Application of fluazinam in the prevention of Mycobacterium tuberculosis infection

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an application of pyridinamide in anti-mycobacterium tuberculosis infection. The present invention finds that fluazinam can effectively inhibit the growth of standard strains and clinical isolates of mycobacterium tuberculosis, and inhibit the proliferation of tuberculosis bacteria in macrophages. In addition, the present invention finds that fluazinam has low cytotoxicity and good safety. It is expected to discover new uses of fluazinam in the treatment of mycobacterium tuberculosis infection diseases.

Description

Technical Field

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

[0002] Tuberculosis is caused by infection with Mycobacterium tuberculosis (M.tb), a major infectious disease that seriously threatens human health. MTB is an intracellular parasite that is mainly transmitted through aerosols. After infecting the host, the degree of infection varies depending on the individual's immunity, such as (1) being completely cleared by the host's immune system; (2) being in the latent infection stage (latent tuberculosis infection, LTBI), where the host's clearance of MTB and MTB's resistance to the host are in a relatively balanced state, which is defined by the WHO as "a state of persistent immune response to MTB, but no clinical evidence of active tuberculosis (ATB)"; (3) MTB rapidly proliferates in the host, the host's resistance is unbalanced, and it develops into ATB. The Global Tuberculosis Report published by the World Health Organization (WHO) pointed out that approximately 2 billion to 3 billion people are infected with M.tb worldwide, most of whom are latently infected with tuberculosis, among which the risk of developing active tuberculosis is 5% to 10%, and approximately 1.3 million people die from tuberculosis-related diseases each year (World Health Organization. Global tuberculosis report 2023. [R] Geneva: World Health Organization, 2023.). In 2019, the number of patients with multidrug-resistant tuberculosis or rifampicin-resistant tuberculosis was 465,000, while the treatment success rate was 59%, and about 180,000 people died from rifampicin-resistant or multidrug-resistant tuberculosis (DEANA S, TOSASAUGUET O, GLAZIOU P, et al. 25 years of surveillance of drug-resistant tuberculosis: achievements, challenges, and way forward [J / OL]. Lancet Infect Dis, 2022, 22(7): e191-e196.). Due to the emergence of drug-resistant tuberculosis and the co-infection of tuberculosis and HIV, the cure rate of tuberculosis is very unsatisfactory.

[0003] Fluazinam is an important variety of phenylpyrroles fungicides used for gray mold control. It was developed by Ishihara Sangyo Co., Ltd. of Japan. It has a unique mechanism of action, can uncouple oxidative phosphorylation, and has high efficiency and broad-spectrum characteristics (Shao Wenyong. Study on the risk of resistance and sensitivity-related gene functions of Botrytis cinerea to fluazinam [D]. Nanjing: Nanjing Agricultural University, 2018.). At present, fluazinam preparations have been registered for the control of a variety of economic crop diseases, and have a good control effect on pathogens of Alternaria, Botrytis, Phytophthora, and Sclerotium (Feng Guangjun, Wang Baolin, Wang Zhenfeng, et al., Clean Synthesis Process of Fluazinam [J]. Pesticides, 2019.58(2):93-95.). Domestic and foreign research on fluazinam has mainly focused on detection methods, residue behavior, dietary risk assessment, and the impact of processing on residues. No research on its antibacterial effect against Mycobacterium tuberculosis has been reported. Summary of the invention

[0004] The present invention has been completed based on the discovery that fluazinam has the effect of inhibiting the activity of Mycobacterium tuberculosis.

[0005] In a first aspect, the present invention provides a use of fluazinam in the preparation of a product for inhibiting Mycobacterium tuberculosis, wherein the fluazinam exerts its effect by inhibiting the activity of Mycobacterium tuberculosis.

[0006] Furthermore, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a Mycobacterium tuberculosis infected person.

[0007] Furthermore, the Mycobacterium tuberculosis inhibition product includes one or more of experimental reagents and / or antibacterial agents.

[0008] Furthermore, the antibacterial agent may be a cleaning agent, including the use of fluazinam and pharmaceutically acceptable substances thereof to inhibit / eliminate Mycobacterium tuberculosis.

[0009] In a second aspect, the present invention provides the use of fluazinam in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection, wherein the fluazinam exerts its effect by inhibiting the activity of Mycobacterium tuberculosis or killing Mycobacterium tuberculosis.

[0010] Furthermore, 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.

[0011] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug.

[0012] Furthermore, the carrier material includes but is not limited to one or more of water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.) and / or enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

[0013] Furthermore, the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0014] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0015] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0016] 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.

