CN118903079B

CN118903079B - Application of dimoxystrobin in resisting infection of mycobacterium abscessus

Info

Publication number: CN118903079B
Application number: CN202411318310.8A
Authority: CN
Inventors: 黄海荣; 齐浩然; 张超; 于霞; 姜广路; 王聪丽
Original assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Current assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Priority date: 2024-09-20
Filing date: 2024-09-20
Publication date: 2025-02-14
Anticipated expiration: 2044-09-20
Also published as: CN118903079A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of dimoxystrobin in resisting infection of mycobacterium abscessus. The MIC of the dimoxystrobin to the standard strain of the mycobacterium abscess ATCC 19977 is 0.0313 mug/mL, the minimum sterilization concentration of the dimoxystrobin to the standard strain of the mycobacterium abscess is 0.0625 mug/mL, and the MBC/MIC ratio of the dimoxystrobin to the standard strain of the mycobacterium abscess is 2, so that the medicament has a sterilization effect on the mycobacterium abscess. When the concentration of the penoxsulam is less than or equal to 5 mu M (namely 2.1 mu g/mL), the penoxsulam is incubated with THP-1 for 24 hours, and the survival rate of the cells is 100%, which indicates that the penoxsulam has lower cytotoxicity. In the anti-abscess mycobacterial infection, the dimoxystrobin can become a novel therapeutic drug.

Description

Application of dimoxystrobin in resisting infection of mycobacterium abscessus

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of dimoxystrobin in resisting infection of mycobacterium abscessus.

Background

Non-tuberculosis mycobacteria (NTM) refers to a generic term for mycobacteria in the genus mycobacterium other than the tuberculosis complex and leprosy mycobacteria. In epidemiological investigation of tuberculosis, it is found that the separation rate of NTM is continuously high, and the proportion of NTM in mycobacterial related diseases is gradually increased as tuberculosis is gradually controlled in a country or region according to epidemiological development rules as high as 22.9％(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.). in investigation in 2010. The number of diseases caused by NTM has exceeded tuberculosis in many countries, such as the united states, france, etc.

Mycobacterium abscesses (Mycobacterium abscessus, mab) account for 20.3% of all reported NTM in China, and up to 46% in Guangdong province, are the most common species (Yu X,Liu P,Liu G,Wang G,Chen S,Huang H.The Prevalence ofNon-tuberculosis mycobacteria infections:Systematic review and meta-analysis[J].Journal of Infection.2016,73(6):558-567).RGM in Mycobacterium fast-growing (Rapid-growing mycobacteria, RGM) and are usually weak in pathogenicity, and Mycobacterium abscesses are a special case. Although Mab infection occurs in patients with underlying disease, more than 30% of patients are well-immunized, indicating that Mab is significantly pathogenic. Mycobacteria abscesses are ineffective against common antitubercular drugs such as first-line drugs (rifampin and isoniazid) and some second-line drugs (frizzled-mycin). The nature of bacteria against most antitubercular drugs and antibiotics makes them one of the most difficult to cure in mycobacterial-induced pulmonary diseases, and even if more than one year of treatment is carried out by using the recommended combined chemotherapy regimen containing amikacin, clarithromycin and imipenem, about 70% of patients fail to treat or relapse (Marion E,Song OR,Christophe T,et al.Mycobacterial Toxin Induces Analgesia in Buruli Ulcerby Targeting theAngiotensin Pathways[J].Cell,2014,157(7):1565-1576.). and in addition, in recent years, the drug resistance of mycobacterium abscesses to clarithromycin is increasingly serious, the reported drug resistance rate is 14％～38％(Lee SH,Yoo HK,Kim SH,KohWJ,Kim CK,ParkYK,et al.The drug resistance profile of Mycobacterium abscessus group strains from Korea.Ann Lab Med.2014;34:31-7.)., however, the research and development report of targeted drugs is still lacking worldwide for mycobacterium abscesses with high isolation rate, strong pathogenicity and serious drug resistance.

