A pharmaceutical composition for resisting mycobacterial infection

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of a pharmaceutical composition in the fight against mycobacterial infections. The combination of juglottisol and bedaquiline significantly enhances the activity against mycobacterial infections. Compared to bedaquiline alone, 2 μg / ml of juglottisol combined with bedaquiline reduced the colony count of MmpL5-MmpS5 strains to varying degrees, from 1 / 16 MIC to 2 MIC of bedaquiline. Juglottisol, as an synergist of bedaquiline, enhances the anti-mycobacterial activity of bedaquiline. The minimum inhibitory concentration (MIC) of juglottisol against the Rv0678 mutant strain in vitro is 16 μg / ml, and the MIC of BDQ against the Rv0678 mutant strain in vitro is 1 μg / mL. The combination of 1 μg / ml zirconia thiazide and BDQ reduced the MIC of the Rv0678 mutant strain against BDQ from 1 μg / ml to 0.03 μg / ml (32-fold increase), which was lower than the MIC of the wild-type strain H37Rv (0.06 μg / ml), reversing BDQ resistance and further enhancing its activity against mycobacterial infections.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a pharmaceutical composition in the treatment of mycobacterial infections. Background Art

[0002] Mycobacteria include the tuberculous mycobacterial complex (including tuberculosis, bovine, African, vole, goat, pinnipedii, suricattae, and mungi mycobacteria), non-tuberculous mycobacteria (NTM), and mycobacterium leprae.

[0003] Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a chronic respiratory infectious disease that seriously endangers human health. The situation regarding drug resistance in TB is severe, with limited drug options for treating drug-resistant TB, long treatment courses, and a low cure rate of approximately 63%. In 2019 and 2022, the WHO listed bedaquiline and clofazimine as Group A and Group B drugs in the core drugs for treating multidrug-resistant TB, respectively. However, bedaquiline-resistant strains were quickly identified. The research team has discovered clinically resistant strains of the new drug bedaquiline, especially a cross-resistant strain (Rv0678 gene mutation) isolated from TB patients who had not been exposed to or used the drug (Xu J, et al. Antimicrob Agents Chemother, 2017). Although mutations in the atpE gene encoding ATP synthase and non-target PepQ gene mutations are associated with bedaquiline resistance, the most clinically reported association with bedaquiline resistance is with the Mycobacterium tuberculosis Rv0678 gene mutation. The Rv0678 mutation can lead to reduced activity against mycobacterial infections in bedaquiline-containing chemotherapy regimens, prolonged treatment cycles, and even treatment failure. Studies have also found that the Rv0678 mutation can lead to resistance to clofazimine, a group B drug in the core treatment of multidrug-resistant tuberculosis, and the new anti-tuberculosis drug PBTZ169, which is in phase II clinical trials. Research has also shown that Rv0678 mutant strains still exhibit partial cross-resistance to next-generation, more potent bedaquiline derivatives (TBAJ-587) and clofazimine derivatives (Xu J, et al. AntimicrobAgents Chemother, 2021; Xu J, et al. AntimicrobAgents Chemother, 2019).

[0004] The Rv0678 gene is a transcriptional repressor of the MmpL5-MmpS5 efflux system, and mutations in it can lead to overexpression of both MmpL5 and MmpS5. Transcriptomic and proteomic studies have also found that mutations in Rv0678 cause more than a two-fold upregulation of MmpL5 and MmpS5 expression in bedaquiline-resistant Mycobacterium tuberculosis (Xu J, et al. Antimicrob Agents Chemother, 2023). One approach to combat drug resistance caused by efflux is to develop drug efflux inhibitors for use in combination with antibiotics. Studies on the roles and functions of MmpL5 and MmpS5 have revealed that co-expression of MmpL5 and MmpS5 is necessary to form an efflux pump in both Mycobacterium smegmae and Mycobacterium tuberculosis, leading to resistance to bedaquiline. MmpL5 alone does not play a role (Li Dongshuo; Wang Bin; Lu Yu; Xu Jian. Expression and function of Mycobacterium tuberculosis membrane proteins MmpS5-MmpL5. Chinese Journal of Antituberculosis, 2022; Xu J, et al. Antimicrob Agents Chemother, 2023). This research lays the foundation for the interaction between MmpL5 and MmpS5 as a novel target.

