A synergistic pharmaceutical composition for resisting mycobacterial infection

Abstract

This invention belongs to the field of biomedicine and specifically relates to the use of a combined synergistic pharmaceutical composition for the treatment of mycobacterial infections. Pimozide (PMZ) described in this invention can be used as an active ingredient in a combined anti-Mycobacterium tuberculosis drug, opening up new applications for PMZ. Combining PMZ with anti-tuberculosis drugs (such as bedaquiline (BDQ) and clofazimine (CFZ)) can enhance anti-tuberculosis activity. PMZ's use as a synergist is expected to shorten treatment courses and reduce drug resistance, representing a novel therapeutic strategy. The combination of PMZ (0.5µg / ml) and BDQ reduced the MIC of susceptible Mycobacterium tuberculosis strain H37Rv to BDQ from 0.06µg / ml to 0.0038µg / ml (a 16-fold increase in efficacy). The combination of pimozide (0.5µg / ml) and BDQ reduced the MIC of the Rv0678 mutant to BDQ from 1µg / ml to 0.0313µg / ml (a 32-fold increase in efficacy), which is lower than the MIC of wild-type H37Rv (0.06µg / ml). This reversed BDQ resistance and further enhanced anti-mycobacterial activity. A 1.5µg / ml PMZ combined with 0.03µg / ml BDQ treatment for 14 days completely killed Mycobacterium tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an application of a combined synergistic pharmaceutical 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). Mycobacterial infections can affect multiple organ systems, including the lungs, skin, lymph nodes, and bones. Mycobacterium tuberculosis is the primary pathogen that causes tuberculosis and is primarily transmitted through the respiratory tract. After infection, patients may develop latent infection or active tuberculosis. Under certain conditions (such as immunodeficiency and diabetes), latent infection may turn into active disease (Lu Lunshan. Diagnosis and prevention measures of Mycobacterium tuberculosis infection [J]. World Latest Medical Information Digest, 2016).

[0003] Tuberculosis is caused by Mycobacterium tuberculosis ( Mycobacterium tuberculosisTuberculosis is a chronic infectious disease caused by tuberculosis (TB), which is primarily transmitted through the air, especially when patients cough, sneeze, or spit, releasing droplets containing the pathogen (SeunNurudeen Akorede, Ayodotun Edward Ajayi et al. “Knowledge of Tuberculosis among Tuberculosis Patients Attending Federal Medical Centre, Owo, Ondo State, Nigeria.” Unnes Journal of Public Health (2021). Tuberculosis can affect multiple organs, but the most common infection is the lungs, known as pulmonary tuberculosis. Tuberculosis remains a serious public health problem worldwide. The main treatment for 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 consists of an intensive phase and a consolidation phase. The intensive phase involves twice-daily treatment to reduce bacterial counts, while the consolidation phase involves longer treatment sessions at lower doses. However, due to the prevalence of drug resistance in Mycobacterium tuberculosis, treatment has become complex and challenging (Xu Yin, Meng Xianmin, Zhang Yongxin. Current Status of Drug-Resistant Tuberculosis and Advances in Anti-TB Drug Research [J]. Shanghai Pharmaceuticals, 2013). Drug-resistant tuberculosis (especially multidrug-resistant tuberculosis (MDR-TB)) has become a major global public health challenge (Shen Xin, Song Shenchao, Lei Shiguang. Current Status of Drug-Resistant Tuberculosis and Prevention and Control Strategies [J]. Occupation and Health, 2017). The treatment of drug-resistant tuberculosis faces numerous challenges. Although chemotherapy remains the mainstay of treatment, its effectiveness is suboptimal for patients with multidrug-resistant and extensively drug-resistant tuberculosis (Zhou Wenqiang, Chu Naihui. Advances in the Treatment of Drug-Resistant Tuberculosis [J]. Chinese Journal of Clinicians, 2020). The situation of drug-resistant tuberculosis is serious. The drug options for treating drug-resistant tuberculosis are limited, the treatment course is long, and the cure rate is as low as 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 or 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 drug-resistant strains reported in Pakistan, the Rv0678 gene mutant strain 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, studies have found that some Mycobacterium tuberculosis strains carry Rv0678 mutations even in the absence of previous treatment with CFZ or BDQ, suggesting 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] Treatment for drug-resistant tuberculosis requires longer time and more expensive medications, placing a significant burden on both the healthcare system and patients' families. Therefore, there is an urgent need to develop new drugs. The R&D cycle for anti-tuberculosis drugs is long and challenging, so developing anti-tuberculosis drug potentiators to enhance the anti-tuberculosis activity of existing drugs and reverse their resistance is a very important treatment strategy. Antimicrobial drug potentiators are not antibiotics themselves, but when used in combination with antibiotics, they can reverse bacterial resistance and restore the sensitivity of resistant bacteria to antibiotics, while slowing the development of bacterial resistance. They are an effective and sustainable strategy for combating drug-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).), marketed in 1970. It is an antipsychotic used to treat acute and chronic schizophrenia, Tourette syndrome, and other conditions. It is a specific central dopamine receptor antagonist. Pimozide's chemical structure is 1-(1-[4,4-bis(4-fluorophenyl)butanyl]-4-piperidinyl)benzo[1,2-a:5,4-e']dibenzopiperidine; there are currently no reports of its enhanced anti-mycobacterial activity in combination with anti-tuberculosis drugs. Summary of the Invention