The penoxsulam (SYP-14288) is a novel bactericide developed by Shenyang chemical industry institute, belongs to the diarylamine family, and is mainly used for preventing and treating fungal infection caused by various plant diseases at present. The main action mechanism of the bixafen is to destroy the normal functions of mitochondria by uncoupling the ATP generation process and electron transfer, thereby reducing the productivity of fungi and affecting the normal biological functions of the fungi. The domestic research on the dimoxystrobin is mainly focused on the aspects of the growth inhibition effect, the antimycotic action mechanism and the like of the economic crop mould, and the research on the antibacterial effect of the mycobacterium abscessus is not reported. There are no studies reported in foreign countries on the dimoxystrobin.

Disclosure of Invention

The present invention has been completed based on the finding that dimoxystrobin (SYP-14288) has an effect of inhibiting the activity of Mycobacterium abscessum.

In a first aspect, the invention provides the use of dimoxystrobin for the preparation of a mycobacterium abscessus inhibiting product.

Further, the mycobacterium abscessus comprises a standard strain of mycobacterium abscessus, a clinical isolate of mycobacterium abscessus, or a mycobacterium abscessus carried by a mycobacterium abscessus infected person.

Further, the mycobacterium abscessus inhibition product is an experimental reagent or a bacteriostatic agent.

Still further, the bacteriostat may be a cleanser, including the use of the composition in the form of dimoxystrobin and pharmaceutically acceptable substances thereof to inhibit/eliminate mycobacterium abscessus.

In a second aspect, the present invention provides the use of dimoxystrobin in the manufacture of a medicament for the prevention and/or treatment of a disease caused by infection with mycobacterium abscessus, said dimoxystrobin acting by inhibiting the activity of mycobacterium abscessus.

Further, the mycobacterium abscessus comprises a standard strain of mycobacterium abscessus, a clinical isolate of mycobacterium abscessus or mycobacterium abscessus carried by a mycobacterium abscessus infected person.

Further, one or more pharmaceutically acceptable carriers or excipients can be added into the medicine for preventing and/or treating diseases caused by the infection of the mycobacterium abscessus.

Still further, 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.

Further, the medicament for preventing and/or treating diseases caused by mycobacterium tuberculosis infection can be prepared into various dosage forms, including but not limited to one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, transdermal agents, suppositories and/or freeze-dried powder injection.

Still further, the formulation may be one or more of a general formulation, a slow release formulation, a controlled release formulation, and/or various microparticle delivery systems.

Further, the various formulations may also incorporate colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials into the pharmaceutical formulation as desired.

Further, the agent for preventing and/or treating a disease caused by infection with mycobacterium abscess may be administered by injection, by luminal administration, by respiratory administration or by mucosal administration.

Still further, the administration of injections includes subcutaneous injections, intravenous injections, intramuscular injections, and intra-luminal injections.

Advantageous effects

The bixafen has good antibacterial activity on standard strains and clinical isolates of the mycobacterium abscessus. The MIC of the dimoxystrobin to the standard strain of the mycobacterium abscess ATCC 19977 is 0.0313 mug/mL, the minimum sterilization concentration of the dimoxystrobin to the standard strain of the mycobacterium abscess is 0.0625 mug/mL, and the MBC/MIC ratio of the dimoxystrobin to the standard strain of the mycobacterium abscess is 2, so that the medicament has a sterilization effect on the mycobacterium abscess. When the concentration of the penoxsulam is less than or equal to 5 mu M (namely 2.1 mu g/mL), the penoxsulam is incubated with THP-1 for 24 hours, and the survival rate of the cells is 100%, which indicates that the penoxsulam has lower cytotoxicity. In the anti-abscess mycobacterial infection, the dimoxystrobin can become a novel therapeutic drug.

Drawings

FIG. 1 shows the MIC value distribution of dimoxystrobin to 43 clinical isolates of Mycobacterium abscessum.

FIG. 2 is a graph showing the effect of penoxsulam on bacterial survival in THP-1 cells.

FIG. 3 is the effect of penoxsulam on THP-1 cytotoxicity.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments described below may be combined with each other as long as they do not collide with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available. The quantitative experiments in the following examples were all set up for 3-5 replicates and the results averaged.

