ml combined with 0.03 µg / ml BDQ reduced intracellular CFU by 0.3 and 0.5 log, respectively, compared with BDQ alone. 10 CFU, all showed statistically significant differences ( p <0.01). 1 µg / ml and 1.5 µg / ml PMZ combined with 0.03 µg / ml BDQ could reduce intracellular CFU by 0.4 and 0.6 log, respectively, compared with BDQ alone. 10 CFU, all showed statistically significant differences ( p <0.01). The results showed that PMZ could enhance the intracellular anti-tuberculosis activity of BDQ.

[0110] Example 5 Evaluation of the antibacterial activity of PMZ combined with BDQ by time-kill curve method 5.1 Test methods In order to investigate the dynamic bactericidal activity of pimozide combined with bedaquiline, the bactericidal curve of the combined action was determined.

[0111] The evaluation of the two-drug combination of antimicrobial drugs included single-drug holes and two-drug combination holes. 1 / 8MIC (0.375μg / ml), 1 / 4MIC (0.75μg / ml), 1 / 2MIC (1.5μg / ml) of PMZ combined with 1 / 2MIC (0.03μg / ml) and 1MIC (0.06μg / ml) of BDQ were selected for time-killing experimental evaluation.

[0112] The method of adding drugs in the 24-well plate is as follows: 5 μl drug diluent + 5 μl DMSO are added to 990 μl 7H9 culture medium in the single drug wells; 5 μl PMZ drug diluent + 5 μl BDQ drug diluent are added to 990 μl 7H9 culture medium in the combination drug wells; 2000 μl 7H9 culture medium is added to the negative control wells without adding drugs; and 1000 μl 7H9 culture medium is added to the positive control wells.

[0113] Add 1000 μl of diluted bacterial solution to each well of the 24 wells to make the final concentration of 2×10 5 CFU / ml, cultured.

[0114] On the day of culture (D0) and on the 3rd, 7th, 10th, and 14th days of culture, 100 μl of the original bacterial solution or the bacterial solution diluted with sterile PBS was inoculated on the 7H10 solid medium containing 10% OADC and 0.5% glycerol and evenly spread. The colonies were counted after 4 weeks of culture. The time-killing curve of the drug was drawn with the culture time point of Mycobacterium tuberculosis as the horizontal axis and the logarithm of the colony number at different culture time points as the vertical axis.

[0115] 5.2 Test results 0.03 μg / ml BDQ and 0.375-1.5 μg / ml PMZ had no bactericidal activity because their concentrations were lower than their respective MICs. However, 0.375, 0.75, and 1.5 μg / ml PMZ combined with 0.03 μg / ml BDQ showed dose-dependent bactericidal activity. 1.5 μg / ml PMZ combined with 0.03 μg / ml BDQ could completely kill Mycobacterium tuberculosis after 14 days of action. 0.06 μg / ml BDQ had certain antibacterial activity, and 0.375, 0.75, and 1.5 μg / ml PMZ combined with 0.06 μg / ml BDQ showed stronger bactericidal activity, and could completely kill Mycobacterium tuberculosis after 14 days of action.

Claims

1. A synergistic combination pharmaceutical composition for resisting mycobacterial infection, the synergistic combination pharmaceutical composition comprising pimozide in combination with another drug for resisting mycobacterial infection, the other drug for resisting mycobacterial infection being bedaquiline and / or clofazimine, and the pharmaceutical composition having at least one of the following effects: a) inhibiting mycobacterial activity; b) protection against mycobacterial infections; c) Prevention and / or treatment of diseases caused by mycobacteria.

2. The synergistic pharmaceutical composition for resisting mycobacterial infection as claimed in claim 1, wherein the synergistic pharmaceutical composition can be prepared into various forms such as tablets, powders, granules, capsules, oral liquids, injection preparations or aerosols; the drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field; the various preparations can also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed; the pharmaceutical composition can also be added with one or more pharmaceutically acceptable carriers; the pimozide also includes pharmaceutically acceptable salts or esters; the pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous or intravenous by physical or chemical methods.

3. The combined synergistic pharmaceutical composition for resisting mycobacterial infection according to claim 1, wherein the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria and / or Mycobacterium leprae; the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection includes: Primary infection, secondary infection, extrapulmonary infection, pulmonary infection; The diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

4. A use of pimozide and bedaquiline in combination for preparing a combined synergistic pharmaceutical composition for resisting mycobacterial infection, wherein the pimozide and bedaquiline are used in combination to exert an anti-mycobacterial infection effect, and the pimozide can enhance the anti-mycobacterial infection activity of bedaquiline.

5. The use of pimozide and bedaquiline in combination for preparing a combined synergistic pharmaceutical composition for resisting mycobacterial infection as claimed in claim 4, wherein the combined synergistic pharmaceutical composition can be prepared in various forms such as tablets, powders, granules, capsules, oral liquids, injection preparations or aerosols; the drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field; the various preparations can also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed; the pharmaceutical composition can also be added with one or more pharmaceutically acceptable carriers; the pimozide also includes pharmaceutically acceptable salts or esters; the pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous or intravenous by physical or chemical methods.

6. The use of pimozide combined with bedaquiline in the preparation of a combined synergistic pharmaceutical composition for anti-mycobacterial infection as claimed in claim 4, wherein the mycobacteria are selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria and / or Mycobacterium leprae; the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection includes: Primary infection, secondary infection, extrapulmonary infection, pulmonary infection.

7. A use of pimozide as a synergist, wherein the synergist has the effect that pimozide can increase the anti-mycobacterial infection effect of bedaquiline and / or clofazimine, that is, when pimozide is used in combination with bedaquiline and / or clofazimine, pimozide can enhance the anti-mycobacterial infection activity of bedaquiline and / or clofazimine.

8. The use of pimozide as a synergist as claimed in claim 7, wherein the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria and / or Mycobacterium leprae; the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection includes: Primary infection, secondary infection, extrapulmonary infection, pulmonary infection.

9. The use of pimozide as a synergist as claimed in claim 7, wherein the pimozide further comprises a pharmaceutically acceptable salt or ester.

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