[0007] The present invention has been found to enhance the anti-mycobacterial activity of bedaquiline and / or clofazimine when used in combination with pimozide. The combination of pimozide, bedaquiline, and / or clofazimine has an anti-mycobacterial effect. Based on this, the present invention has been completed.

[0008] In a first aspect, the present invention provides a synergistic combination pharmaceutical composition for use against mycobacterial infection, comprising pimozide in combination with another anti-mycobacterial infection drug, wherein the other anti-mycobacterial infection drug is bedaquiline and / or clofazimine, and the pharmaceutical composition has at least one of the following effects:

[0009] a) inhibiting mycobacterial activity;

[0010] b) protection against mycobacterial infections;

[0011] c) Prevention and / or treatment of diseases caused by mycobacteria.

[0012] Furthermore, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculosis mycobacteria and / or Mycobacterium leprae.

[0013] 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.

[0014] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0015] 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.

[0016] Furthermore, the drug-resistant tuberculosis includes but is not limited to monodrug-resistant tuberculosis, multidrug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0017] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, negative pulmonary tuberculosis, etc.

[0018] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0019] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0020] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0021] Furthermore, the non-tuberculosis mycobacteria include but are not limited to Mycobacterium avium ( M. avium ), Mycobacterium intracellulare ( Intracellular M. ), Mycobacterium kansasii ( M. kansasii ), Mycobacterium fortuitum ( M. chance ), Mycobacterium abscessus ( M. abscess ), Mycobacterium ulcerans ( Ulcerative muscle ) and / or Mycobacterium marinum ( Marine M. )wait.

[0022] Furthermore, the disease caused by non-tuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphoma, disseminated NTM disease and / or other NTM diseases.

[0023] Furthermore, the pharmaceutical composition may also contain other active ingredients against mycobacterial infection.

[0024] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0025] Furthermore, the pharmaceutical composition can be prepared into various forms such as tablets, powders, granules, capsules, oral solutions, injection preparations or aerosols; the above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Furthermore, the wetting agent and adhesive include but are not limited to one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0030] 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 lauryl sulfate, methyl cellulose and / or ethyl cellulose.

[0031] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0032] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Furthermore, the injectable 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-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0037] Furthermore, in order to prepare an isotonic injection, the injectable preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0038] Furthermore, the various preparations may also contain colorants, preservatives, spices, flavorings, sweeteners or other materials as needed.

[0039] Furthermore, the pharmaceutical composition can be introduced into the body through physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0040] Furthermore, the pimozide also includes pharmaceutically acceptable salts or esters.

[0041] In a second aspect, the present invention provides a use of pimozide as a synergist, wherein the synergist functions as pimozide increasing 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.

[0042] Furthermore, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculosis mycobacteria and / or Mycobacterium leprae.

[0043] 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.

[0044] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0045] 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.

[0046] Furthermore, the drug-resistant tuberculosis includes but is not limited to monodrug-resistant tuberculosis, multidrug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0047] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, negative pulmonary tuberculosis, etc.

