Application of GSK1292263 in the preparation of drugs for inhibiting Mycobacterium tuberculosis

Abstract

The present invention relates to the field of medical technology, and in particular to the use of small molecule compounds in the preparation of drugs for inhibiting mycobacterium tuberculosis, wherein the small molecule compound is GSK1292263 or a composition containing GSK1292263, and the combination of GSK1292263 is a composition comprising GSK1292263 and other active ingredients, wherein the other active ingredients are active substances that can promote the body's anti-tuberculosis and / or drugs that improve tuberculosis symptoms. The GSK1292263 of the present invention exhibits good antibacterial activity against tuberculosis bacilli and can significantly inhibit mycobacterium tuberculosis. In addition, GSK1292263 has a relatively high survival rate for U937 and THP-1 cells.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a small molecule compound GSK1292263 in the preparation of a drug for inhibiting mycobacterium tuberculosis. Background Art

[0002] Tuberculosis (TB) is one of the most lethal chronic infectious diseases in the world caused by Mycobacterium tuberculosis (Mtb). It is also one of the major challenges facing global public health, the "number one killer" of people infected with human immunodeficiency virus (HIV), and the main lethal infectious disease associated with antimicrobial resistance. The mortality rate is very high among HIV carriers who are also infected with multidrug-resistant Mycobacterium tuberculosis. This mixed infection is also the cause of the prevalence of multidrug-resistant tuberculosis. According to estimates by the World Health Organization, in 2021, there will be more than 10.6 million new cases of tuberculosis and more than 1.6 million deaths worldwide. Only 36% of patients who need treatment will receive treatment. The prevalence of drug-resistant strains and co-infection with HIV have further exacerbated the global TB burden. The number of patients with rifampicin resistance-tuberculosis (RR-TB) and multiple drug resistant tuberculosis (MDR-TB) who started treatment in 2021 was 161 and 746, covering only one-third of all patients in need of treatment. However, 187,000 people infected with HIV died of tuberculosis. Effective control of tuberculosis remains a major public health challenge facing the world.

[0003] Although the drugs for the treatment of tuberculosis are continuously being improved and developed, and the world has been actively developing a vaccine that can effectively prevent the spread of Mycobacterium tuberculosis, it is still challenging to develop a new vaccine that is more applicable and durable than Bacillus Calmette-Guerin (BCG). Mycobacterium tuberculosis has the ability to regulate the host immune response and evade drugs, which can lead to the failure of BCG vaccination and eventually form drug-resistant Mycobacterium tuberculosis.

[0004] In recent years, great progress has been made in the treatment of tuberculosis. For example, isoniazid (INH), rifampicin (RFP), bedaquiline, delamanid, etc. are very effective against Mycobacterium tuberculosis, but tuberculosis cannot be completely cured. When INH becomes resistant, it usually leads to the emergence of multidrug-resistant strains, which has become a major obstacle to the control of tuberculosis. Bedaquiline and delamanid have limited activity in killing persisters, and both drugs have strong cardiotoxicity. More importantly, clinically, mutant strains of Mycobacterium tuberculosis that are resistant to these two drugs were quickly discovered. Fluoroquinolones are effective second-line anti-tuberculosis drugs for the treatment of drug-resistant tuberculosis and are widely used to treat multidrug-resistant tuberculosis. However, it has been found that fluoroquinolones are easily resistant to multidrug-resistant tuberculosis. Although fluoroquinolones have a good inhibitory effect on DNA gyrase, mutations in the gyrA domain produce resistance to these drugs during the treatment of tuberculosis. With the emergence of drug resistance in Mycobacterium tuberculosis, new therapies that can shorten the treatment cycle are crucial for the prevention and treatment of tuberculosis, especially some therapies with new mechanisms of action provide effective methods for the treatment of drug-resistant tuberculosis. Based on the above problems, the urgent need to develop new, highly effective and low-toxic anti-tuberculosis drugs to effectively control tuberculosis has become an important research direction for many researchers. Summary of the invention

[0005] In a first aspect, the present invention provides a use of a small molecule compound in the preparation of an anti-Mycobacterium tuberculosis preparation.

[0006] Furthermore, the small molecule compound is GSK1292263 or a composition containing GSK1292263.

[0007] Furthermore, the structural formula of GSK1292263 is as shown in I:

[0008]

[0009] Furthermore, the combination of GSK1292263 is a composition comprising GSK1292263 and other active ingredients.

[0010] Furthermore, the other active ingredients are active substances that can promote the body's anti-tuberculosis and / or drugs that improve tuberculosis symptoms.