[0005] Nontuberculous mycobacteria (NTM) have been known by various names, including atypical mycobacteria, atypical acid-fast bacilli, non-classified mycobacteria, unclassified mycobacteria, unnamed mycobacteria, wild mycobacteria, opportunistic mycobacteria, paratuberculosis bacilli, and pseudotuberculosis bacteria. NTM disease refers to human infection with NTM, which causes lesions in related tissues and organs (Daley CL, Iaccarino JM, Lange C, et al. Treatment of nontuberculous mycobacterial pulmonary disease: anovulatory ATS / ERS / ESCMID / IDSA clinical practice guideline[J]. Eur Respir J, 2020, 56(1): 2000-53.). NTM disease is a systemic disease that mainly affects lung tissue, but can affect various organ systems throughout the body, including systemic poisoning symptoms and local damage (Tang Shenjie, Gao Wen. Clinical Tuberculosis[M]. 2nd ed. Beijing: People's Medical Publishing House, 2019: 1026-1046.). Without proper identification of the pathogen, nontuberculous mycobacterial pulmonary disease (NTM) can be misdiagnosed as tuberculosis or bronchiectasis for a long time. The clinical manifestations of NTM vary depending on the infecting pathogen, affected tissues, and organs. In recent years, NTM has shown a rapid increase in cases, becoming a significant public health problem threatening human health (Furuuchi K, Morimoto K, Yoshiyama T, et al. Interrelational changes in the epidemiology and clinical features of nontuberculous mycobacterial pulmonary disease and tuberculosis in a referral hospital in Japan[J]. Respir Med, 2019, 152:74-80.).

[0006] In the diagnostic field, GeneXpert MTB / RIF has become the primary method for rapid diagnosis of tuberculosis and rapid detection of rifampicin resistance; in the therapeutic field, the all-oral short-course treatment regimen for multidrug-resistant / rifampicin-resistant tuberculosis (MDR / RR-TB) has become a guideline-recommended treatment. However, the proportion of drug-resistant diseases is still rising, and the lack of rapid diagnostic methods, long treatment courses, and the inclusion of injectable drugs in treatment regimens fail to meet the needs for rapid diagnosis and affect patient adherence. Therefore, new treatment methods are urgently needed.

[0007] Beanchlorothiazol is a thioxanthracene derivative that works by blocking dopamine receptors, exhibiting significant antipsychotic and sedative effects. Discovered in 1962, benzchlorothiazol has been used for antipsychotic purposes and is marketed in over 30 countries, including Europe, Latin America, and Canada. Currently, there are no reports on its activity against Mycobacterium tuberculosis or its synergistic antimycobacterial activity. With the increasing severity of tuberculosis drug resistance, developing novel treatment strategies to enhance the antimycobacterial activity of bedaquiline and reduce the occurrence of new drug resistance is urgently needed. Summary of the Invention

[0008] In this invention, it was discovered that the combined use of zuclothiasol and bedaquiline enhances the anti-mycobacterial activity of bedaquiline, thus the combined drug of zuclothiasol and bedaquiline exhibits anti-mycobacterial activity. Based on this, this invention was completed.

[0009] In a first aspect, the present invention provides a pharmaceutical composition for combating mycobacterial infections, the pharmaceutical composition comprising zuclothiasol and bedaquiline, the pharmaceutical composition having at least one of the following effects:

[0010] a) Inhibits mycobacterial activity;

[0011] b) Antibacterial infection;

[0012] c) Prevention and / or treatment of diseases caused by mycobacteria.

[0013] Furthermore, the mycobacteria are selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria, and / or Mycobacterium leprae.

[0014] 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.

[0015] Furthermore, the aforementioned Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0016] Furthermore, the diseases caused by Mycobacterium tuberculosis include, but are not limited to, drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, and extrapulmonary tuberculosis.

[0017] Furthermore, the drug-resistant tuberculosis mentioned includes, but is not limited to, single-drug resistant tuberculosis, multidrug resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0018] Furthermore, the pulmonary tuberculosis mentioned includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, and sputum-negative pulmonary tuberculosis.

[0019] Furthermore, the extrapulmonary tuberculosis includes, but is not limited to, lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, and bone and joint tuberculosis.

[0020] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0021] Furthermore, the tuberculosis mycobacteria include Mycobacterium humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannerae, and Mycobacterium villiformis.

[0022] Furthermore, the nontuberculous mycobacteria include, but are not limited to, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium ulcerans, and / or Mycobacterium marinum.

[0023] Furthermore, the disease caused by the nontuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphoma, disseminated NTM disease and / or other NTM diseases.

[0024] Furthermore, the pharmaceutical composition may also contain other active ingredients that combat mycobacterial infections.