[0048] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0049] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0050] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0051] 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. chance ), Mycobacterium abscessus ( M. abscess ), Mycobacterium ulcerans ( Ulcerative muscle ) and / or Mycobacterium marinum ( Marine M. )wait.

[0052] Furthermore, the disease caused by non-tuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphoma, disseminated NTM disease and / or other NTM diseases.

[0053] Furthermore, the pimozide also includes pharmaceutically acceptable salts or esters.

[0054] 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, bedaquiline and / or clofazimine are used in combination to exert an anti-mycobacterial infection effect, and the pimozide can enhance the anti-mycobacterial infection activity of bedaquiline and / or clofazimine.

[0055] Furthermore, the mycobacterium is selected from Mycobacterium tuberculosis, non-tuberculosis mycobacteria and / or Mycobacterium leprae.

[0056] 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.

[0057] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0058] 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.

[0059] Furthermore, the drug-resistant tuberculosis includes but is not limited to monodrug-resistant tuberculosis, multidrug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0060] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, negative pulmonary tuberculosis, etc.

[0061] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0062] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0063] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0064] 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.

[0065] Furthermore, the disease caused by non-tuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphoma, disseminated NTM disease and / or other NTM diseases.

[0066] Furthermore, the pharmaceutical composition may also contain other active ingredients against mycobacterial infection.

[0067] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0068] Furthermore, the pharmaceutical composition can be prepared into various forms such as tablets, powders, granules, capsules, oral solutions, injection preparations or aerosols; the above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Furthermore, the wetting agent and adhesive include but are not limited to one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0073] 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 lauryl sulfate, methyl cellulose and / or ethyl cellulose.

[0074] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0075] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] Furthermore, the injectable 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-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0080] Furthermore, in order to prepare an isotonic injection, the injectable preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0081] Furthermore, the various preparations may also contain colorants, preservatives, spices, flavorings, sweeteners or other materials as needed.

[0082] Furthermore, the pharmaceutical composition can be introduced into the body through physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0083] Furthermore, the pimozide also includes pharmaceutically acceptable salts or esters.

[0084] Beneficial effects

[0085] The PMZ described in the present invention can be used as an active ingredient in a drug used in combination with anti-tuberculosis drugs, opening up new uses for PMZ. It can be used in combination with anti-tuberculosis drugs (such as bedaquiline and clofazimine) to enhance anti-tuberculosis activity. PMZ can be used as a synergist, which is expected to shorten the course of treatment and reduce drug resistance, and will be a new treatment strategy. 0.5 μg / ml of pimozide combined with BDQ reduced the MIC of Mycobacterium tuberculosis-sensitive strain H37Rv to BDQ from 0.06μg / ml to 0.0038μg / ml (a 16-fold increase in efficacy); 0.5 μg / ml of pimozide combined with BDQ reduced the MIC of Mycobacterium tuberculosis-sensitive strain H37Rv to BDQ from 0.06μg / ml to 0.0038μg / ml (a 16-fold increase in efficacy); Rv0678 The mutant strain's MIC for BDQ decreased from 1µg / ml to 0.0313µg / ml (a 32-fold increase in potency), which is lower than the wild-type H37Rv MIC (0.06µg / ml), reversing BDQ resistance and further enhancing anti-mycobacterial activity. A 1.5µg / ml PMZ combined with 0.03µg / ml BDQ for 14 days completely killed M. tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The anti-tuberculosis activity of different concentrations of PMZ combined with BDQ in macrophages infected with Mycobacterium tuberculosis H37Rv.

[0087] Figure 2 This is the time-kill curve of PMZ combined with BDQ against Mycobacterium tuberculosis. DETAILED DESCRIPTION

[0088] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0089] 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.

[0090] the term

[0091] Bedaquiline (BDQ): chemically known as 1-(6-bromo-2-methoxyquinolin-3-yl)-4-dimethylamino-1-phenyl-2-(1-naphthyl)-2-butanol, and marketed as Sirturo, is a novel diarylquinoline antimycobacterial drug that inhibits the proton pump activity of the ATP synthase of Mycobacterium tuberculosis, thereby affecting ATP synthesis in the bacterium and exerting its antibacterial and bactericidal effects. It is clinically used to treat multidrug-resistant pulmonary tuberculosis (MDR-PTB) in adults.