[0011] Furthermore, the preparation includes a biological preparation or a pharmaceutical preparation.

[0012] Furthermore, the dosage forms of the anti-tuberculosis mycobacterium preparation include: sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, powders, wine preparations, preparations, drops, injections, powder injections, creams, sustained-release preparations, targeted agents, etc.

[0013] Furthermore, the administration methods of the anti-tuberculosis mycobacterium preparation include oral administration, injection, implantation, external application, spraying, and inhalation.

[0014] Furthermore, the anti-Mycobacterium tuberculosis preparation can be used to prevent or treat tuberculosis caused by Mycobacterium tuberculosis infection.

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

[0016] Furthermore, the tuberculosis includes but is not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

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

[0018] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

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

[0020] In a second aspect, the present invention provides an application of a small molecule compound in the preparation of an anti-tuberculosis preparation.

[0021] Furthermore, the small molecule compound is GSK1292263 or a composition containing GSK1292263.

[0022] Furthermore, the structural formula of GSK1292263 is as shown in I:

[0023]

[0024] Furthermore, the combination of GSK1292263 is a composition comprising GSK1292263 and other active ingredients.

[0025] Furthermore, the other active ingredients are active substances that can promote the body's anti-tuberculosis and / or drugs that improve tuberculosis symptoms.

[0026] Furthermore, the preparation includes a biological preparation or a pharmaceutical preparation.

[0027] Furthermore, the anti-tuberculosis preparations and / or drug dosage forms include: sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, powders, wine preparations, preparations, drops, injections, powder injections, creams, sustained-release agents, targeted agents, etc.

[0028] Furthermore, the administration of the anti-tuberculosis preparation includes oral administration, injection, implantation, external application, spraying, inhalation and / or a combination thereof.

[0029] Furthermore, the tuberculosis includes but is not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0030] Furthermore, the drug-resistant tuberculosis includes but is not limited to mono-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0031] Furthermore, the pulmonary tuberculosis is not limited to primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

[0032] Furthermore, the extrapulmonary tuberculosis includes but is not limited to intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 MIC test results of GSK1292263 on standard strains of Mycobacterium tuberculosis and drug-resistant Mycobacterium tuberculosis

[0034] Figure 2 Effect of GSK1292263 on the survival rate of U937 and THP-1 cells DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with the accompanying drawings and specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention. The experimental methods in the examples, unless otherwise specified, all adopt conventional techniques in the art, and the experimental reagents are all purchased commercially.

[0036] The chemical structural formula of GSK1292263 is as shown in I:

[0037]

[0038] It is a small molecule compound that targets the Wnt / β-catenin pathway to disrupt the stability of β-catenin and Ras, inhibiting a series of reactions including epithelial-mesenchymal transition (EMT) and apoptosis. Abnormal activation of the Wnt / β-catenin and Ras pathways has been found to be associated with metastatic colorectal cancer and to act synergistically in the process of tumor metastasis. GSK1292263 disrupts the binding affinity of Ras and β-catenin complex by binding to the G protein signaling domain (Regulator of G protein Signaling, RGS) domain of axin, causing β-catenin and Ras to lose stability simultaneously through GSK3β activation. This mechanism of action is an ideal strategy for the treatment of metastatic colorectal cancer.

[0039] Cell lines: The standard strain of Mycobacterium tuberculosis H37Rv was from the cell bank (ATCC: 27294), the drug-resistant strain MDR-1 was from the clinical isolate of Beijing Chest Hospital (MDR-1 strain was resistant to rifampicin, isoniazid, and streptomycin; human histiocytic lymphoma cells (U937 cells), and human monocytic leukemia cells (THP-1 cells).

[0040] Monoresistant tuberculosis: Monoresistant tuberculosis refers to the Mycobacterium tuberculosis infected in tuberculosis patients, which has been confirmed to be resistant to one anti-tuberculosis drug in vitro.

[0041] Multidrug-resistant tuberculosis: Multidrug resistance means that the Mycobacterium tuberculosis infected by tuberculosis patients has been confirmed in vitro to be resistant to more than one anti-tuberculosis drug, but does not include cases where the Mycobacterium tuberculosis is resistant to both isoniazid and rifampicin at the same time.

[0042] Multidrug-resistant tuberculosis: Tuberculosis that is resistant to both isoniazid and rifampicin is called multidrug-resistant tuberculosis.