[0017] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0018] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0019] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0020] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0021] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0022] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0023] Furthermore, the pills may be prepared using a wide variety of carriers known in the art, including diluents and absorbents, binders and / or disintegrants.

[0024] Furthermore, the diluent and absorbent include, but are not limited to, one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin and / or talc.

[0025] Furthermore, the adhesive includes, but is not limited to, one or more of gum arabic, gum tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste and / or batter.

[0026] Furthermore, the disintegrant includes, but is not limited to, one or more of agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0027] Furthermore, the suppository may be made of a wide variety of carriers known in the art, including but not limited to one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and / or semi-synthetic glycerides.

[0028] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0029] Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0030] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0031] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0032] Furthermore, the drug can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

[0033] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0034] Beneficial Effects

[0035] 1. The present invention found that fluazinam can effectively inhibit the growth of Mycobacterium tuberculosis standard strain, and the MIC of fluazinam to Mycobacterium tuberculosis standard strain ATCC 27294 is 1 μg / mL.

[0036] 2. The present invention found that fluazinam can effectively inhibit the growth of clinical isolates of Mycobacterium tuberculosis, and the MIC of clinical isolates of Mycobacterium tuberculosis is 0.125-4 μg / mL. 50 1μg / mL.

[0037] 3. The present invention found that fluazinam can effectively inhibit the proliferation of tuberculosis in macrophages; when the concentration of fluazinam is 1 μg / mL, the bacterial load is reduced by 44.46%±9.23% after 1 day of treatment, and the bacterial load is reduced by 63.81%±9.94% after 3 days of treatment; when the concentration of fluazinam is 2 μg / mL, the bacterial load is reduced by 55.00%±11.42% after 1 day of treatment, and the bacterial load is reduced by 73.92%±11.51% after 3 days of treatment; when the concentration of fluazinam is 4 μg / mL, the bacterial load is reduced by 65.82%±13.66% after 1 day of treatment, and the bacterial load is reduced by 87.47%±5.24% after 3 days of treatment; when the concentration of fluazinam is 8 μg / mL, the bacterial load is reduced by 75.00%±13.45% after 1 day of treatment, and the bacterial load is reduced by 93.92%±8.84% after 3 days of treatment.

[0038] 4. The present invention found that fluazinam has low cytotoxicity; when fluazinam ≤5 μM (i.e. 2.33 μg / mL), the cell survival rate was 90-100% after incubation with THP-1 for 24 hours; when fluazinam was 10 μM (i.e. 4.66 μg / mL), the cell survival rate was 80.36% after 24 hours of incubation, and the cell survival rate was 62.85% after 48 hours of incubation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Figure 2 shows the distribution of MIC values ​​of fluazinam against 37 clinical isolates of Mycobacterium tuberculosis.

[0040] Figure 2 The inhibitory activity of fluazinam against tuberculosis in macrophages.

[0041] Figure 3 The effect of fluazinam on THP-1 cell cytotoxicity. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0044] Material Description:

[0045] Mycobacterium tuberculosis standard strain ATCC 19977.

[0046] Fluazinam: Molecular formula: C 13 H4C l2 F6N4O4, CAS No.:79622-59-6, has the following structural formula:

[0047]

[0048] Example 1: Detection of antibacterial activity of fluazinam against standard strains of Mycobacterium tuberculosis

[0049] Detectable substance: Fluazinam

[0050] 1. Add Mueller Hinton (MH) medium to each well of the plate;

[0051] 2. After completing step 1, take the well plate, add a 16 μg / mL drug solution to be tested (prepared with DMSO) to the second column, mix well, aspirate an equal amount and add to the third column, dilute in sequence to the 11th column, draw out an equal amount and discard, the first and 12 columns do not contain drugs, and are the negative control wells and positive control wells, respectively.

[0052] 3. After completing step 2, take the well plate and add an equal amount of Mycobacterium tuberculosis standard strain suspension to each well, with a final concentration of 2.5×10 5 CFU / mL; the final drug concentration in each well is shown in Table 1. Preparation method of Mycobacterium tuberculosis standard strain bacterial suspension: The Mycobacterium tuberculosis standard strain is inoculated in neutral Roche medium. After culturing for about 3 days, the strain in the logarithmic growth phase on the neutral Roche medium is scraped, the bacteria are ground and turbidized, and then diluted with Mueller Hinton (MH) medium.

[0053] Table 1 Final drug concentration in each column well

[0054]

[0055] 4. After completing step 3, the well plate is cultured for 6-8 days.

[0056] 5. After completing step 4, take the well plate, add ALamar blue and 5% Tween 80 to each well, and then continue culturing for 24 hours.

