Application of diphenylamine in the preparation of medicines for preventing or treating diseases caused by Mycobacterium tuberculosis infection

Abstract

The invention relates to the use of diphenylpyralid in preparing a product for inhibiting mycobacterium tuberculosis, which has a good antibacterial effect on mycobacterium tuberculosis, wherein the MIC of diphenylpyralid to a standard strain of mycobacterium tuberculosis (ATCC25618) is 0.125 μg / mL, and the MBC of diphenylpyralid to a standard strain of mycobacterium tuberculosis is 0.25 μg / mL. Diphenylpyralid has low cytotoxicity and good safety, and can be used as a candidate drug for the development of drugs for anti-mycobacterium tuberculosis infection.

Description

Technical Field

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

[0002] Tuberculosis (TB) is a public health problem that seriously threatens human life and health. It is caused by infection with Mycobacterium tuberculosis and is a highly contagious and potentially fatal infectious disease. At present, for sensitive tuberculosis, a combination of four first-line anti-tuberculosis drugs, such as isoniazid, rifampicin, pyrazinamide and ethambutol, is commonly used in clinical treatment, and the treatment cycle is usually six to nine months. Due to the long anti-tuberculosis treatment cycle, when the patient's compliance is poor, treatment interruption and irregular medication often occur, which is one of the important reasons for tuberculosis resistance. In recent years, multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have appeared in large numbers, making the tuberculosis prevention and control situation face more severe challenges (Gandhi NR, Nunn P, Dheda K, et al. Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis [J]. Lancet, 2010, 375 (9728): 1830-1843). The cure rate of multidrug-resistant tuberculosis patients is low, requiring more expensive drugs and longer treatment cycles (Ding P, Li X, Jia Z, et al. MuLtidrug-resistant tuberculosis (MDR-TB) disease burden in China: a systematic review and spatio-temporal analysis [J]. BMC Infect Dis, 2017, 17 (1): 57.). Studies have shown that the mortality rate of AIDS patients with multidrug-resistant tuberculosis or extensively drug-resistant tuberculosis exceeds 90% (Ding P, Li X, Jia Z, et al. MuLtidrug-resistant tuberculosis (MDR-TB) disease burden in China: a systematic review and spatio-temporal analysis [J]. BMC Infect Dis, 2017, 17 (1): 57.). Therefore, it is urgent to find and discover new drugs to treat Mycobacterium tuberculosis.

[0003] Diphenylpyridamole (SYP-14288) is a new fungicide developed by Shenyang Institute of Chemical Industry. It belongs to the diarylamine family and is currently mainly used to prevent and treat fungal infections caused by various plant diseases. Diphenylpyridamole is an uncoupler of mitochondrial oxidative phosphorylation. Its main mechanism of action is to destroy the normal function of mitochondria by uncoupling the ATP production process and electron transfer, thereby causing a decrease in fungal production capacity and affecting its normal biological function. Domestic research on diphenylpyridamole mainly focuses on the inhibitory effect on the growth of economic crop molds and the antifungal mechanism of action. There are no reports on the antibacterial effect of Mycobacterium tuberculosis. There are no reports on research on diphenylpyridamole abroad. Summary of the invention

[0004] The present invention finds that bifenthrin has good antibacterial activity against Mycobacterium tuberculosis, has low cytotoxicity and good safety. Based on this, the present invention is completed.

[0005] In a first aspect, the present invention provides a use of diphenylpyralid in preparing a product for inhibiting Mycobacterium tuberculosis, wherein the product contains diphenylpyralid.

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

[0007] Furthermore, the product for inhibiting Mycobacterium tuberculosis is an experimental reagent or an antibacterial agent.

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

[0009] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the product.

[0010] Furthermore, 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.

[0011] Furthermore, the water-soluble carrier material includes, but is not limited to, one or more of polyethylene glycol, polyvinyl pyrrolidone and / or organic acid.

[0012] Furthermore, the poorly soluble carrier material includes but is not limited to one or more of ethyl cellulose and / or cholesterol stearate.

[0013] Furthermore, the enteric carrier material includes but is not limited to one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0014] Furthermore, the product 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0021] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0022] 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.

[0023] 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.

[0024] Furthermore, the pills may be prepared using a wide variety of carriers known in the art, including diluents and absorbents, binders and / or disintegrants.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0033] In a second aspect, the present invention provides a use of diphenylpyralid in the preparation of a drug for preventing and / or treating a disease caused by Mycobacterium tuberculosis infection, wherein the drug contains diphenylpyralid, and the diphenylpyralid exerts its effect by inhibiting the activity of Mycobacterium tuberculosis.

