A compound for resisting mycobacterium tuberculosis infection and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a compound in anti-mycobacterium tuberculosis infection. The application finds that Haloxon has good bacteriostatic activity on mycobacterium tuberculosis standard strains and multi-drug resistant mycobacterium tuberculosis clinical isolates, the MIC of Haloxon on the mycobacterium tuberculosis standard strains can reach 10 muM, the MIC distribution of Haloxon on the multi-drug resistant mycobacterium tuberculosis clinical isolates is 1.25-20 muM, and Haloxon does not have obvious toxicity, and is expected to become a new anti-mycobacterium tuberculosis infection drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a compound in anti-Mycobacterium tuberculosis infection. BACKGROUND

[0002] Mycobacterium tuberculosis (MTB) is a kind of obligate parasite in human, and as the main pathogen of global tuberculosis, its infection leads to long-term high incidence and mortality of human. Tuberculosis (TB) is a kind of chronic infectious disease with serious harm, which is mainly caused by Mycobacterium tuberculosis complex (MTBC). As the most important extrapulmonary tuberculosis pathogen, MTB infection widely exists in the world and has high infectivity. In recent years, with the wide use of vaccines and anti-tuberculosis drugs, the overall incidence of pulmonary tuberculosis has decreased and the cure rate has increased, but the incidence of drug-resistant pulmonary tuberculosis has been rising. Due to the long treatment time, non-compliance with medication, non-standard medication, drug target gene mutation, cross drug resistance and other factors in the treatment of tuberculosis (Tuberculosis, TB) (Chinese Anti-Tuberculosis Association. Guidelines for the chemotherapy of drug-resistant tuberculosis (2019 edition) Chinese Journal of Anti-Tuberculosis [J]. Chinese Journal of Anti-Tuberculosis, 2019, 41(10): 1025-1073.), the emergence of drug-resistant Mycobacterium tuberculosis (DR-MTB) has brought great challenges to the global tuberculosis control.

[0003] Studies have shown that the drug resistance mechanism of mycobacterium mainly occurs in the cell wall. Unlike other bacteria, MTB has only one drug resistance mechanism, which is to escape the action of drugs by producing MTBs that inhibit the synthesis of MTB cell wall (Shan L, Wang Z, Wu L, et al. Statistical and network analyses reveal mechanisms for the enhancement of macrophage immunity by manganese in Mycobacterium tuberculosis infection [J]. Biochem Biophys Rep. 2023 Dec 20; 37: 101602.). However, the ability of MTB to produce MTBs will decrease with the extension of drug action time. Mutations in multiple MTBs genes can lead to decreased sensitivity to antituberculosis drugs. In addition to producing drugs that inhibit MTBs, Jayaraman M et al. (Jayaraman M, Gosu V, Kumar R, et al. Computational insights into potential marine natural products as selective inhibitors of Mycobacterium tuberculosis InhA: A structure-based virtual screening study [J]. Comput Biol Chem. 2024 Feb; 108: 107991.) found that rifampicin-resistant, rifampicin-sensitive strains, and isoniazid-resistant strains also produce resistance to rifampicin. In clinical practice, some patients may have multiple drug resistance mechanisms at the same time. Multidrug-resistant tuberculosis (MDR-TB) refers to tuberculosis that is resistant to at least two first-line antituberculosis drugs, isoniazid and rifampicin. It is currently a threat to global TB treatment, and its incidence in previously treated patients is significantly higher than that in untreated patients. Drug-resistant tuberculosis has a high incidence, a long infectious period, and a variety of drug resistance factors such as high-frequency mutations and efflux pump activation, which pose a huge challenge to global public health security. Therefore, it is urgent to develop new antituberculosis drugs.

