Application of difenac in the prevention of Mycobacterium abscessus infection

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an application of diphenylamine in anti-mycobacterium abscess infection. The MIC of diphenylamine to the standard strain of Mycobacterium abscess ATCC 19977. is 0.0313μg / mL; the minimum bactericidal concentration of diphenylamine to the standard strain of Mycobacterium abscess is 0.0625μg / mL, and the MBC / MIC ratio of diphenylamine to the standard strain of Mycobacterium abscess is 2, indicating that the drug has a bactericidal effect on Mycobacterium abscess. When diphenylamine is ≤5μM (i.e., 2.1μg / mL), the survival rate of cells is 100% after incubation with THP-1 for 24h, indicating that it has low cytotoxicity. In anti-mycobacterium abscess infection, diphenylamine can become a new therapeutic drug.

Description

Technical Field

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

[0002] Nontuberculous mycobacteria (NTM) refers to the general term for mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae in the genus Mycobacterium. In previous epidemiological surveys on tuberculosis, it was found that the isolation rate of NTM continued to rise, accounting for as high as 22.9% in the 2010 survey (Zhou L, Xu D, Liu HC, Wan KL, Wang RB, Yang ZC. Trends in the Prevalence and Antibiotic Resistance of Non-tuberculous Mycobacteria, 2000-2019. Systematic Review and Meta-Analysis. Front Public Health. 2020, 8: 295.). According to the law of epidemiological development, as tuberculosis is gradually controlled in a country or region, the proportion of NTM disease in mycobacterium-related diseases will gradually increase. At present, in many countries, such as the United States and France, the number of people with diseases caused by NTM has exceeded that of tuberculosis.

[0003] Mycobacterium abscessus (Mab) accounts for 20.3% of all reported NTM in my country, and up to 46% in Guangdong Province. It is the most common species among rapid-growing mycobacteria (RGM) (Yu X, Liu P, Liu G, Wang G, Chen S, Huang H. The Prevalence of Non-tuberculosis mycobacteria infections: Systematic review and meta-analysis [J]. Journal of Infection. 2016, 73 (6): 558-567). RGM is usually weak in pathogenicity, but Mycobacterium abscessus is a special case. Although Mab infection often occurs in patients with underlying diseases, more than 30% of patients have normal immunity, indicating that Mab has obvious pathogenicity. Mycobacterium abscessus is ineffective against common anti-tuberculosis drugs, such as first-line drugs (rifampicin and isoniazid) and some second-line drugs (capreomycin). The bacteria's natural resistance to most anti-tuberculosis drugs and antibiotics makes it the most difficult type of mycobacterial lung disease to be cured. Even if the recommended combination chemotherapy regimen containing amikacin, clarithromycin and imipenem is used for more than one year, about 70% of patients fail or relapse (Marion E, Song OR, Christophe T, et al. Mycobacterial Toxin Induces Analgesia in Buruli Ulcer by Targeting the Angiotensin Pathways [J]. Cell, 2014, 157 (7): 1565-1576.). In addition, in recent years, the resistance of Mycobacterium abscessus to clarithromycin has become increasingly serious, with a reported resistance rate of 14% to 38% (Lee SH, Yoo HK, Kim SH, Koh WJ, Kim CK, Park YK, et al. The drug resistance profile of Mycobacterium abscessus group strains from Korea. Ann Lab Med. 2014; 34: 31-7.). However, there is still a lack of reports on the development of targeted drugs for Mycobacterium abscessus, which has a high isolation rate, strong pathogenicity and severe drug resistance.

[0004] 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 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 abscessus. There are no reports on research on diphenylpyridamole abroad. Summary of the invention

[0005] The present invention is completed based on the discovery that SYP-14288 has the effect of inhibiting the activity of Mycobacterium abscessus.

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

[0007] Furthermore, the Mycobacterium abscessus includes a standard strain of Mycobacterium abscessus, a clinical isolate of Mycobacterium abscessus, or Mycobacterium abscessus carried by a Mycobacterium abscessus infected person.

[0008] Furthermore, the product for inhibiting Mycobacterium abscessus is an experimental reagent or an antibacterial agent.

[0009] Furthermore, the antibacterial agent may be a cleaning agent, including the use of bifenthrin and its pharmaceutically acceptable substances to inhibit / eliminate Mycobacterium abscessus.

[0010] In a second aspect, the present invention provides a use of diphenylpyralid in the preparation of a medicament for preventing and / or treating a disease caused by Mycobacterium abscessus infection, wherein the diphenylpyralid exerts its effect by inhibiting the activity of Mycobacterium abscessus.

[0011] Furthermore, the Mycobacterium abscessus includes a standard strain of Mycobacterium abscessus, a clinical isolate of Mycobacterium abscessus, or Mycobacterium abscessus carried by a Mycobacterium abscessus infected person.

[0012] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection.

[0013] 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.

[0014] 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, granules, transdermal agents, 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 various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0017] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

[0018] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.