[0043] Extensively drug-resistant tuberculosis: In addition to multidrug-resistant tuberculosis, tuberculosis is also resistant to fluoroquinolones and one of the second-line injectable anti-tuberculosis drugs, including kanamycin, amikacin, capreomycin and streptomycin. It is called extensively drug-resistant tuberculosis.

[0044] Example 1 GSK1292263 in vitro drug sensitivity test for inhibiting tuberculosis bacillus

[0045] The in vitro anti-Mycobacterium tuberculosis standard strain H37Rv activity of GSK1292263 was determined by 96-well plate microdilution method.

[0046] Add 200 μL of sterile PBS twice around the perimeter of a sterile 96-well plate to prevent evaporation. 120 μL of 7H9 liquid culture medium containing 10% OADC was added to wells C3 and D3 (except rows A and H), and 100 μL of 7H9 liquid culture medium containing 10% OADC was added to the wells in the remaining columns C4-C10. 80 uL of GSK1292263 (powder dissolved in DMSO and stored at -80°C) with a diluted concentration of 100 μM was added to the experimental wells C3 and D3. The liquid in the wells in the third column was pipetted evenly with a pipette, and 100 μL was drawn into the corresponding wells in the fourth column. Then, the liquid was pipetted evenly and the above operation was repeated to the corresponding wells in the tenth column until all drugs were diluted to a final concentration of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, and 0.156 μM (200 μL volume). Add INH and RIF stock solutions with a final concentration of 1 μg / ml to the positive drug wells in row E, and set up 4 replicate wells. Add 200 μL of 7H9 liquid culture medium containing 10% OADC to rows F3-F6 as negative control wells, and add 100 μL of 7H9 liquid culture medium containing 10% OADC to rows F7-F10 as positive control wells (see Table 1).

[0047] Table 1 Distribution of concentrations of MTB standard strains in 96-well plates

[0048] 1 2 3 4 5 6 7 8 9 10 11 12 A PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS B PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS C PBS PBS 20μM 10μM 5μM 2.5μM 1.25μM 0.625μM 0.312μM 0.156μM PBS PBS D PBS PBS 20μM 10μM 5μM 2.5μM 1.25μM 0.625μM 0.312μM 0.156μM PBS PBS E PBS PBS RIF RIF RIF RIF INH INH INH INH PBS PBS F PBS PBS Positive Positive Positive Positive Negative Negative Negative Negative PBS PBS G PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS H PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

[0049] The activity of GSK1292263 against MDR-resistant strains was determined by 96-well plate microdilution assay.

[0050] Add 200 μl sterile PBS (to prevent evaporation) around a sterile 96-well plate, add 120 μl of 7H9 liquid culture medium containing 10% OADC to each well of B2 and C2, and add 100 μl of 7H9 liquid culture medium containing 10% OADC to each well of the remaining wells, and add 80 μl of GSK1292263 stock solution (powder dissolved in DMSO and stored at -80°C) with a diluted concentration of 100 μM to each well of B2 and C2. Use a pipette to blow the liquid in the wells of B2 and C2 evenly, then draw 100 μl into the wells corresponding to the third column, and then continue to blow evenly and repeat the above operation to the wells corresponding to the ninth column until all drugs are diluted, so that the final drug concentration is 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, 0.156 μM (200 μl volume). B2-11 / C2-11 is the MDR group, 100 μL of 7H9 liquid culture medium containing 10% OADC is added to the 12th column as a negative control well, the 10th column is a positive control well, and there are 2 positive control wells for each of the 2 drug-resistant bacteria (see Table 2).

[0051] Table 2 Distribution of concentrations of MTB resistant strains in 96-well plates

[0052]

[0053]

[0054] The Roche culture medium of the two MTB strains cultured to the logarithmic growth phase was taken out from the 37°C constant temperature incubator, and the colonies were scraped and placed in a 2mL ultrasonic dispersion tube containing 10% OADC 7H9 liquid culture medium. The tube was ultrasonicated for 1 minute in an ultrasonic dispersion instrument, and the ratio of the bacterial solution to the liquid culture medium was adjusted in a colorimetric cup, and the cup was placed in a spectrophotometer. The detection wavelength was set to 600nm, and the absorbance value of each strain was adjusted to 0.3. The concentration of the bacterial solution of each strain was calculated based on the absorbance value. Then, the two bacterial solutions were diluted 10 times with 7H9 liquid culture medium, and 100 μL of the diluted bacterial solution was pipetted and added to two 96-well plates, and the sterile 96-well culture plates with the bacterial solution were sealed with sealing film and placed in a 37°C constant temperature incubator for static culture for 10 days.