[0004] Haloxon (Haloxon), the chemical name is bis (2-chloroethyl) -3-chloro-4-methyl coumarin-7-yl phosphate, is an organic phosphorus anthelmintic drug for the treatment of bovine parasites, which has a dual inhibitory effect on cholinesterase. It can be used to study the infection of horse ascarid, horse sharp tail nematode and common roundworm. As a broad-spectrum antiparasitic drug, it can effectively kill parasitic worms and other internal parasites, while causing no significant damage to the host animal. As an anthelmintic reported in the 1960s, haloxon can still be used as a model for the interaction of organic phosphorus compounds with cholinesterase. There is no report that haloxon inhibits Mycobacterium tuberculosis. SUMMARY

[0005] The present application finds that Haloxon has the effect of inhibiting Mycobacterium tuberculosis, and accordingly completes the present application.

[0006] In a first aspect, the present application provides the use of Haloxon in the preparation of a product for inhibiting Mycobacterium tuberculosis.

[0007] Further, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

[0008] Further, the product for inhibiting Mycobacterium tuberculosis includes a medical product or a non-medical product.

[0009] Further, the medical product refers to a medical drug or a pharmaceutical excipient, and the non-medical product refers to an experimental reagent or a bacteriostatic agent.

[0010] Further, one or more pharmaceutically acceptable carriers or excipients can be added to the product.

[0011] Further, the carrier material includes one or more of water-soluble carrier material, poorly soluble carrier material, and / or enteric carrier material.

[0012] Further, the water-soluble carrier material includes, but is not limited to, one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acid.

[0013] Further, the poorly soluble carrier material includes, but is not limited to, one or more of ethyl cellulose and / or cholesteryl stearate.

[0014] Further, the enteric carrier material includes, but is not limited to, one or more of cellulose acetate phthalate and / or carboxymethyl cellulose.

[0015] In a second aspect, the present application provides the use of Haloxon in the preparation of a medicament for preventing and / or treating a disease caused by M. tuberculosis infection, wherein the Haloxon exerts its effects by inhibiting the activity of M. tuberculosis.

[0016] Further, the M. tuberculosis includes M. tuberculosis standard strain, M. tuberculosis clinical isolate, or M. tuberculosis carried by a patient infected with M. tuberculosis.

[0017] Further, one or more pharmaceutically acceptable carriers or excipients can be added to the medicament.

[0018] Further, the carrier material includes one or more of water-soluble carrier material, poorly soluble carrier material, and / or enteric soluble carrier material.

[0019] Further, the medicament can be prepared into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations, and / or suppositories.

[0020] Further, the preparation can be one or more of ordinary preparation, sustained-release preparation, controlled-release preparation, and / or various microparticle drug delivery systems.

[0021] Further, the various preparations can also add coloring agents, preservatives, fragrances, flavoring agents, sweeteners, or other materials to the pharmaceutical preparation, if necessary.

[0022] Further, the medicament is introduced into the body, such as muscle, intradermal, subcutaneous, intravenous, or mucosal tissue, by injection, penetration, absorption, physical or chemical mediation; or is introduced into the body after being mixed or wrapped with other substances.

[0023] In a third aspect, the present application provides a pharmaceutical composition comprising Haloxon and another anti-M. tuberculosis infection drug,

[0024] The pharmaceutical composition has at least one of the following effects:

[0025] a) inhibiting the activity of M. tuberculosis;

[0026] b) anti-M. tuberculosis infection;

[0027] c) preventing and / or treating diseases caused by M. tuberculosis.

[0028] Further, the M. tuberculosis includes M. tuberculosis standard strain, M. tuberculosis clinical isolate, or M. tuberculosis carried by a patient infected with M. tuberculosis.

[0029] Further, the other anti-Mycobacterium tuberculosis infection drugs include one or more of antibiotics and other drugs capable of helping to inhibit or kill Mycobacterium tuberculosis or provide resistance to patients.

[0030] Further, the antibiotics include one or more of rifampicin, streptomycin, ethambutol, moxifloxacin, clarithromycin and / or amikacin; and the other drugs include one or more of vitamins, amino acids, proteins and / or minerals.

[0031] Further, one or more pharmaceutically acceptable carriers can be added to the pharmaceutical composition.

[0032] Further, the pharmaceutical composition can be prepared into tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations and / or suppositories, etc. The above-mentioned various forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0033] Further, the pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or be mixed or wrapped with other substances and then introduced into the body.