[0034] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a Mycobacterium tuberculosis infected person.

[0035] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

[0036] Furthermore, 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.

[0037] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection 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, lozenges, suppositories and / or lyophilized powder injections.

[0038] 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.

[0039] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0040] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

[0041] 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.

[0042] Beneficial Effects

[0043] The diphenylpyralid in the present invention has a good antibacterial effect on Mycobacterium tuberculosis, the MIC of diphenylpyralid to the standard strain of Mycobacterium tuberculosis (ATCC25618) is 0.125 μg / mL, and the MBC of diphenylpyralid to the standard strain of Mycobacterium tuberculosis is 0.25 μg / mL. Diphenylpyralid has low cytotoxicity and good safety, and can be used as a candidate drug for the development of anti-Mycobacterium tuberculosis infection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The MIC distribution of diphenylamine against clinical isolates of Mycobacterium tuberculosis.

[0045] Figure 2 This is the effect of diphenylamine on the bacterial survival in THP-1 cells.

[0046] Figure 3 For the cytotoxicity assay of difenac. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] Material Description:

[0050] Bifenthion: synthesized from Shanghai Runnuo Biotechnology Co., Ltd. Bifenthion molecular formula: C 13 H7Cl3N4O6, CAS: No.1334790-94-1. The structural formula is as follows:

[0051]

[0052] Mycobacterium tuberculosis standard strain: H37Rv (ATCC 25618)

[0053] Example 1: Detection of antibacterial activity of diphenylamine against standard strains of Mycobacterium tuberculosis

[0054] 1.1 Test steps

[0055] (1) Add Mueller Hinton (MH) medium into the well plate;

[0056] (2) Add the drug solution to be tested (prepared with DMSO) to column 2 of the well plate and dilute it to column 11. Column 1 is the positive control and column 12 is the negative control.

[0057] (3) Add a suspension of Mycobacterium tuberculosis standard strains into the well plate. The final drug concentration in each well is shown in Table 1.

[0058] (4) Cultivation, adding Alamar-Blue and 5% Tween 80, and continuing cultivation;

[0059] (5) Read the minimum inhibitory concentration (MIC) and calculate the inhibition rate.

[0060] Table 1 Final drug concentration in each column well

[0061]

[0062] 1.2 Results

[0063] 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 generation of reduced Alamar-Blue by fluorescence detection (E× / Em, 530nm / 600nm).

[0064] Inhibition rate % = 100% - (fluorescence value of the detection well - background fluorescence value) / (fluorescence value of the growth control well - background fluorescence value) × 100%.

[0065] 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.

[0066] The negative control was a culture medium without drugs and bacterial solution, and the positive control was a culture medium containing bacteria without drugs.

[0067] The MIC of diphenylpyridamole against the standard strain of Mycobacterium tuberculosis H37Rv (ATCC 25618) was 0.125 μg / mL.

[0068] Example 2: Detection of antibacterial activity of diphenylamine against clinically isolated Mycobacterium tuberculosis strains

[0069] Clinical isolates: 81 strains were isolated and cultured from sputum specimens of patients infected with Mycobacterium tuberculosis and identified as Mycobacterium tuberculosis by sequencing of 16SrRNA, hsp65, rpoB, and 16-23S rRNA intergenic region.

[0070] According to the method in Example 1, the antibacterial activity of the tested drug against 81 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 Tables 3 and Figure 1 shown.

[0071] Table 281 MIC results of clinical Mycobacterium tuberculosis strains

[0072]

[0073]

[0074]

[0075] Table 3 Statistical results of MIC concentration distribution of diphenylamine against clinically isolated Mycobacterium tuberculosis strains

[0076]

[0077] Example 3. Determination of the minimum bactericidal concentration (MBC) of diphenylamine against standard strains of Mycobacterium tuberculosis 3.1 Experimental procedures

[0078] (1) According to Example 1, the MIC value was determined;

[0079] (2) According to the MIC results, the holes with 1×, 2×, 4×, 8×, 16×, 32×, 64×, and 128× MIC were delineated.

[0080] Aspirate the medium from the MIC wells and make multiple dilutions;

[0081] (3) The dilutions were spotted on Mueller Hinton (MH) solid culture plates;

[0082] (4) Count the CFU of the colonies.

[0083] 3.2 Test results

[0084] According to the CLSI guidelines, MBC is the drug concentration that can achieve a 99.9% kill rate against the final inoculum. MBC is defined as the minimum effective drug concentration in CFU, which is at least 3 log lower than the initial CFU. 10 When the MBC / MIC ratio is ≤4, the antibiotic is considered to have a bactericidal effect; otherwise, it is considered to have an antibacterial effect.