[0025] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0026] Furthermore, the pharmaceutical composition can be formulated into various forms such as injection, tablets, powder, granules, capsules, oral liquid, injectable preparations, or aerosols; all of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0027] Furthermore, the formulation may be one or more of a conventional formulation, a sustained-release formulation, a controlled-release formulation, and / or a variety of microparticle delivery systems.

[0028] Furthermore, the tablets can widely utilize a variety of carriers known in the art, including one or more of diluents and absorbents, humectants and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0029] 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.

[0030] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinylpyrrolidone.

[0031] Furthermore, the disintegrant includes, but is not limited to, one or more of the following: dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose and / or ethylcellulose.

[0032] Furthermore, the disintegration inhibitors include, but are not limited to, sucrose, tristearate, cocoa butter, and / or hydrogenated oils.

[0033] Furthermore, the absorption enhancer includes, but is not limited to, one or more of quaternary ammonium salts and / or sodium dodecyl sulfate.

[0034] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silica, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0035] Furthermore, the tablets can be further formulated into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, bilayer tablets, and multilayer tablets.

[0036] Furthermore, the injectable formulation includes, but is not limited to, one or more of the following: solutions, emulsions, lyophilized powder for injection, and / or suspensions.

[0037] Furthermore, the injectable formulation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and / or polyoxyethylene sorbitan fatty acid esters.

[0038] Furthermore, in order to prepare an isotonic injection, appropriate amounts of one or more of sodium chloride, glucose, glycerol, conventional solubilizers, buffers, and / or pH adjusters may be added to the injectable formulation.

[0039] Furthermore, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary.

[0040] Furthermore, the pharmaceutical composition can be delivered into the body, such as through muscles, intradermis, subcutaneous tissue, or veins, via physical or chemical means.

[0041] Furthermore, the benzylchlorothiazol also includes pharmaceutically acceptable salts or esters.

[0042] Furthermore, the pharmaceutical salts mentioned include, but are not limited to, pharmaceutical salts such as acetates and hydrochlorides.

[0043] Furthermore, the pharmaceutically defined esters include, but are not limited to, decanoates.

[0044] Secondly, the present invention provides the application of juglottisol as an enhancer of the anti-mycobacterial activity of bedaquiline. When juglottisol is used in combination with bedaquiline, juglottisol can enhance the anti-mycobacterial activity of bedaquiline.

[0045] Furthermore, the mycobacteria are selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria, and / or Mycobacterium leprae.

[0046] 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.

[0047] Furthermore, the aforementioned Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0048] Furthermore, the diseases caused by Mycobacterium tuberculosis include, but are not limited to, drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, and extrapulmonary tuberculosis.

[0049] Furthermore, the drug-resistant tuberculosis mentioned includes, but is not limited to, single-drug resistant tuberculosis, multidrug resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0050] Furthermore, the pulmonary tuberculosis mentioned includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, and sputum-negative pulmonary tuberculosis.

[0051] Furthermore, the extrapulmonary tuberculosis includes, but is not limited to, lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, and bone and joint tuberculosis.

[0052] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0053] Furthermore, the tuberculosis mycobacteria include Mycobacterium humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannerae, and Mycobacterium villiformis.

[0054] Furthermore, the nontuberculous mycobacteria include, but are not limited to, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium ulcerans, and / or Mycobacterium marinum.

[0055] Furthermore, the disease caused by the nontuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphoma, disseminated NTM disease and / or other NTM diseases.

[0056] Furthermore, the benzylchlorothiazol also includes pharmaceutically acceptable salts or esters.

[0057] Furthermore, the pharmaceutical salts mentioned include, but are not limited to, pharmaceutical salts such as acetates and hydrochlorides.

[0058] Furthermore, the pharmaceutically defined esters include, but are not limited to, decanoates.

[0059] Thirdly, the present invention provides the application of juglottisol in combination with bedaquiline in the preparation of an anti-mycobacterial infection drug composition, wherein the combination of juglottisol and bedaquiline exerts an anti-mycobacterial infection effect, and juglottisol can enhance the anti-mycobacterial infection activity of bedaquiline.

[0060] Furthermore, the mycobacteria are selected from Mycobacterium tuberculosis, non-tuberculous mycobacteria, and / or Mycobacterium leprae.

[0061] 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.

[0062] Furthermore, the aforementioned Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0063] Furthermore, the diseases caused by Mycobacterium tuberculosis include, but are not limited to, drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, and extrapulmonary tuberculosis.

[0064] Furthermore, the drug-resistant tuberculosis mentioned includes, but is not limited to, single-drug resistant tuberculosis, multidrug resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0065] Furthermore, the pulmonary tuberculosis mentioned includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, and sputum-negative pulmonary tuberculosis.