[0092] The synergist in the present invention refers to pimozide. When pimozide and bedaquiline are used in combination, the anti-mycobacterial activity of bedaquiline can be enhanced. The anti-tuberculosis ability of the pharmaceutical composition after the combination of pimozide and bedaquiline is enhanced compared with the use of bedaquiline alone.

[0093] The minimum inhibitory concentration (MIC) is a measure of the antimicrobial activity of an antimicrobial drug. It refers to the lowest drug concentration that inhibits the growth of Mycobacterium tuberculosis in culture for 7 to 10 days in vitro. In vitro pharmacodynamic interactions involve evaluating the interactions and effective doses of investigational drugs to determine their feasibility for further development as pulmonary tuberculosis treatment options. A commonly used quantitative method is the checkerboard dilution method. For example, the interaction between two drugs is first determined for each drug. Based on the MIC values, the highest concentration is set at 2× the MIC of the individual drugs. Two-fold dilutions are then performed (in both vertical and horizontal rows of the matrix), with each tube (well) containing a mixture of different concentrations of the two drugs. Six to eight dilutions are typically designed. The initial inoculum is 5 × 105 CFU / mL. The cells are incubated at 37°C for 7 days. 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 Joint / MIC A Single + MIC B Joint / MIC B alone

[0094] MICA alone and MICB alone represent the MICs of drug A and drug B, respectively, against Mycobacterium tuberculosis when applied alone. MICA combined and MICB combined represent the lowest concentrations of drug A and drug B that prevent the color change from blue to pink after combined application. The in vitro activity of two-drug combinations is determined by the following criteria: FICI ≤ 0.5 indicates synergism; FICI 0.5-4 indicates indifference; and FICI > 4 indicates antagonism.

[0095] Time-kill curves allow dynamic observation of drug (compound)-M. tuberculosis interactions. They compare the effects of different compound concentrations and exposure times on M. tuberculosis growth, providing additional insight into evaluating a compound's antimycobacterial activity. A time-kill curve experiment typically lasts 14 days. M. tuberculosis is cultured continuously in drug-containing medium for 14 days. During this period, aliquots of the culture fluid are plated on solid medium every 3-4 days, and colony counts are performed after 3-4 weeks of culture. Time-kill curves are plotted with the time of culture fluid collection as the x-axis and the log10 of the colony count as the y-axis. Time-kill curves are also used to evaluate in vitro interactions between drugs in drug combinations.

[0096] The Microplate Alamar Blue Assay (MAB) is a rapid, simple, and low-cost chromatographic method for determining the minimum inhibitory concentration (MIC) of microorganisms. This method uses Alamar Blue dye as an indicator, assessing bacterial growth by measuring color changes. In drug resistance studies of Mycobacterium tuberculosis, MABA is widely used to assess the inhibitory effects of different drugs on tuberculosis.

[0097] Example

[0098] Example 1 Screening of Pimozide

[0099] 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.

[0100] From a library of 4,000 compounds, we found that pimozide, which has the best activity against mycobacterial infection by enhancing bedaquiline, has the following structural formula:

[0101] Example 2 Determination of Minimum Inhibitory Concentration and Combined Synergistic Activity of Pimozide by MABA Method

[0102] 2.1 Test method

[0103] In this part, the microplate Alamar Blue assay (MABA) was used to determine the MICs of PMZ and immobilized PMZ combined with BDQ against Mycobacterium tuberculosis.

[0104] 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.

[0105] Take a culture medium of Mycobacterium tuberculosis in the logarithmic growth phase and place it in a well plate. Measure the OD value of each well at a wavelength of 570nm, where OD (strain) = OD (culture medium) - OD (7H9 medium). OD = 0.1 is equivalent to 1 × 10 7 CFU / mL, and dilute each bacterial solution so that the final concentration of each strain is set to 1×10 5 CFU / mL.

[0106] Add culture medium and drug stock solution, dilute the drug 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 seventh day of incubation, Tween-80 and resazurin indicator were added to each microwell and incubation continued. The next day, color changes were observed and fluorescence values ​​of each well were measured at excitation wavelengths of 530 nm and 590 nm, respectively. The color of each well was recorded: blue indicates no growth, and red indicates bacterial growth. The MIC indicates the lowest drug concentration at which the blue color changes to red.

