Compound for resisting mycobacterium tuberculosis infection and application thereof

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to application of a compound in resisting Mycobacterium tuberculosis infection. Background Art

[0002] Mycobacterium tuberculosis (MTB) is a pathogen that parasitizes humans. As the main pathogen of tuberculosis worldwide, its infection causes high morbidity and mortality in humans. Tuberculosis (TB) is a chronic infectious disease with serious hazards, mainly caused by Mycobacterium tuberculosis complex (MTBC). As the most important pathogen of extrapulmonary tuberculosis, MTB infection is widely present in the world and is highly contagious. In recent years, with the widespread use of vaccination 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 continued to rise. Due to factors such as long treatment time, non-compliance with medication, irregular medication, drug target gene mutation, and cross-resistance in the treatment of tuberculosis (Chinese Anti-Tuberculosis Association. Guidelines for chemotherapy of drug-resistant tuberculosis (2019 simplified version). 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 global tuberculosis control.

[0003] Studies have shown that the drug resistance mechanism of mycobacteria mainly occurs in the cell wall. Different from other bacteria, MTB has only one drug resistance mechanism, that is, by producing MTBs that inhibit the synthesis of the MTB cell wall to evade the action of drugs (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 decreases with the prolongation of drug action time. Multiple MTBs gene mutations can lead to a decrease in the sensitivity to anti-tuberculosis drugs. In addition to 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 strains resistant to isoniazid can also develop resistance to rifampicin. In clinical practice, some patients may have multiple drug resistance mechanisms simultaneously. Multidrug-resistant tuberculosis (MDR-TB) refers to tuberculosis that is resistant to at least two first-line anti-tuberculosis drugs, isoniazid and rifampicin. It currently poses a threat to global TB treatment and has a significantly higher incidence in previously treated patients than in untreated patients. The incidence of drug-resistant tuberculosis is high and the infectious period is long. With the emergence of multiple drug resistance factors such as high-frequency mutations and efflux pump activation, it poses a huge challenge to global public health security. Therefore, there is an urgent need to develop new anti-tuberculosis drugs.

[0004] Haloxon, chemically named bis(2-chloroethyl)-3-chloro-4-methylcoumarin-7-yl phosphate, is an organophosphorus anthelmintic used for the treatment of bovine parasites and has a dual inhibitory effect on cholinesterase. It can be used to study the infections of Parascaris equorum, Oxyuris equi, and Strongylus vulgaris. As a broad-spectrum antiparasitic drug, it can effectively kill parasitic worms and other endoparasites without causing significant damage to the host animals. As an anthelmintic reported in the 1960s, Haloxon can still be used as a research model for the interaction between organophosphorus compounds and cholinesterase. There is currently no report on the inhibition of Mycobacterium tuberculosis by Haloxon. Summary of the Invention

[0005] The present invention discovers that Haloxon has the effect of inhibiting the activity of Mycobacterium tuberculosis, and thus completes the present invention.

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

[0007] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strains, Mycobacterium tuberculosis clinical isolates, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

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

[0009] Even further, the medical products refer to medical drugs or pharmaceutical excipients, and the non-medical products refer to experimental reagents or bacteriostatic agents.

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

[0011] Even further, the carrier materials include one or more of water-soluble carrier materials, poorly water-soluble carrier materials, and / or enteric-soluble carrier materials.

[0012] Furthermore, the water-soluble carrier materials include, but are not limited to, one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acids.

[0013] Furthermore, the poorly water-soluble carrier materials include, but are not limited to, one or more of ethyl cellulose and / or cholesterol stearate.

[0014] Furthermore, the enteric-soluble carrier materials include, but are not limited to, one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

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

[0016] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strains, Mycobacterium tuberculosis clinical isolates, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

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

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

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

[0020] Even further, the preparation can be one or more of an ordinary preparation, a sustained-release preparation, a controlled-release preparation, and / or various particulate drug delivery systems.

[0021] Furthermore, if necessary, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, or other materials can also be added to the pharmaceutical preparation.

[0022] Furthermore, the medicament is introduced into the body, such as muscle, intradermal, subcutaneous, intravenous, or mucosal tissues, by injection, penetration, absorption, physical or chemical-mediated methods; or is introduced into the body after being mixed or encapsulated with other substances.