[0034] Advantages

[0035] Haloxon has good bacteriostatic activity on standard strains of Mycobacterium tuberculosis and multi-drug resistant clinical isolates of Mycobacterium tuberculosis, and the MIC of Haloxon on standard strains of Mycobacterium tuberculosis can reach 10 μM, and the MIC distribution of Haloxon on multi-drug resistant clinical isolates of Mycobacterium tuberculosis is 1.25-20 μM.

[0036] Haloxon has no obvious toxicity and is expected to become a new anti-Mycobacterium tuberculosis infection drug. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Survival rate of THP-1 cells treated with Haloxon. DETAILED DESCRIPTION

[0038] The specific embodiments of the present application are described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0039] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0040] MATERIALS

[0041] 1. Test strains and drugs

[0042] Mycobacterium tuberculosis standard strain H37Rv: ATCC 27294.

[0043] Haloxon: purchased from MedchemExpress, CAS No. 321-55-1; molecular formula is C 14 H 14 Cl3O6P, the structure is as follows:

[0044]

[0045] 2. Conversion of test drugs

[0046] The molecular weight of Haloxon is 415.59 g / mol, 5 mg of Haloxon is added to 1.2031 mL of DMSO to prepare a drug stock solution with a final concentration of 10 mM, and when used, it is diluted to a working solution with a concentration of 320 μM.

[0047] Example 1: Detection of the bacteriostatic activity of Haloxon on Mycobacterium tuberculosis standard strain

[0048] 1.1 Method

[0049] 1. Preparation of standard strain suspension: inoculate Mycobacterium tuberculosis standard strain on neutral L-J medium and culture for 2-3 weeks. When the strain grows to the logarithmic phase, take the strain from the neutral L-J medium. After grinding the bacteria, use a spectrophotometer to adjust the optical density (OD 600 ) of the bacterial solution to 0.3, and the bacterial content is about 3 x 10 7 CFU / mL. Dilute the bacterial solution with Middlebrooks 7H9 medium at a ratio of 1:20 for standby use.

[0050] 2. Drug dilution: add 100 μL of Middlebrooks 7H9 medium to each well of the well plate. Add 100 μL of Haloxon drug working solution with a concentration of 320 μM to the wells in column 3, mix well, and then transfer 100 μL to the subsequent wells for two-fold gradient dilution until column 12. The final concentration of the drug in each well is shown in Table 1.

[0051] 3. Inoculation of bacterial solution: column 1 is used as negative control (containing only medium without drug and bacterial solution). Add the diluted Mycobacterium tuberculosis standard strain bacterial suspension to each well in columns 2 to 12 to make the final bacterial solution concentration reach 7.5 x 10 5 CFU / mL. Another plate is set as a positive control plate: add 100 μL of medium without drug to each well, and then add 100 μL of bacterial-containing medium. Culture all the plates for about one week.

[0052] 4. Add color reagent: judge the state of the test plate after incubation: add 30 μL of reagent solution of resazurin to each well of the positive control plate (add 1 day in advance), if the color of the positive well changes (from blue to pink), then add 30 μL of the same color reagent to each well of the test plate, continue to incubate for 24 hours, and observe the color change.

[0053] 4. Read the results: read the minimal inhibitory concentration (MIC).

[0054] Record the color change of each well. Blue indicates no bacterial growth, and pink indicates bacterial growth. The MIC (minimal inhibitory concentration) is the lowest drug concentration that can prevent the color change of the bacterial solution (keep blue), which is defined as the drug concentration that can inhibit 90% of the growth of bacterial colonies.

[0055] The negative control is the medium without drugs and bacteria, i.e. the first column; the positive control is the bacterial culture medium without drugs, i.e. the second column. When the positive control is pink and the negative control is blue, the MIC data of this batch is valid.

[0056] Table 1. Drug concentration of Haloxon

[0057]

[0058] 1.2 Results

[0059] The MIC of Haloxon for the standard strain of Mycobacterium tuberculosis is 10 μM.