[0107] Combined activity was determined using the MIC assay, except that PMZ was added to the culture medium at final concentrations of 2 μg / ml, 1 μg / ml, and 0.5 μg / ml, and this medium was used for subsequent assays. The first six wells served as negative controls, and the last six wells were inoculated with diluted bacterial culture as positive controls. Subsequently, 198 μL of PMZ-containing culture medium was added to the first column of wells in rows BH of the 96-well plate, and 100 μL of PMZ-containing culture medium was added to the remaining wells. 2 μL of BDQ stock solution was added to the first column of wells in rows BH. After mixing the drug solution and PMZ-containing culture medium, this procedure was repeated using a two-fold dilution method until the last column of wells. The MIC of bedaquiline in combination with a fixed concentration of PMZ was determined, as well as the fold reduction.

[0108] 2.2 Test results

[0109] As shown in Table 1, pimozide has an in vitro Rv0678 The minimum inhibitory concentration of the mutant strains was 3.13µg / ml. Rv0678 The minimum inhibitory concentration of the mutant strain was 1µg / mL (as shown in Table 1). 0.5µg / ml of pimozide combined with BDQ reduced the MIC of susceptible Mycobacterium tuberculosis H37Rv to BDQ from 0.06µg / ml to 0.0038µg / ml (16-fold increase in efficacy). Rv0678 The mutant strain's MIC for BDQ decreased from 1µg / ml to 0.0313µg / ml (a 32-fold increase in potency), which is lower than the wild-type H37Rv MIC (0.06µg / ml), reversing BDQ resistance and further enhancing anti-mycobacterial activity. Increasing pimozide concentrations significantly reduced the BDQ MIC, demonstrating a dose-response relationship between 0.5-2µg / ml.

[0110]

[0111] Example 3 Determination of the combined activity of pimozide and different anti-tuberculosis drugs against different strains by checkerboard assay

[0112] 3.1 Test method

[0113] The checkerboard assay is a method based on MIC for determining the interaction between two drug combinations.

[0114] The checkerboard method was used to determine the FIC values ​​of PMZ in combination with BDQ, CFZ, INH, RFP, LVX, and PBTZ169 against the standard strain H37Rv.

[0115] Based on the MIC of each drug when used alone, a total of seven concentration gradients were set up, with a concentration range of 2×MIC-1 / 32×MIC. Drugs were distributed in a checkerboard pattern, as shown in Table 2. B2-H9 represents drug A (PMZ) at 2×MIC-1 / 32×MIC, and A3-H9 represents drug B (BDQ, CFZ, INH, RFP, LVX, PBTZ169) at 2×MIC-1 / 32×MIC. Drug B was diluted twofold horizontally and twofold vertically. Columns B10-H10 and B11-H11 represent negative and positive control wells, respectively. The MIC of each drug was determined by observing color changes, and drug interactions were determined using FICI.

[0116] After 7 days of culture, Alamar Blue indicator was added. After 24 hours, the color changes of drug A and drug B alone 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:

[0117] FICI=MIC A Joint / MIC A Single + MIC B Joint / MIC B alone

[0118] MICA alone and MICB alone represent the MICs of drug A and drug B, respectively, against Mycobacterium tuberculosis when applied alone. MICA combined and MICB combined represent the lowest concentrations of drug A and drug B that prevent the color change from blue to pink after combined application. The in vitro activity of two-drug combinations is determined by the following criteria: FICI ≤ 0.5 indicates synergism; FICI 0.5-4 indicates indifference; and FICI > 4 indicates antagonism.

[0119]

[0120] Note: X is drug A, i.e., pimozide: PMZ; X1-X7 are 7 concentration gradients of PMZ: 2×MIC-1 / 32×MIC;

[0121] Y represents drug B, which are bedaquiline: BDQ; isoniazid: INH; rifampicin: RFP; levofloxacin: LVX and PBTZ169; Y1-Y7 represent the 7 concentration gradients of drug B: 2×MIC-1 / 32×MIC.

[0122] 3.2 Test results

[0123] As shown in Table 3, the checkerboard assay showed that pimozide and BDQ had the best in vitro synergistic anti-mycobacterial activity against H37Rv, with an FICI of 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 other anti-tuberculosis drugs, including INH, RFP, levofloxacin (LVX), and PBTZ-169.