[0019] Beneficial Effects

[0020] Diphenylpyrazone has good antibacterial activity against standard strains and clinical isolates of Mycobacterium abscessus. The MIC of diphenylpyrazone against the standard strain of Mycobacterium abscessus ATCC 19977. is 0.0313μg / mL; the minimum bactericidal concentration of diphenylpyrazone against the standard strain of Mycobacterium abscessus is 0.0625μg / mL, and the MBC / MIC ratio of diphenylpyrazone against the standard strain of Mycobacterium abscessus is 2, indicating that the drug has a bactericidal effect on Mycobacterium abscessus. When diphenylpyrazone is ≤5μM (i.e., 2.1μg / mL), the cell survival rate is 100% after incubation with THP-1 for 24h, indicating that it has low cytotoxicity. Diphenylpyrazone can become a new therapeutic drug in the fight against Mycobacterium abscessus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure shows the distribution of MIC values ​​of difenac against 43 clinical isolates of Mycobacterium abscessus.

[0022] Figure 2 This is the effect of diphenylamine on the bacterial survival in THP-1 cells.

[0023] Figure 3 This is the effect of diphenylamine on the cytotoxicity of THP-1 cells. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are all commercially available unless otherwise specified. The quantitative experiments in the following examples were repeated 3-5 times, and the results were averaged.

[0026] the term

[0027] Alamar-Blue: Cell Viability Reagent is a ready-to-use resazurin-based solution that uses the reducing ability of living cells to quantitatively detect viability as an indicator of cell health. Resazurin, the active ingredient in the alamarBlue reagent, is a cell-permeable, non-toxic compound that is blue in color and virtually non-fluorescent. Once inside living cells, resazurin is reduced to resorufin, a red, highly fluorescent compound. Changes in cell viability can be easily detected using an absorbance- or fluorescence-based microplate reader. The alamarBlue Cell Viability Reagent has broad applicability and can be used for cell viability assays in a variety of human and animal cell lines, bacteria, plants, and fungi.

[0028] MBC (Minimal Bactericidal Concentration): refers to the minimum drug concentration required to kill 99.9% (reduced by 3 orders of magnitude) of the tested microorganisms. According to the CLSI guidelines, MBC is defined as the lowest effective drug concentration in CFU, which is at least 3 log10 lower than the initial CFU. 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.

[0029] Material Description

[0030] 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:

[0031]

[0032] Mycobacterium abscessus standard strain: ATCC 19977.

[0033] Bacterial suspension of Mycobacterium abscessus standard strain: inoculate the Mycobacterium abscessus standard strain into neutral Roche medium, scrape the strain in the logarithmic growth phase after cultivation, grind the bacteria for turbidity measurement and dilute with Mueller Hinton (MH) medium.

[0034] Clinical isolates: 43 strains isolated and cultured from sputum specimens of patients infected with Mycobacterium abscessus were identified as Mycobacterium abscessus by sequencing of 16SrRNA, hsp65, rpoB, and 16-23S rRNA intergenic region.

[0035] THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0036] PMA (phorbol ester): purchased from Sigma.

[0037] CCK-8 solution: purchased from Solebol.

[0038] Example 1 Detection of the antibacterial activity of diphenylamine against standard strains of Mycobacterium abscessus

[0039] 1.1 Test steps

[0040] (1) Add Mueller Hinton (MH) medium into the well plate;

[0041] (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.

[0042] (3) Add the standard strain of Mycobacterium abscessus to the well plate. The final drug concentration in each well is shown in Table 1.

[0043] (4) Cultivation, adding Alamar-Blue and 5% Tween 80, and continuing cultivation;

[0044] (5) Read the minimum inhibitory concentration (MIC) and calculate the inhibition rate.

[0045] Table 1. Final drug concentrations in each column

[0046]

[0047] 1.2 Test results

[0048] Minimum inhibitory concentration (MIC) reading method: Minimum inhibitory concentration (MIC) is the drug concentration that can inhibit 90% bacterial growth.

[0049] The minimum drug concentration that inhibits >90% of the reduced Alamar-Blue production as measured by fluorescence (E× / Em, 530nm / 600nm).

[0050] Inhibition rate % = 100% - (fluorescence value of the detection well - background fluorescence value) / (fluorescence value of the growth control well - background fluorescence value) × 100%.

[0051] 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.

[0052] The negative control was a culture medium without drugs and bacterial solution, and the positive control was a culture medium containing bacteria without drugs.

[0053] The results showed that the MIC of difenac against the standard strain of Mycobacterium abscessus ATCC 19977 was 0.0313 μg / mL.

[0054] Example 2 Detection of antibacterial activity of diphenylamine against clinically isolated Mycobacterium abscessus strains

[0055] According to the method in Example 1, the antibacterial activity of the drug to be tested against 43 clinical Mycobacterium abscessus strains was detected.

[0056] The MIC results are shown in Table 2.