[0066] Furthermore, the extrapulmonary tuberculosis includes, but is not limited to, lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, and bone and joint tuberculosis.

[0067] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0068] Furthermore, the tuberculosis mycobacteria include Mycobacterium humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannerae, and Mycobacterium villiformis.

[0069] Furthermore, the nontuberculous mycobacteria include, but are not limited to, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium ulcerans, and / or Mycobacterium marinum.

[0070] Furthermore, the disease caused by the nontuberculous mycobacteria is selected from one or more of NTM lung disease, NTM lymphoma, disseminated NTM disease and / or other NTM diseases.

[0071] Furthermore, the pharmaceutical composition may also contain other active ingredients that combat mycobacterial infections.

[0072] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0073] Furthermore, the pharmaceutical composition can be formulated into various forms such as injection, tablets, powder, granules, capsules, oral liquid, injectable preparations, or aerosols; all of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0074] Furthermore, the formulation may be one or more of a conventional formulation, a sustained-release formulation, a controlled-release formulation, and / or a variety of microparticle delivery systems.

[0075] Furthermore, the tablets can widely utilize a variety of carriers known in the art, including one or more of diluents and absorbents, humectants and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0076] 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.

[0077] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinylpyrrolidone.

[0078] Furthermore, the disintegrant includes, but is not limited to, one or more of the following: dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose and / or ethylcellulose.

[0079] Furthermore, the disintegration inhibitors include, but are not limited to, sucrose, tristearate, cocoa butter, and / or hydrogenated oils.

[0080] Furthermore, the absorption enhancer includes, but is not limited to, one or more of quaternary ammonium salts and / or sodium dodecyl sulfate.

[0081] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silica, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0082] Furthermore, the tablets can be further formulated into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, bilayer tablets, and multilayer tablets.

[0083] Furthermore, the injectable formulation includes, but is not limited to, one or more of the following: solutions, emulsions, lyophilized powder for injection, and / or suspensions.

[0084] Furthermore, the injectable formulation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and / or polyoxyethylene sorbitan fatty acid esters.

[0085] Furthermore, in order to prepare an isotonic injection, appropriate amounts of one or more of sodium chloride, glucose, glycerol, conventional solubilizers, buffers, and / or pH adjusters may be added to the injectable formulation.

[0086] Furthermore, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary.

[0087] Furthermore, the pharmaceutical composition can be delivered into the body, such as through muscles, intradermis, subcutaneous tissue, or veins, via physical or chemical means.

[0088] Furthermore, the benzylchlorothiazol also includes pharmaceutically acceptable salts or esters.

[0089] Furthermore, the pharmaceutical salts mentioned include, but are not limited to, pharmaceutical salts such as acetates and hydrochlorides.

[0090] Furthermore, the pharmaceutically defined esters include, but are not limited to, decanoates.

[0091] Beneficial effects

[0092] In this invention, the combination of juglone and bedaquiline exhibits a significant enhancement of activity against mycobacterial infections:

[0093] Compared to bedaquiline alone, 2 μg / ml of zirconia thiamethoxam combined with bedaquiline reduced the colony count of MmpL5-MmpS5 strains to varying degrees, from 1 / 16 MIC to 2 MIC of bedaquiline.

[0094] As a potentiator of bedaquiline, zirconia thiamethoxam can enhance the antibacterial activity of bedaquiline.

[0095] The minimum inhibitory concentration (MIC) of ziclothiasol against the Rv0678 mutant strain in vitro was 16 μg / mL, while the MIC of BDQ against the Rv0678 mutant strain in vitro was 1 μg / mL. The combination of 1 μg / mL ziclothiasol and BDQ reduced the MIC of the Rv0678 mutant strain against BDQ from 1 μg / mL to 0.03 μg / mL (a 32-fold increase), which was lower than the MIC of the wild-type strain H37Rv (0.06 μg / mL), reversing BDQ resistance and further enhancing its activity against mycobacterial infections. Attached Figure Description

[0096] Figure 1 Time-killing curves of zirconia thiamethoxam (ZUC) in combination with bedaquiline (BDQ) on Mycobacterium tuberculosis H37Rv.

[0097] Figure 2 Time-killing curves of zirconia thiabendazole (ZUC) in combination with bedaquiline (BDQ) on Mycobacterium tuberculosis MmpL5-mmpS5 overexpression strain.

[0098] Figure 3 Time-kill curves of different concentrations of bedaquiline (BDQ) on MmpL5-MmpS5 overexpressing strains.