[0023] In a third aspect, the present invention provides a pharmaceutical composition, which comprises Haloxon and another anti-Mycobacterium tuberculosis infection drug.

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

[0025] a) inhibiting the activity of Mycobacterium tuberculosis;

[0026] b) anti-Mycobacterium tuberculosis infection;

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

[0028] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strains, Mycobacterium tuberculosis clinical isolates, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0029] Further, the other anti - Mycobacterium tuberculosis infection drug includes one or more of antibiotics and other drugs that can help inhibit or kill Mycobacterium tuberculosis or provide resistance to the patient.

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

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

[0032] Further, the pharmaceutical composition can be made into various forms such as tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents and / or suppositories, etc.; the drugs in the above various dosage forms can all be prepared according to the 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 tissues by injection, penetration, absorption, physical or chemical mediated methods; or be introduced into the body after being mixed or encapsulated by other substances.

[0034] Beneficial effects

[0035] Haloxon has good antibacterial activity against both the standard strain of Mycobacterium tuberculosis and multi - drug - resistant clinical isolates of Mycobacterium tuberculosis. The MIC of Haloxon against the standard strain of Mycobacterium tuberculosis can reach 10 μM, and the MIC distribution of Haloxon against 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. Description of the drawings

[0037] Figure 1 The survival rate of Haloxon on THP - 1 cells. Detailed implementation manners

[0038] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following described implementation manners 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, and the test materials used in the following examples are all available through conventional commercial channels unless otherwise specified.

[0040] Material description

[0041] 1. Test Strains and Drugs

[0042] Standard strain of Mycobacterium tuberculosis H37Rv: ATCC 27294.

[0043] Haloxon: Purchased from MedchemExpress, CAS number 321 - 55 - 1; molecular formula C 14 H 14 Cl3O6P, and the structural formula 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 diluted to a working solution with a concentration of 320 μM when in use.

[0047] Example 1 Detection of the Bacteriostatic Activity of Haloxon against Standard Strains of Mycobacterium tuberculosis

[0048] 1.1 Method

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

[0050] 2. Drug dilution: Add 100 μL of Middlebrooks 7H9 medium to each well of the microplate. Add 100 μL of the Haloxon drug working solution with a concentration of 320 μM to the wells in the 3rd column. After mixing, take 100 μL and transfer it successively to the subsequent wells for two - fold serial dilution until the 12th column. The final concentrations of the drugs in each well are shown in Table 1.

[0051] 3. Inoculation of bacterial solution: The 1st column serves as a negative control (containing only medium, without drugs and bacterial solution). Add the diluted standard strain bacterial suspension of Mycobacterium tuberculosis to each well in the 2nd to 12th columns to make the final bacterial solution concentration reach 7.5×10 5 CFU / mL. Set up another plate as a positive control plate: Add 100 μL of medium without drugs to each well, and then add 100 μL of medium containing bacteria. Incubate all culture plates for about one week.

[0052] 4. Add the chromogenic agent: Predict the status of the experimental plate after culturing: Add 30 μL of resazurin chromogenic solution to each well of the positive control plate (add it 1 day in advance). If the positive wells change color (from blue to pink), then 30 μL of the same chromogenic agent can be added to each well of the experimental plate, and continue to incubate for 24 hours to observe the color change.

[0053] 4. Read the result: 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 (Minimum Inhibitory Concentration) is the lowest drug concentration that can prevent the bacterial solution from changing color (remaining blue), and is defined as the drug concentration that can inhibit the growth of 90% of the colonies.

[0055] The negative control is the medium without drugs and bacterial solution added, that is, the first column; the positive control is the bacterial-containing medium without drugs added, that is, the second column. When the positive control is pink and the negative control is blue, the MIC data measured in this batch is valid.