[0057] The statistical results of MIC concentration distribution are shown in Table 3 and Figure 1 As shown, bifenthrin has good antibacterial activity against clinically isolated Mycobacterium abscessus.

[0058] Table 2. MIC results of 43 clinical Mycobacterium abscessus strains

[0059]

[0060]

[0061] Table 3. Statistical results of MIC concentration distribution of diphenylamine against clinically isolated Mycobacterium abscessus strains

[0062]

[0063]

[0064] Example 3. Determination of the minimum bactericidal concentration (MBC) of diphenylpyrazone against standard strains of Mycobacterium abscessus

[0065] 3.1 Test steps

[0066] (1) According to Example 1, the MIC value was determined;

[0067] (2) According to the MIC results, the holes with 1×, 2×, 4×, 8×, 16×, 32×, 64×, and 128× MIC were delineated.

[0068] Aspirate the medium from the MIC wells and make multiple dilutions;

[0069] (3) The dilutions were spotted on Mueller Hinton (MH) solid culture plates;

[0070] (4) Count the CFU of the colonies.

[0071] 3.2 Test results

[0072] 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.

[0073] The minimum bactericidal concentration of diphenylamine against the standard strain of Mycobacterium abscessus is 0.0625 μg / mL. Therefore, the MBC / MIC ratio of diphenylamine against the standard strain of Mycobacterium abscessus is 2, indicating that the drug has a bactericidal effect on Mycobacterium abscessus. Example 4. Evaluation of the effect of diphenylamine on bacterial survival in THP-1 cells by CFU counting method

[0074] 4.1 Cell culture

[0075] THP-1 cells in the logarithmic growth phase were inoculated and incubated with PMA to induce differentiation into macrophages.

[0076] 4.2 Infection of THP-1 cells with Mycobacterium abscessus standard strain

[0077] (1) Preparation of a suspension of Mycobacterium abscessus standard strain: inoculate the Mycobacterium abscessus standard strain into a neutral Roche medium.

[0078] In the middle, after culturing, the strains in the logarithmic growth phase were scraped and ground to measure the turbidity. Then, the strains were diluted with 1640 medium at an MOI of 5:1. (2) After washing the cells, the diluted suspension of Mycobacterium abscessus standard strain was added and infected.

[0079] (3) After washing the cells, the cells were treated with 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL

[0080] The 1640 cell culture medium containing 0.1% DMSO was used as the control group; 4.3 Determination of the inhibitory activity of 1640 against bacteria in THP-1 cells

[0081] (1) Cell lysis: After washing the cells, add 0.01% Triton X-100, incubate, and mix;

[0082] (2) diluting the cell lysate and spotting it on a Mueller Hinton (MH) solid culture plate;

[0083] (3) Cultivation and counting of CFU of the colonies on the culture plate;

[0084] 4.4 Test results

[0085] from Figure 2 It can be seen that bifenthrin can reduce the survival of the standard strain of Mycobacterium abscessus in THP-1 cells. Example 5. Detection of the cytotoxicity of bifenthrin on THP-1 cells by CCK-8 method

[0086] 5.1 Cell culture

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

[0088] 5.2 Verification of the cytotoxicity of difenac on THP-1 cells

[0089] (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 cultured in 1640 cell culture medium containing 0.1% DMSO;

[0090] (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.

[0091] 5.3 Test results

[0092] Viability calculation: The cell viability of THP-1 cells cultured at different concentrations of diphenylpyridamole was calculated according to the following formula.

[0093] Cell viability (%) = [A(drug addition) - A(blank)] / [A(0 drug addition) - A(blank)] × 100;

[0094] 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.

[0095] from Figure 3 It 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. Use of diphenylamine in preparing a product for inhibiting Mycobacterium abscessus, wherein the product is an experimental reagent or an antibacterial agent.

2. The use according to claim 1, wherein the antibacterial agent is a detergent.

3. Use of diphenylpyralid in the preparation of a drug for treating diseases caused by Mycobacterium abscessus infection, wherein the diphenylpyralid exerts its effect by inhibiting the activity of Mycobacterium abscessus.

4. The use according to any one of claims 1 to 3, wherein the Mycobacterium abscessus is selected from a standard strain of Mycobacterium abscessus, a clinical isolate of Mycobacterium abscessus, or Mycobacterium abscessus carried by a Mycobacterium abscessus infected person.

5. The use as claimed in claim 3, wherein the drug may further be added with one or more pharmaceutically acceptable carriers or excipients.

6. The use according to claim 5, wherein the carrier is selected from one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

7. The use according to claim 3, wherein the dosage form of the drug is selected from tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, transdermal preparations, suppositories or freeze-dried powder injections.

8. The use as claimed in claim 3, wherein the drug can be a common preparation, a sustained-release preparation, a controlled-release preparation or one of various microparticle delivery systems.

9. The use according to claim 3, wherein the drug can be administered by injection, cavity administration or mucosal administration.

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