[0057] 6. After completing step 5, take the well plate, read the minimum inhibitory concentration (MIC) and calculate the inhibition rate.

[0058] Minimum inhibitory concentration (MIC) reading method: Minimum inhibitory concentration (MIC) is the drug concentration that can inhibit 90% of bacterial colony growth. The minimum drug concentration that inhibits >90% of the production of reduced Alamar blue by fluorescence detection (E× / Em, 530nm / 600nm).

[0059] Inhibition rate % = 100% - (fluorescence value of the detection well - background fluorescence value) / (fluorescence value of the growth control well - background fluorescence value) × 100%.

[0060] The background fluorescence value is the fluorescence value of the negative control, and the fluorescence value of the growth control well is the fluorescence value of the positive control.

[0061] The negative control was a culture medium without drugs and bacterial solution, and the positive control was a culture medium containing bacteria without drugs.

[0062] The results showed that the MIC of fluazinam against the standard strain of Mycobacterium tuberculosis ATCC 27294 was 1 μg / mL.

[0063] Example 2: Detection of antibacterial activity of fluazinam against clinically isolated Mycobacterium tuberculosis strains

[0064] Clinical isolates: 62 strains isolated and cultured from sputum specimens of patients infected with Mycobacterium tuberculosis were identified as Mycobacterium tuberculosis by sequencing of 16SrRNA, hsp65, rpoB, and 16-23S rRNA intergenic region.

[0065] Drug to be tested: Fluazinam

[0066] According to the method in Example 1, the antibacterial activity of the tested drug against 62 clinical Mycobacterium tuberculosis strains was detected. The MIC results are shown in Table 2. The statistical results of the MIC concentration distribution are shown in Figures 3 and Figure 1 shown.

[0067] Table 1 Antibacterial activity of the tested drugs against 62 clinical strains of Mycobacterium tuberculosis

[0068]

[0069]

[0070] Table 3 shows the statistical results of the MIC concentration distribution of fluazinam against clinically isolated Mycobacterium tuberculosis strains

[0071]

[0072] The results showed that fluazinam had good antibacterial activity against 62 clinically isolated Mycobacterium tuberculosis strains, with the MIC of fluazinam being 0.125-4 μg / mL and the MIC 50 It is expected to discover new uses of fluazinam in the treatment of Mycobacterium tuberculosis infection.

[0073] Example 3: Evaluation of the antibacterial activity of fluazinam against tuberculosis in macrophages

[0074] Detectable substance: Fluazinam

[0075] 3.1 Cell preparation

[0076] 1. Cultivate cells: Prepare 1640 complete medium (0.1% double antibody + 10% fetal bovine serum (FBS)) and culture THP-1 cells in a cell culture flask.

[0077] 2. Collect, count and dilute cells: THP-1 cells are suspension cells. The cultured cells are collected and centrifuged, and the supernatant is discarded. The cells are resuspended in complete cell culture medium containing double antibodies and the cells are counted. The cells are diluted to 1×10 according to the counting results. 6 Pieces / mL.

[0078] 3. Add phorbol ester (PMA): Prepare PMA, a macrophage differentiation stimulator, and add it to the cell suspension to a final concentration of 100 ng / mL.

[0079] 4. Plate: Prepare a culture plate and plate the above cells into the culture plate. Make sure that the number of cells in each well is 5×10 5 The monocytes were cultured statically to allow them to differentiate into macrophages.

[0080] 5. Wash away PMA: After culture, macrophages adhere to the wall and grow. Discard the supernatant, wash with buffer, add complete cell culture medium without double antibody, and continue incubation.

[0081] 3.2 Bacterial preparation

[0082] 1. Centrifuge the collected bacterial suspension and discard the supernatant. Resuspend with complete cell culture medium without double antibody, measure OD600 value, adjust the bacterial OD600 value to 0.6, corresponding to a bacterial concentration of about 1×10 8 Pieces / mL.

[0083] 2. Infect cells at MOI = 10:1. The bacterial suspension with OD600 = 0.6 needs to be diluted 100 times by adding complete cell culture medium before being used to infect cells.

[0084] 3.3 Bacterial infection of cells

[0085] 1. Discard the supernatant of macrophages that have grown on the wall, and discard the supernatant after washing with the solution;

[0086] 2. Add the prepared bacterial suspension;

[0087] 3. Incubate in a cell culture incubator, discard the supernatant, wash, and remove extracellular bacteria.

[0088] 3.4 Target drug incubation

[0089] The macrophage wells from which extracellular bacteria had been washed were added with 1, 2, 4, and 8 pairs of clinically isolated Mycobacterium tuberculosis strains with MICs of 0.125-4 μg / mL, respectively. 50 Add 1 μg / mL fluazinam and the same volume of DMSO in 1640 complete medium and continue incubation.