[0124] Note: When the MIC of BDQ alone is 0.06 μg / mL and the MIC of the combination is 0.0075 μg / mL, the PMZ concentration is 0.75 μg / mL; when the MIC of CFZ alone is 0.125 μg / mL and the MIC of the combination is 0.03125 μg / mL, the PMZ concentration is 0.75 μg / mL; when the MIC of INH alone is 0.0625 μg / mL and the MIC of the combination is 0.03125 μg / mL, the PMZ concentration is 1.5 μg / mL; when the MIC of RFP alone is 0.03125 μg / mL and the MIC of the combination is 0.0156 μg / mL, the PMZ concentration is 0.75 μg / mL; when the MIC of LVX alone is 0.5 μg / mL and the MIC of the combination is 0.25 μg / 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.

[0125] As shown in Table 4, the activity of PMZ combined with BDQ / CFZ was further determined against different clinically isolated Mycobacterium tuberculosis strains. Strains 17851 and 13385 were sensitive to BDQ and CFZ, while strains 11492 and 12657 were resistant to BDQ and CFZ.

[0126] Checkerboard assays demonstrated in vitro synergistic effects between pimozide and BDQ / CFZ against both BDQ-susceptible (17851, 13385) and BDQ-resistant (11492, 12657) clinical isolates of Mycobacterium tuberculosis. The synergistic anti-TB effect of pimozide and BDQ was superior to that of CFZ, with a smaller FICI.

[0127]

[0128] Example 4 Determination of the anti-tuberculosis activity of PMZ combined with BDQ in macrophages

[0129] 4.1 Test method

[0130] Mycobacterium tuberculosis is an intracellular parasite, and macrophages are its parasitic site.

[0131] This application uses mouse mononuclear macrophage J774A.1 infected with Mycobacterium tuberculosis H37Rv as a research model.

[0132] J774A.1 cells were cultured in DMEM complete medium containing 10% FBS. The cells were digested and a cell suspension was obtained, which was diluted to a final concentration of 4×10 5 Add diluted J774A.1 cells to the cell culture plate at a volume of 1 ml / well and incubate. Add H37Rv in the logarithmic growth phase and set the final concentration of the bacterial solution to 2×10 6 CFU / mL, continue incubation, discard the culture medium, and wash out the uninfected H37Rv in J774 A.1 cells with PBS.

[0133] Add 2 ml of drug-containing culture medium, PMZ at 1 and 1.5 µg / ml, BDQ at 0.3 and 0.6 µg / ml, and a combination of PMZ and BDQ, setting up two parallel drug control groups and six negative controls, and incubate. Add 0.1% SDS to 0.2 mL / well to lyse the cells, and replenish with complete DMEM medium to 0.8 mL / well. Dilute the cells 10-fold, then spread 100 µL onto 7H10 medium and incubate for 4 weeks before counting CFUs. Paired t-tests were used to determine the bactericidal activity of the two-drug combination on macrophages. If the P value for the reduction in CFU counts compared to either drug alone was < 0.05, the combination was considered to have enhanced activity.

[0134] 4.2 Test results

[0135] In macrophages infected with Mycobacterium tuberculosis H37Rv, PMZ at 1.5µg / ml had no intracellular anti-TB activity. 1 µg / ml and 1.5µg / ml PMZ combined with 0.03µg / ml BDQ reduced intracellular CFU by 0.3 and 0.5 log, respectively, compared to 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 reduced intracellular CFU by 0.4 and 0.6 log, respectively, compared to 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.

[0136] Example 5 Evaluation of the antibacterial activity of PMZ combined with BDQ using the time-kill curve method

[0137] 5.1 Test method

[0138] In order to investigate the dynamic bactericidal activity of pimozide combined with bedaquiline, a bactericidal curve study of the combined action was conducted.

[0139] The evaluation of the two-drug combination of antimicrobial drugs included single-drug wells and two-drug combination wells. 1 / 8 MIC (0.375 μg / ml), 1 / 4 MIC (0.75 μg / ml), and 1 / 2 MIC (1.5 μg / ml) of PMZ were combined with 1 / 2 MIC (0.03 μg / ml) and 1 MIC (0.06 μg / ml) of BDQ for time-killing experimental evaluation.