[0085] The minimum bactericidal concentration of diphenylamine against the standard strain of Mycobacterium tuberculosis is 0.25 μg / mL. Therefore, the MBC / MIC ratio of diphenylamine against the standard strain of Mycobacterium tuberculosis is 2, indicating that the drug has a bactericidal effect on Mycobacterium tuberculosis. Implementation Case 4: CFU counting method to evaluate the effect of diphenylamine on bacterial survival in THP-1 cells

[0086] 4.1 Cell culture

[0087] THP-1 cells in the logarithmic growth phase were inoculated and incubated with PMA to induce differentiation into macrophages.

[0088] 4.2. Infection of THP-1 cells with standard strain of Mycobacterium tuberculosis

[0089] (1) Preparation of a bacterial suspension of a standard strain of Mycobacterium abscessus: inoculate the standard strain of Mycobacterium abscessus in a neutral Roche medium, scrape the strain in the logarithmic growth phase after cultivation, grind the bacteria for turbidity measurement, and then dilute it with 1640 medium at an MOI of 5:1;

[0090] (2) After washing the cells, add a diluted suspension of standard strains of Mycobacterium abscessus for infection;

[0091] (3) After washing the cells, THP-1 cells were treated with diphenylpyridamole solutions at concentrations of 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, respectively. The control group was incubated with 1640 cell culture medium containing 0.1% DMSO.

[0092] 4.3 Determination of the inhibitory activity of difenac against bacteria in THP-1 cells

[0093] (1) Cell lysis: After washing the cells, add Triton X-100, incubate, and mix;

[0094] (2) Dilute the cell lysate and spot on MiddleBrook 7H10 Broth Agar solid culture plates;

[0095] (3) Cultivation and counting of CFU of the colonies on the culture plate;

[0096] 4.4 Results

[0097] from Figure 2 It can be seen that bifenthrin can reduce the survival of the standard strain of Mycobacterium tuberculosis in THP-1 cells.

[0098] Example 5: Detection of the cytotoxicity of diphenylamine to THP-1 cells by CCK-8 method

[0099] 5.1 Cell culture

[0100] THP-1 cells in the logarithmic growth phase were inoculated and incubated with PMA to induce differentiation into macrophages.

[0101] 5.2. Verification of the cytotoxicity of diphenylamine on THP-1 cells

[0102] (1) THP-1 cells were treated with 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.16 μM, and 0.08 μM diphenylamine solutions, respectively, and the control group was incubated with 1640 cell culture medium containing 0.1% DMSO;

[0103] (2) Aspirate the culture medium (to remove the influence of the original drug), add incomplete culture medium and CCK-8 solution, incubate, and measure the absorbance.

[0104] 5.3 Results

[0105] The cell viability of THP-1 cells cultured at different concentrations of diphenylpyridamole was calculated according to the following formula: Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100, where 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.

[0106] from Figure 3It can be seen that when the concentration of diphenylpyridamole was ≤5 μM (ie, 2.1 μg / mL), the cell survival rate was 100% after incubation with THP-1 for 24 h, indicating that it has low cytotoxicity.

Claims

1. A use of diphenylamine in the preparation of an antibacterial agent for inhibiting Mycobacterium tuberculosis, wherein the antibacterial agent is a detergent.

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

3. Use of diphenylamine as claimed in claim 2 in the preparation of a medicament for preventing or treating diseases caused by Mycobacterium tuberculosis infection, wherein the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis and a clinical isolate of Mycobacterium tuberculosis.

4. The use of bifenthrin as claimed in claim 2 in the preparation of a medicament for preventing or treating diseases caused by Mycobacterium tuberculosis infection, wherein the dosage form of the medicament is selected from one or more of tablets, capsules, pills, aerosols, powders, solutions, suspensions, transdermal agents, suppositories and / or lyophilized powder injections.

5. The use of diphenylpyrazone as claimed in claim 2 in the preparation of a medicament for preventing or treating diseases caused by Mycobacterium tuberculosis infection, wherein the medicament is administered by injection, respiratory tract administration or mucosal administration.

6. Use of the diphenylamine as claimed in claim 2 in the preparation of a medicament for preventing or treating a disease caused by Mycobacterium tuberculosis infection, wherein the medicament comprises one or more pharmaceutically acceptable carriers or excipients.

7. Use of diphenylamine as claimed in claim 6 in the preparation of a medicament for preventing or treating a disease caused by Mycobacterium tuberculosis infection, wherein the carrier is selected from one or more of a water-soluble carrier, a poorly soluble carrier and / or an enteric-soluble carrier.

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