[0099] Figure 4 Time-kill curves of different concentrations of zirconia thiabendazole (ZUC) combined with bedaquiline (BDQ) against MmpL5-MmpS5 overexpressing strains. DETAILED DESCRIPTION

[0100] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, 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.

[0101] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0102] the term

[0103] Glucothiasol: Glucothiasol is a thioxanthracene derivative, the cis isomer of chloropentathione. It works by blocking dopamine receptors, exhibiting significant antipsychotic and specific sedative effects, making it particularly suitable for patients with schizophrenia. Glucothiasol has low acute and chronic toxicity, and no toxicity was observed at therapeutic doses. There are no special precautions required when used in women of childbearing age. It has no mutagenic or carcinogenic properties, making it particularly suitable for elderly patients.

[0104] Bedaquiline (BDQ): Chemically named 1-(6-bromo-2-methoxyquinoline-3-yl)-4-dimethylamino-1-phenyl-2-(1-naphthyl)-2-butanol, traded as Sirturo, is a novel diarylquinoline antibacterial drug. It exerts its antibacterial and bactericidal effects by inhibiting the proton pump activity of ATP synthase in Mycobacterium tuberculosis. Clinically, it is used to treat multidrug-resistant pulmonary tuberculosis (MDR-PTB) in adults.

[0105] In this invention, the synergist refers to juglothiol. When juglothiol and bedaquiline are used in combination, the activity of bedaquiline against mycobacterial infection is enhanced. The anti-tuberculosis ability of the drug composition after the combination of juglothiol and bedaquiline is enhanced compared with the use of bedaquiline alone.

[0106] MIC (Minimum Inhibitory Concentration): This is an indicator of the antibacterial activity of an antimicrobial drug, referring to the lowest drug concentration that can inhibit the growth of pathogenic bacteria 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 research drugs to determine their feasibility for further development as a treatment for pulmonary tuberculosis. Currently, the commonly used quantitative method is the checkerboard dilution method. Taking the interaction of two drugs as an example, the MIC of each research drug against Mycobacterium tuberculosis is first determined. Based on the MIC values, the maximum concentration is set as 2×MIC of a single drug. Then, each drug is diluted twofold (in both the vertical and horizontal columns of a checkerboard array), with each tube (well) containing a mixture of different concentrations of the two drugs. Generally, 6–8 dilutions are designed. The initial inoculum is 5×10⁵ CFU / mL, incubated at 37°C for 7 days, and the results are observed and the fractional inhibitory concentration index (FICI) is calculated. The FICI index is calculated as follows: (MIC of drug A in combination / MIC of drug A alone) + (MIC of drug B in combination / MIC of drug B alone). Based on the FICI value, effects are categorized as synergistic (≤0.5), additive (0.5–1), irrelevant (1–2), and antagonistic (>2). FICI is one of the pharmacodynamic (PD) parameters for antimicrobial drugs, and it is an indicator of drug sensitivity in combination of two antimicrobial drugs (when two antimicrobial drugs are used simultaneously, four scenarios can occur: synergistic, additive, irrelevant, and antagonistic).

[0107] Time-kill curves allow for the dynamic observation of the interaction between drugs (compounds) and Mycobacterium tuberculosis. They compare the effects of different concentrations of compounds on Mycobacterium tuberculosis growth at different treatment times, providing more evidence for evaluating the anti-mycobacterial activity of compounds. The experimental period for time-kill curves is typically 14 days. Mycobacterium tuberculosis is continuously cultured in drug-containing medium for 14 days, with a sample taken every 3-4 days and cultured on solid medium. Colony counting is performed after 3-4 weeks. The time-kill curves are plotted with the time point of bacterial collection as the x-axis and the Log10 of the colony count as the y-axis. Time-kill curves are also used to evaluate the interactions between drugs in drug combinations in vitro.

[0108] Verapamil: A derivative of papaverine, commonly used as its hydrochloride salt, is a white powder; odorless. It is readily soluble in methanol, ethanol, or chloroform, and soluble in water. Melting point 140℃~145℃. It was used as a coronary vasodilator in 1962. Verapamil, also known as isopranosine or valproic acid, is a calcium channel blocker. In recent years, it has been used to treat hypertension, angina pectoris, arrhythmia, cerebrovascular disease, finger vasospasm, abdominal pain, achalasia, migraine, pulmonary hypertension, and to prevent premature birth.

[0109] Example

[0110] Example 1: Screening of benzylchlorothiazide

[0111] Using Mycobacterium tuberculosis with Rv0678 mutant strain (MmpL5-MmpS5 overexpression) (Rv0678 mutant strain: BDQMIC = 1 μg / ml) as a functional screening model, compounds that can reduce bedaquiline MIC were screened from a drug library.