Terminology

Alamar-Blue cell viability reagent is a ready-to-use resazurin-based solution, and can be used as a cell health index by quantitatively detecting viability by utilizing the reducing capability of living cells. Resazurin is an active ingredient of alamarBlue reagent, a nontoxic compound with cell permeability, and is blue in color with little fluorescence. After entry into living cells, resazurin is reduced to resorufin, a red, strongly fluorescent compound. Cell viability changes can be easily detected using absorbance or fluorescence based enzyme-labeled instruments. The alamarBlue cell viability reagent has wide applicability and can be used for cell viability detection of various human and animal cell lines, bacteria, plants and fungi.

MBC (minimum bactericidal concentration, minimal Bactericidal Concentration) refers to the minimum drug concentration required to kill 99.9% (3 orders of magnitude reduction) of the test microorganisms. MBC is defined as the lowest effective drug concentration in the CFU, at least 3log10 lower than the initial CFU, according to the CLSI guidelines. When the MBC/MIC ratio is less than or equal to 4, the antibiotics are considered to have bactericidal effect, otherwise, the antibiotics are considered to have bacteriostatic effect.

Description of materials

The bixafen is synthesized from Shanghai run Nuo Biotechnology Co. The molecular formula of the dimoxystrobin is C ₁₃H₇Cl₃N₄O₆, the CAS is No.1334790-94-1, and the structural formula is as follows:

The standard strain of mycobacterium abscessus, ATCC 19977.

Standard strain of mycobacterium abscessus bacterial suspension, which is to inoculate standard strain of mycobacterium abscessus into neutral rogowski culture medium, scrape strain in logarithmic phase after culturing, dilute with Mueller Hinton (MH) culture medium after grinding.

Clinical isolation of strains 43 strains isolated and cultured from sputum specimens of patients infected with Mycobacterium abscessum were identified as Mycobacterium abscessum by sequencing of 16SrRNA, hsp65, rpoB and 16-23S rRNA interarea.

THP-1 cells purchased from cell banks of the national academy of sciences.

PMA (phorbol ester) from Sigma company.

CCK-8 solution, available from Soy pal.

Example 1 detection of antibacterial Activity of Bifenamid against Mycobacterium abscessum Standard Strain

1.1 Test procedure

(1) Mueller Hinton (MH) medium was added to the well plate;

(2) Adding a drug solution to be tested (prepared by DMSO) into the 2 nd column of the pore plate, and carrying out gradient dilution until the drug solution is 11 th column, wherein the 1 st column is positive control, and the 12 th column is negative control;

(3) Adding standard bacterial suspension of mycobacterium abscessus into the pore plate, wherein the final drug concentration in each pore is shown in table 1;

(4) Culturing, namely adding Alamar-Blue and 5% Tween 80, and continuing culturing;

(5) Minimum Inhibitory Concentration (MIC) was read and inhibition was calculated.

TABLE 1 final drug concentration in each column of wells

1.2 Test results

The Minimum Inhibitory Concentration (MIC) is the concentration of the drug capable of inhibiting the growth of 90% of bacterial colonies.

The minimum drug concentration generated by >90% reduced Alamar-Blue was inhibited by fluorescence detection (E×/Em,530nm/600 nm).

Inhibition% = 100% - (detection Kong Yingguang value-background fluorescence value)/(growth control Kong Yingguang value-background fluorescence value) ×100%.

The background fluorescence value is the fluorescence value of the negative control, and the fluorescence value of the growth control hole is the fluorescence value of the positive control.

The negative control is culture medium without adding medicine and bacterial liquid, and the positive control is bacterial culture medium without adding medicine.

The results showed that the MIC of the dimoxystrobin was 0.0313 μg/mL for the mycobacterium abscessus standard strain ATCC 19977.

Example 2 detection of antibacterial Activity of Bifenamid against clinically isolated Mycobacterium abscessum Strain

The bacteriostatic activity of the test drug against 43 clinical Mycobacterium abscessum strains was examined as in example 1.

MIC results are shown in table 2.

The statistical results of MIC concentration distribution are shown in Table 3 and FIG. 1, and the dimoxystrobin has better antibacterial activity on clinically isolated mycobacterium abscessus.