[0055] After 10 days, take out the two 96-well culture plates from the constant temperature incubator, carefully remove the sealing film in the biosafety cabinet, and add 70 μL of resazurin indicator to each well. The sterile 96-well culture plates with the indicator added are placed in a 37°C constant temperature incubator for 24-48 hours.

[0056] After the incubation, take out the two 96-well culture plates, observe and record the color changes of each well in the two 96 microplates (blue indicates no strain growth, pink indicates strain growth), and determine the MIC (minimum inhibitory concentration is defined as the lowest drug concentration that inhibits visible bacterial growth). The MIC value is defined as the drug concentration value at which the color changes from blue to pink after adding the resazurin indicator 24-48 hours after culturing the 96-well plate in a 37°C constant temperature incubator for 10 days, and pink indicates bacterial growth.

[0057] GraphPad 8.0.2 software was used to draw graphs and perform statistical analysis on the results.

[0058] The experimental results showed that the MIC of GSK1292263 against MTB H37Rv was 5 μM, and the MIC against clinical drug-resistant bacteria MDR-1 (resistant to isoniazid and rifampicin) was ≥2.5 μM.

[0059] The MIC of clinical multidrug-resistant strains is 10 μM. GSK1292263 has a good inhibitory effect on Mycobacterium tuberculosis in vitro (see Figure 1 ).

[0060] Example 2 Experimental study on the inhibition of Mycobacterium tuberculosis cytotoxicity by GSK1292263

[0061] The present invention adopts CCK-8 method to determine the cell viability. The CCK-8 method utilizes WST-8 [(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt], which can be reduced to a water-soluble orange-yellow formazan dye by dehydrogenase in the mitochondria of living cells under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate, and the amount of the generated formazan is proportional to the number of living cells.

[0062] Human histiocytic lymphoma cells (U937 cells) and human monocytic leukemia cells (THP-1 cells) were taken out of liquid nitrogen and revived to the logarithmic phase. The concentration of the cell suspension was adjusted to about 2×10 5 / ml, add to 96-well cell culture plates, 100μL per well, add phorbol ester (Phorbol 12-myristate 13-acetate, PMA) to differentiate the cells into macrophages, the final concentration is 50ng / mL, and place in a cell culture incubator (5% CO2, 37°C) for overnight culture. Then, discard the old culture medium, add culture medium to dilute GSK1292263 with a final concentration of 10μM, 5μM, 2.5μM, and 1.25μM to the culture plate, and set up 2 replicates for each concentration. The negative control well does not contain drugs, and the cell plate is placed in the incubator for another 24h. Then discard the culture medium, and follow the instructions of the CCK-8 Cell Proliferation / Cytotoxicity Kit (Cat. No.: CK04, Japan Dojin Chemical Research Institute) to detect the cytotoxicity of the drug.

[0063] The experimental results showed that the survival rates of U937 and THP-1 cells were 117.5% and 71.9% at a concentration of 10 μM GSK1292263, and 128.7% and 84.9% at a concentration of 1.25 μM GSK1292263, respectively. When the drug concentration was 10 μM and 1.25 μM, the cell survival rate was not significantly different from that of the control group (see Figure 2 ).

[0064] In summary, the experimental results show that GSK1292263 exhibits good antibacterial activity against Mycobacterium tuberculosis and can significantly inhibit Mycobacterium tuberculosis. In addition, GSK1292263 has a relatively high survival rate for U937 and THP-1 cells. Therefore, GSK1292263 is of great significance in the preparation of anti-Mycobacterium tuberculosis drugs.

Claims

1. Use of a small molecule compound in the preparation of an anti-Mycobacterium tuberculosis preparation, wherein the small molecule compound is GSK1292263, and the structural formula of GSK1292263 is as shown in I: （I）。 2. The use according to claim 1, wherein the preparation further comprises other active ingredients.

3. The use according to claim 2, characterized in that The other active ingredients are drugs that promote the body's resistance to tuberculosis and / or improve tuberculosis symptoms.

4. The use according to claim 1, characterized in that The preparation can be used for treating tuberculosis caused by Mycobacterium tuberculosis infection.

5. Use of a small molecule compound in the preparation of a preparation for treating tuberculosis, wherein the small molecule compound is GSK1292263, and the structural formula of GSK1292263 is as shown in I: （I）。 6. The use according to claim 5, characterized in that The formulations may also contain other active ingredients.

7. The use according to claim 6, characterized in that The other active ingredients are drugs that promote the body's resistance to tuberculosis and / or improve tuberculosis symptoms.

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