[0112] A screening of 4000 compounds revealed zirconia thiasol, which exhibits the best synergistic activity against mycobacterial infections with bedaquiline. Its structural formula is as follows:

[0113]

[0114] Example 2: Determination of minimum inhibitory concentration and synergistic activity of benzylchlorothiazol using the MABA method

[0115] 2.1 Test Content

[0116] This section uses the microplate Alamar Blue assay (MABA) to determine the minimum inhibitory concentration.

[0117] Add culture medium and drug stock solution to the microwells:

[0118] Weigh the drug powder and dissolve the lipid-soluble drug in DMSO to achieve a stock solution concentration of 2 mg / ml. Take a logarithmic growth phase Mycobacterium tuberculosis bacterial suspension and culture medium in a well plate. Measure the OD value of each well at 570 nm, where OD(strain) = OD(bacterial suspension) - OD(7H9 medium). OD = 0.1 is equivalent to 1 × 10⁻⁶. 7 CFU / mL, dilute each bacterial suspension to achieve a final concentration of 1×10⁻⁶ for each strain. 5 CFU / mL.

[0119] The drug in the first column of microwells was diluted to the tenth column using a two-fold dilution method, with a final concentration of 1×10⁻⁶ microbial solution in each microwell. 5CFU / mL. On day 7, Tween-80 solution and 20 Alamar blue indicator were added to each well, and incubation continued. The color change was observed the following day. Fluorescence values ​​of each well were measured at excitation wavelengths of 530 nm and 590 nm, and the color of each well was recorded. Blue indicated no bacterial growth, and red indicated bacterial growth. MIC represents the minimum drug concentration required for the color to change from blue to red.

[0120] The combined activity was determined using the MIC method, but with a difference in that after adding 1 μg / mL zuclothiasol or 10 μg / mL verapamil to the culture medium, subsequent operations were performed using culture medium containing 1 μg / mL zuclothiasol or 10 μg / mL verapamil. The first 6 wells served as negative controls, and the last 6 wells contained diluted bacterial suspension as positive controls. Then, 198 μL of culture medium containing 1 μg / mL zuclothiasol or 10 μg / mL verapamil was added to the first column of wells in row BH of a 96-well plate, and 100 μL of culture medium containing 1 μg / mL zuclothiasol or 10 μg / mL verapamil was added to the remaining wells. 2 μL of BDQ stock solution was added to the first column of wells in row BH. After mixing the drug solution with the culture medium containing 1 μg / mL zuclothiasol or 10 μg / mL verapamil, this operation was repeated using a two-fold dilution method until the last column of wells. Therefore, the MIC of bedaquiline and the fold reduction were determined when it was treated with a fixed concentration of benzylchlorothiazide or verapamil.

[0121] 2.2 Test Results

[0122] The minimum inhibitory concentration (MIC) of ziclothiasol against the Rv0678 mutant strain in vitro was 16 μg / ml, and the MIC of BDQ against the Rv0678 mutant strain in vitro was 1 μg / ml (as shown in Table 1). The combination of 1 μg / ml ziclothiasol and BDQ reduced the MIC of the Rv0678 mutant strain against BDQ from 1 μg / ml to 0.03 μg / ml (a 32-fold increase), which was lower than the MIC of the wild-type strain H37Rv (0.06 μg / ml), reversing BDQ resistance and further enhancing its activity against mycobacterial infections. In contrast, 10 μg / ml of the efflux pump inhibitor verapamil had no effect on the MIC of BDQ in the Rv0678 mutant strain (as shown in Table 1).

[0123] Table 1. Activity of betachlorothiazol-enhanced bedaquiline against Rv0678 mutant strain

[0124]

[0125] Note: BDQ's MIC (0.06 μg / ml) against wild-type H37Rv

[0126] Example 3: Determination of the combined activity of benzylchlorothiazol and different anti-tuberculosis drugs against different strains using the checkerboard method.

[0127] 3.1 Test Content

[0128] The checkerboard method was used to determine the in vitro activity of benzylchlorothiazol in combination with different anti-tuberculosis drugs, as well as its activity in combination with bedaquiline for Mycobacterium tuberculosis with different sensitivities.