[0056] Table 1. Drug concentrations of Haloxon

[0057]

[0058] 1.2 Results

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

[0060] Example 2 Detection of the antibacterial activity of Haloxon against multi-drug resistant clinically isolated Mycobacterium tuberculosis strains

[0061] 2.1 Test samples and methods

[0062] Clinically isolated strains: 20 strains isolated and cultured from sputum specimens of patients infected with multi-drug resistant Mycobacterium tuberculosis, identified as Mycobacterium tuberculosis by 16S rRNA, hsp65, rpoB, and 16 - 23S rRNA intergenic region sequencing, and Mycobacterium tuberculosis strains that are simultaneously 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, detect the in vitro antibacterial activity of Haloxon against 20 clinically isolated strains of multi-drug resistant Mycobacterium tuberculosis.

[0064] 2.2 Test results

[0065] The MIC results of haloxon against clinical isolates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) are shown in Table 2. The best MIC of haloxon against clinical isolates of MDR-TB is only 1.25 μM.

[0066] Table 2. MIC of 20 clinical isolates of multidrug-resistant Mycobacterium tuberculosis

[0067] Strain number 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 Cytotoxicity experiment of haloxon against Mycobacterium tuberculosis

[0069] The CCK-8 (Cell Counting Kit-8) experiment is a colorimetric method based on WST-8 (tetrazolium salt) to detect cell proliferation and viability. Its core principle is that mitochondrial dehydrogenase in 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 seeding: Collect suspension 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 5×10 5 / mL. Add phorbol myristate acetate (PMA) to stimulate cell differentiation into macrophages, with a final concentration of 100 ng / mL, and culture for 24 hours. Add 100 μL / well of the cell suspension to the well plate.

[0072] 2. Drug addition and incubation: After the cells adhere to the wall, aspirate and discard the culture medium. Set up blank control group, positive control group and experimental group, and incubate in the incubator for 24 h and 48 h respectively; among them, blank control: only RPMI-1640 medium without drug; positive control: add RPMI-1640 medium without drug and cell suspension; experimental group: add culture medium containing different concentrations of drug (final concentrations of haloxon are 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM).

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

[0074] Cell survival rate calculation formula

[0075] Calculate the survival rate (%) of THP - 1 cells after stimulation with Haloxon = (OD 450nm [experimental group] - OD 450nm [negative control group] / OD 450nm [positive control group] - OD 450nm [negative control group]) × 100%.

[0076] 3.2 Experimental results

[0077] Haloxon has no toxicity to THP - 1 cells at concentrations of 0.3125 - 40 μM for 24 hours, and the cell viability is about 100%; after co - incubation for 48 hours, slight cytotoxicity begins to appear only at a drug concentration of 40 μM (cell viability is about 90%), indicating that Haloxon drug has good safety within a concentration of 20 μM ( Figure 1 ).

Claims

1. Use of haloxon in the preparation of a product for inhibiting Mycobacterium tuberculosis.

2. The use according to claim 1, wherein the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinically isolated strain of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

3. The use according to claim 1, wherein the product for inhibiting Mycobacterium tuberculosis includes a medical product or a non-medical product; the medical product refers to a medical drug or a pharmaceutical excipient, and the non-medical product refers to a laboratory reagent or an antibacterial agent.

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

5. The use according to claim 4, wherein the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinically isolated strain of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

6. The use according to claim 4, wherein one or more pharmaceutically acceptable carriers or excipients can be added to the drug.

7. The use according to claim 4, wherein the drug can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, and / or suppositories.

8. A pharmaceutical composition, the pharmaceutical composition comprising haloxon and another anti-Mycobacterium tuberculosis infection drug, The pharmaceutical composition has at least one of the following effects: a) Inhibiting the activity of Mycobacterium tuberculosis; b) Anti-Mycobacterium tuberculosis infection; c) Preventing and / or treating diseases caused by Mycobacterium tuberculosis.

9. The pharmaceutical composition according to claim 8, wherein the other anti-Mycobacterium tuberculosis infection drug includes one or more of antibiotics and other drugs that can help inhibit or kill Mycobacterium tuberculosis or provide resistance to the patient.

10. The pharmaceutical composition according to claim 8, wherein the antibiotics include one or more of rifampicin, streptomycin, ethambutol, moxifloxacin, clarithromycin, and / or amikacin; the other drugs include one or more of vitamins, amino acids, proteins, and / or minerals.

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