[0140] Drugs were added to the 24-well plate as follows: single-drug wells were treated with 5 μl of drug diluent + 5 μl of DMSO added to 990 μl of 7H9 medium; combination drug wells were treated with 5 μl of PMZ drug diluent + 5 μl of BDQ drug diluent added to 990 μl of 7H9 medium; negative control wells without drug addition were treated with 2000 μl of 7H9 medium; and positive control wells were treated with 1000 μl of 7H9 medium.

[0141] 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, and culture.

[0142] On the day of incubation (D0) and on days 3, 7, 10, and 14 of incubation, 100 μl of the original bacterial solution or the bacterial solution diluted in sterile PBS was aspirated from a 24-well plate and evenly inoculated onto 7H10 solid medium containing 10% OADC and 0.5% glycerol. Colonies were counted after 4 weeks of incubation. Time-kill curves of the drugs were plotted, with the incubation time of M. tuberculosis as the abscissa and the logarithm of the colony count at different incubation time points as the ordinate.

[0143] 5.2 Test results

[0144] 0.03 μg / ml BDQ and 0.375-1.5 μg / ml PMZ had no bactericidal activity, as their concentrations were below their respective MICs. However, 0.375, 0.75, and 1.5 μg / ml PMZ combined with 0.03 μg / ml BDQ exhibited dose-dependent bactericidal activity. 1.5 μg / ml PMZ combined with 0.03 μg / ml BDQ completely killed M. tuberculosis after 14 days of treatment. 0.06 μg / ml BDQ exhibited some antibacterial activity, while 0.375, 0.75, and 1.5 μg / ml PMZ combined with 0.06 μg / ml BDQ exhibited even stronger bactericidal activity, achieving complete killing of M. tuberculosis after 14 days of treatment.

Claims

1. A synergistic combination pharmaceutical composition for use against mycobacterial infection, comprising pimozide in combination with another anti-mycobacterial infection drug, wherein the other anti-mycobacterial infection drug is bedaquiline and / or clofazimine, and wherein 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.

2. The synergistic combination pharmaceutical composition for combating mycobacterial infection according to claim 1, wherein the dosage form of the combination pharmaceutical composition is selected from tablets, powders, granules, capsules, oral solutions, injections, or aerosols; the pimozide comprises a pharmaceutically acceptable salt; and the pharmaceutical composition is introduced into the body via intramuscular, intradermal, subcutaneous, or intravenous routes.

3. The synergistic combination pharmaceutical composition for treating mycobacterial infection according to claim 1, wherein the mycobacterium is selected from Mycobacterium tuberculosis; the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and / or standard strains of Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection; and the diseases caused by Mycobacterium tuberculosis include drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis and extrapulmonary tuberculosis.

4. Use of pimozide in combination with bedaquiline and / or clofazimine in the preparation of a combined synergistic pharmaceutical composition for anti-mycobacterial infection, wherein the pimozide, bedaquiline and / or clofazimine are used in combination to exert an anti-mycobacterial infection effect, and the pimozide enhances the anti-mycobacterial infection activity of bedaquiline and / or clofazimine.

5. The use according to claim 4, wherein the dosage form of the combined synergistic pharmaceutical composition is selected from tablets, powders, granules, capsules, oral solutions, injections or aerosols; the pimozide comprises a pharmaceutically acceptable salt; and the pharmaceutical composition is introduced into the body by intramuscular, intradermal, subcutaneous or intravenous administration.

6. The use according to claim 4, wherein the mycobacterium is selected from Mycobacterium tuberculosis; the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and / or standard strains of Mycobacterium tuberculosis; and the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection.

7. Use of pimozide in the preparation of a synergist for an anti-mycobacterial infection drug, wherein the synergist acts to enhance the anti-mycobacterial infection activity of bedaquiline and / or clofazimine. That is, when pimozide is used in combination with bedaquiline and / or clofazimine, pimozide enhances the anti-mycobacterial infection activity of bedaquiline and / or clofazimine.

8. The use according to claim 7, wherein the mycobacterium is selected from Mycobacterium tuberculosis; the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and / or standard strains of Mycobacterium tuberculosis; and the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection.

9. The use according to claim 7, wherein the pimozide comprises a pharmaceutically acceptable salt.

Images