[0129] Based on the MIC of each drug when used alone, a total of 7 concentration gradients were set up, with a concentration range of 4×MIC⁻¹ / 16×MIC. The drugs were distributed in a checkerboard pattern, as shown in Table 2. In the 96-well plate, X1-X7 were drug A (zirconiazid: ZUC) at 4×MIC⁻¹ / 16×MIC, and Y1-Y7 were drug B (bedaquiline: BDQ; isoniazid: INH; PA-824; moxifloxacin: MXF and linezolid: LZD) at 4×MIC⁻¹ / 16×MIC. Drug A was diluted vertically and drug B was diluted horizontally. Columns A9-H9 and A10-H10 were negative control wells and positive control wells, respectively (as shown in Table 2).

[0130] After 7 days of incubation, Alamar Blue indicator was added. 24 hours later, 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 formula:

[0131] FICI = MICA combined / MICA alone + MICB combined / MICB alone

[0132] Among them, MICA alone and MICB alone represent the MIC of Mycobacterium tuberculosis when used alone as drugs A and B, respectively. MICA combined and MICB combined represent the minimum concentrations of drugs A and B that prevent the color from changing from blue to pink when used in combination as drugs A and B, respectively.

[0133] Table 2 Two-dimensional chessboard design

[0134]

[0135] Note: X represents drug A, i.e., zirconia: ZUC; X1-X7 represent 7 concentration gradients of zirconia: 4×MIC-1 / 16×MIC;

[0136] Y represents drug B, namely bedaquiline (BDQ), isoniazid (INH), PA-824, moxifloxacin (MXF), and linezolid (LZD); Y1-Y7 represent the seven concentration gradients of drug B: 4×MIC-1 / 16×MIC.

[0137] 3.2 Test Results

[0138] The checkerboard method experiment showed that juglottiol and BDQ had the best synergistic activity against mycobacterial infection in vitro against the Rv0678 mutant strain, with a FICI of 0.156. In addition to the synergistic anti-tuberculosis effect with BDQ, juglottiol did not interact with other anti-tuberculosis drugs such as isoniazid (INH), PA-824, moxifloxacin (MXF), and linezolid (LZD) (as shown in Table 3).

[0139] Table 3. The combined effects of zirconia thiazide and anti-tuberculosis drugs on the Rv0678 mutant strain.

[0140]

[0141] Note: When BDQ alone has a MIC of 1 μg / mL and a combined MIC of 0.03125 μg / mL, the concentration of globulin is 2 μg / mL; when INH alone has a MIC of 0.04 μg / mL and a combined MIC of 0.00125 μg / mL, the concentration of globulin is 16 μg / mL; when PA-824 alone has a MIC of 0.16 μg / mL and a combined MIC of 16 μg / mL, the concentration of globulin is 16 μg / mL; when MFX alone has a MIC of 1 μg / mL and a combined MIC of 0.03 μg / mL, the concentration of globulin is 16 μg / mL; when LZD alone has a MIC of 0.29 μg / mL and a combined MIC of 0.009 μg / mL, the concentration of globulin is 16 μg / mL.

[0142] Further investigation was conducted to determine the activity of betachlorothiazol combined with bedaquiline in different bedaquiline-resistant strains. The combined synergistic activity was studied on the constructed genetically engineered strain MmpL5-MmpS5 overexpression strain, the sensitive strain H37Rv, different Rv0678 mutant strains, and the bedaquiline target gene atpE mutant strain.

[0143] The checkerboard method was used to determine the interaction and found that benzylchlorothiazol and bedaquiline had synergistic activity in all tested strains, but the synergistic activity was better in the Rv0678 mutant and the MmpL5-MmpS5 overexpression strain than in the H37Rv strain and the atpE mutant (as shown in Table 4).

[0144] Table 4. Activity of piracetam in combination with bedaquiline against mycobacterial infections in different bacterial strains.

[0145]

[0146] Note: For the H37Rv standard strain, the concentration of juglottisol was 2 μg / mL when the MIC of BDQ alone was 0.06 μg / mL and the combined MIC was 0.015 μg / mL; for the MmpL5-MmpS5 overexpression strain, the concentration of juglottisol was 2 μg / mL when the MIC of BDQ alone was 0.5 μg / mL and the combined MIC was 0.03125 μg / mL; for the Rv0678 mutant strain 2, the concentration of juglottisol was 2 μg / mL when the MIC of BDQ alone was 1 μg / mL and the combined MIC was 0.0625 μg / mL; and for the atpE mutant strain, the concentration of juglottisol was 8 μg / mL when the MIC of BDQ alone was 1.25 μg / mL and the combined MIC was 0.3125 μg / mL.

[0147] Example 4: Evaluation of the antibacterial activity of the two-drug combination containing piracetam using the time-based bactericidal curve method.