TABLE 2.43 MIC results of clinical Mycobacterium abscessum strains

TABLE 3 statistical results of MIC concentration distribution of Bifenamid on clinically isolated Mycobacterium abscessum strains

Example 3 determination of Minimum Bactericidal Concentration (MBC) of Bifenamid against Mycobacterium abscessum Standard Strain

3.1 Test procedure

(1) According to example 1, MIC values are determined;

(2) From the MIC results, wells were defined 1×, 2×,4×,8×, 16×, 32×, 64×, 128× MIC, from the differences

Sucking out the culture medium from the MIC well, and diluting the culture medium in a multiple ratio;

(3) The dilutions were spotted onto Mueller Hinton (MH) solid plates;

(4) Colonies CFU were counted.

3.2 Test results

MBC refers to the drug concentration that achieves a 99.9% kill rate for the final inoculum size, defined as the lowest effective drug concentration in the CFU, 3log ₁₀ lower than the initial CFU, according to the CLSI guidelines. When the MBC/MIC ratio is less than or equal to 4, the antibiotics are considered to have bactericidal effect, otherwise, the antibiotics are considered to have bacteriostatic effect.

The minimum bactericidal concentration of the bixafen on the standard strain of the mycobacterium abscess is 0.0625 mug/mL, so that the MBC/MIC ratio of the bixafen on the standard strain of the mycobacterium abscess is 2, which shows that the medicine has bactericidal effect on the mycobacterium abscess. EXAMPLE 4 CFU counting to evaluate the Effect of Bifenamid on THP-1 intracellular bacterial survival

4.1 Cell culture

THP-1 cells in the logarithmic phase of growth were inoculated, incubated with PMA, and induced to differentiate into macrophages.

4.2 Infection of THP-1 cells with Mycobacterium abscessum Standard Strain

(1) Preparation of Mycobacterium abscessum Standard Strain A Mycobacterium abscessum Standard Strain is inoculated in neutral Roche Medium

After culturing, scraping the strain in the growth log phase, diluting with 1640 culture medium according to MOI=5:1 after the strain is ground to be specific to turbidity;

(3) After washing the cells, the cells were washed with a concentration of 0.125. Mu.g/mL, 0.25. Mu.g/mL, 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, respectively

The bixafen solution acts on THP-1 cells, a control group uses 1640 cell culture medium of 0.1% DMSO, and the inhibition activity of 4.3 bixafen on bacteria in THP-1 cells is measured

(1) Cell lysis, namely after cleaning cells, adding 0.01% Triton X-100, incubating and uniformly mixing;

(2) Cell lysates were diluted and spotted onto Mueller Hinton (MH) solid plates;

(3) Culturing, counting colony CFU on a culture plate;

4.4 test results

As can be seen from FIG. 2, dimoxystrobin can reduce survival of the THP-1 intracellular abscess Mycobacterium standard strain. EXAMPLE 5 CCK-8 detection of the cytotoxicity of Bifenamid on THP-1 cells

5.1 Cell culture

5.2 Verification of cytotoxicity of Bifenamid against THP-1 cells

(1) THP-1 cells were treated with a concentration of 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.3125. Mu.M, 0.16. Mu.M, 0.08. Mu.M, and a control group of the cells were cultured using 1640 cell culture medium of 0.1% DMSO;

(2) The medium was aspirated (without the effect of the original drug), the incomplete medium and CCK-8 solution were added, incubated, and absorbance was measured.

5.3 Test results

Vitality calculation the cell vitality of THP-1 cells cultured at different concentration of bixafen was calculated according to the following formula.

Cell viability (%) = [ a (dosing) -a (blank) ]/[ a (0 dosing) -a (blank) ]x100;

wherein A (dosing) represents the absorbance of wells with cells, CCK-8 solution and drug solution, A (blank) represents the absorbance of wells with medium and CCK-8 solution without cells, and A (0 dosing) represents the absorbance of wells with cells, CCK-8 solution without drug solution.

As can be seen from FIG. 3, when the amount of the penoxsulam was less than or equal to 5. Mu.M (i.e., 2.1. Mu.g/mL), the penoxsulam was incubated with THP-1 for 24 hours, and the cell viability was 100%, suggesting that the cell viability was low.

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.