[0060] Example 2 Inhibition of Mycobacterium tuberculosis by Haloxon

[0061] 2.1 Test samples and methods

[0062] Clinical isolates: 20 strains of Mycobacterium tuberculosis isolated and cultured from sputum samples of patients infected with multi-drug resistant Mycobacterium tuberculosis, identified by 16S rRNA, hsp65, rpoB, and 16-23S rRNA intergenic region sequencing, and resistant to at least two of the most effective first-line anti-tuberculosis drugs, isoniazid (INH) and rifampicin (RIF).

[0063] According to the method in Example 1, the in vitro inhibition of 20 multi-drug resistant Mycobacterium tuberculosis clinical isolates by Haloxon was detected.

[0064] 2.2 Test results

[0065] The MIC results of Haloxon on MDR-TB clinical isolates are shown in Table 2, and the best MIC of Haloxon on MDR-TB clinical isolates is only 1.25 μM.

[0066] Table 2. MIC of Haloxon on 20 MDR-TB clinical isolates

[0067] Strain No. MIC (μM) 1 20 2 10 3 20 4 20 5 20 6 20 7 20 8 20 9 10 10 20 11 20 12 10 13 10 14 5 15 10 16 5 17 2.5 18 2.5 19 2.5 20 1.25

[0068] Example 3. Haloxon inhibits the cytotoxicity of Mycobacterium tuberculosis

[0069] CCK-8 (Cell Counting Kit-8) assay is a colorimetric method based on WST-8 (tetrazolium salt) to detect cell proliferation and viability. The core principle is that the mitochondrial dehydrogenase of living cells catalyzes WST-8 to produce soluble orange formazan dye, and the metabolic activity of cells is reflected by measuring the absorbance (OD value) at 450 nm.

[0070] 3.1 Experimental method

[0071] 1. Cell plating: Collect the suspension of human monocytic leukemia cells (THP-1 cells) in the logarithmic growth phase and centrifuge. After discarding the culture medium, resuspend with RPMI-1640 medium (containing 10% FBS) and adjust the cell density to 5 x 10 5 / mL. Add phorbol ester (PMA) to stimulate cell differentiation into macrophages, with a final concentration of 100 ng / mL, and culture for 24 hours. Add 100 μL of cell suspension per well to the well plate.

[0072] 2. Drug incubation: After the cells adhere, discard the culture medium and set up the blank control group, positive control group and experimental group, and incubate in the incubator for 24 h and 48 h, respectively; the blank control group only contains drug-free RPMI-1640 medium; the positive control group contains drug-free RPMI-1640 medium and cell suspension; the experimental group contains different concentrations of drug-containing medium (the final concentration of Haloxon is 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, respectively).

[0073] 3. CCK8 treatment: discard the old culture medium, add RPMI-1640 medium (containing 10% FBS) containing CCK8 reagent (CCK8: medium = 1:10) to each well. Incubate in the incubator and measure the OD 450nm absorbance with a microplate reader.

[0074] Cell survival rate calculation formula

[0075] Survival rate (%) of THP-1 cells after stimulation by Haloxon = (OD 450nm [Experimental group] - OD 450nm [Experimental group] - OD 450nm [Experimental group] - OD 450nm [Experimental group] - OD

[0076] 3.2 Experimental results

[0077] Haloxon was not toxic to THP-1 cells at a concentration of 0.3125-40 μΜ for 24 hours, with a cell viability of about 100%; after 48 hours of co-incubation, only slight cytotoxicity began to appear at a drug concentration of 40 μΜ (cell viability of about 90%), indicating that Haloxon drugs have good safety within a concentration of 20 μΜ Figure 1 ).

Claims

1. Application of Haloxon in the preparation of drugs for inhibiting Mycobacterium tuberculosis.

2. The use according to claim 1, wherein the Mycobacterium tuberculosis comprises a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

3. Use of Haloxon in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection, wherein the Haloxon exerts its effect by inhibiting the activity of Mycobacterium tuberculosis.

4. The use according to claim 3, wherein the Mycobacterium tuberculosis comprises a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

5. The use according to claim 3, wherein the drug comprises one or more pharmaceutically acceptable carriers or excipients.

6. The use according to claim 3, wherein the dosage form of the drug is selected from one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents and / or suppositories.

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