[0148] 4.1 Test Content

[0149] To investigate the dynamic bactericidal activity of benzylchlorothiazol combined with bedaquiline, a study was conducted to determine the bactericidal curve of the combined action.

[0150] The evaluation of two-drug combinations of antimicrobial agents included single-drug wells and two-drug combination wells. Time-kill assays were performed using ZUC 10 μg / mL and BDQ 1 / 2×MIC.

[0151] Prepare drug and drug combination solutions of appropriate concentrations, and add H37Rv or Rv0678 mutant bacterial suspensions to achieve a concentration of 2×10⁻⁶ for each bacterial suspension. 5 CFU / mL, cultured. On the day of culture (D0) and on days 3, 7, 10, and 14 of culture, the original or diluted bacterial solution was taken from the 24-well plate and inoculated onto solid culture medium, spread evenly, and cultured for 4 weeks. Colony counts were performed. The culture time points of Mycobacterium tuberculosis were used as the x-axis, and the logarithm of the colony count at different culture time points was used as the y-axis to plot the time-killing curves of the combination of zirconia thiamethoxam and bedaquiline.

[0152] 4.2 Test Results

[0153] Both 10 μg / ml zirconia thiamethoxam and 1 / 2 MIC bedaquiline exhibited antibacterial activity against mycobacterial infections in H37Rv and MmpL5-MmpS5 overexpressing strains, while neither 10 μg / ml zirconia thiamethoxam nor 1 / 2 MIC bedaquiline alone showed bactericidal activity (e.g., ...). Figure 1 (As shown).

[0154] The combination of tebufenozide and bedaquiline showed a more significant bactericidal effect against the Rv0678 mutant strain, achieving sterilization in 10 days, while H37Rv required 14 days to achieve sterilization (e.g., Figure 2 (As shown).

[0155] Further reducing the effective concentration of tebuconazole and treating the MmpL5-MmpS5 strain with different concentrations of bedaquiline revealed that compared to bedaquiline alone (e.g., ... Figure 3 As shown), 2 μg / ml zirconia thiamethoxam combined with bedaquiline reduced the colony count of MmpL5-MmpS5 strains to varying degrees, from 1 / 16 MIC to 2 MIC of bedaquiline, demonstrating significantly enhanced activity against mycobacterial infections (e.g., Figure 4 (As shown).

Claims

1. A pharmaceutical composition for treating Mycobacterium tuberculosis infection, the pharmaceutical composition comprising zuclothiasol and bedaquiline, the pharmaceutical composition having at least one of the following effects: a) Inhibits the activity of Mycobacterium tuberculosis; b) Anti-tuberculosis mycobacterial infection; c) Prevention and / or treatment of diseases caused by Mycobacterium tuberculosis.

2. The pharmaceutical composition of claim 1, wherein the dosage form of the pharmaceutical composition includes injection, tablets, powder, granules, capsules, oral liquid, and aerosol; the zuclothiasol further includes a pharmaceutically acceptable salt, wherein, The pharmaceutical salts include acetates and hydrochlorides.

3. The pharmaceutical composition of claim 1, wherein the Mycobacterium tuberculosis comprises clinical isolates and standard strains of Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection comprises 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. The application of juglottisol in combination with bedaquiline in the preparation of a drug composition for treating Mycobacterium tuberculosis infection, wherein the combination of juglottisol and bedaquiline exerts an anti-Mycobacterium tuberculosis infection effect, and juglottisol enhances the anti-Mycobacterium tuberculosis infection activity of bedaquiline.

5. The application as described in claim 4, wherein the dosage form of the pharmaceutical composition includes injection, tablets, powder, granules, capsules, oral liquid, and aerosol; the zusylchlorothiazol further includes a pharmaceutically acceptable salt, wherein, The pharmaceutical salts include acetates and hydrochlorides.

6. The application as described in claim 4, wherein the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and standard strains of Mycobacterium tuberculosis; and the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

7. The application of pinecone in the preparation of potentiators for anti-tuberculosis mycobacterial infection drugs, wherein the potentiator's function is to increase the anti-tuberculosis mycobacterial infection effect of bedaquiline, that is, when pinecone is used in combination with bedaquiline, pinecone enhances the anti-tuberculosis mycobacterial infection activity of bedaquiline.

8. The application as described in claim 7, wherein the dosage form of the drug includes injection, tablets, powder, granules, capsules, oral liquid, and aerosol; the zucithinol further includes a pharmaceutically acceptable salt, wherein, The pharmaceutical salts include acetates and hydrochlorides.

9. The application as described in claim 7, wherein the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and standard strains of Mycobacterium tuberculosis; and the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

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