[0090] 3.5 Colony count

[0091] 1. Use 0.01% Triton (diluted with PBS) to lyse the cells at 0, 1, and 3 days of drug incubation, and the cells will be completely lysed after incubation.

[0092] 2. Transfer the lysate stock solution to the well plate, and make 2 duplicate wells in each well of the well plate;

[0093] 3. Use a well plate to perform gradient dilution of the stock solution;

[0094] 4. Pipette each gradient dilution of bacterial solution and drop it on a solid agar plate of 7H10 (containing 10% OADC);

[0095] 5. Wait for the liquid on the plate to absorb and dry, then close the lid, seal it with sealing film, and invert and culture for 3-4 weeks.

[0096] The results show that: Figure 2As shown, after 1 day of treatment with different concentrations of fluazitamide, compared with the DMSO control group, when the concentration of fluazitamide was 1 μg / mL, the bacterial load was reduced by 44.46%±9.23%; when the concentration was 2 μg / mL, the bacterial load was reduced by 55.00%±11.42%; when the concentration was 4 μg / mL, the bacterial load was reduced by 65.82%±13.66%; when the concentration was 8 μg / mL, the bacterial load was reduced by 75.00%±13.45%. After 3 days of treatment, compared with the DMSO control group, when the concentration of fluazinam was 1ug / mL, the bacterial load decreased by 63.81%±9.94%; when the concentration was 2μg / mL, the bacterial load decreased by 73.92%±11.51%; when the concentration was 4ug / mL, the bacterial load decreased by 87.47%±5.24%; when the concentration was 8ug / mL, the bacterial load decreased by 93.92%±8.84%.

[0097] Example 4: Detection of the cytotoxicity of fluazinam on THP-1 cells by CCK-8 method

[0098] 4.1 Cell culture

[0099] THP-1 cells in the logarithmic growth phase were inoculated and 100 ng / L PMA was added and incubated for 48 h to induce differentiation into macrophages.

[0100] 4.2 Verification of the cytotoxicity of fluazinam on THP-1 cells

[0101] 1. THP-1 cells were treated with fluazinam solutions at concentrations of 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM, 0.16μM, and 0.08μM, respectively. The control group used 1640 cell culture medium with 0.1% DMSO, 200μL / well, and incubated for 24h and 48h, respectively. Five replicates were performed for each concentration.

[0102] 2. Aspirate the culture medium (to remove the influence of the original drug), add incomplete culture CCK-8 solution to each well, incubate for 2 hours, and measure the absorbance at 450nm using an enzyme reader.

[0103] 4.3 Vitality calculation

[0104] The cell viability of THP-1 cells cultured at different fluazinam concentrations was calculated according to the following formula.

[0105] Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100

[0106] Wherein, A(drug added) represents the absorbance of the wells with cells, CCK-8 solution and drug solution; A(blank) represents the absorbance of the wells with culture medium and CCK-8 solution but no cells; A(0 drug added) represents the absorbance of the wells with cells, CCK-8 solution but no drug solution. The results are shown in Figure 3 shown.

[0107] from Figure 3 It can be seen that when fluazitamide ≤5 μM (i.e., 2.33 μg / mL), the cell survival rate was 90-100% after incubation with THP-1 for 24 hours, indicating that it has low cytotoxicity; when fluazitamide is 10 μM (i.e., 4.66 μg / mL), the cell survival rate was 80.36% after 24 hours of incubation, and the cell survival rate was 62.85% after 48 hours of incubation.

Claims

1. A use of fluazinam in the preparation of a product for inhibiting Mycobacterium tuberculosis, wherein the fluazinam exerts its effect by inhibiting the activity of Mycobacterium tuberculosis.

2. The use according to claim 1, wherein the product for inhibiting Mycobacterium tuberculosis is selected from experimental reagents and / or antibacterial agents.

3. The use according to claim 2, wherein the antibacterial agent can be a detergent, and the detergent inhibits / eliminates Mycobacterium tuberculosis through fluazinam.

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

5. The use according to claim 4, 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.

6. The use according to any one of claims 4 or 5, wherein one or more pharmaceutically acceptable carriers may be added to the drug.

7. The use according to claim 6, wherein the carrier material includes but is not limited to one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

8. The use as claimed in claim 4, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

9. The use according to claim 8, wherein the dosage form can be one or more of a conventional preparation, a sustained-release preparation and / or a controlled-release preparation.

10. The use according to claim 4, wherein the drug can be administered by injection, cavity administration or respiratory tract administration.

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