Pharmaceutical composition for resisting nontuberculous mycobacterium infection

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of Fluazinam in preparation of a medicine for preventing and / or treating diseases caused by mycobacterium abscessus infection. Compared with a conventional anti-mycobacterium abscessus medicine, the Fluazinam disclosed by the invention has a remarkable antibacterial effect, the MIC of the Fluazinam on the mycobacterium abscessus can reach 0.0313 mu g / mL, the Fluazinam is low in cytotoxicity and good in safety, the application field of the Fluazinam is widened, and a new method is provided for treating mycobacterium abscessus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of pyridinamine in resisting non-tuberculous mycobacteria infection. Background Art

[0002] Non-tuberculous mycobacteria (NTM) are a collective term for all mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae. NTM are ubiquitous in nature and are opportunistic pathogens. They can cause lung infections with increasing morbidity and mortality worldwide, posing a serious threat to human health. NTM include both fast-growing and slow-growing mycobacteria. Among these, Mycobacterium abscessus, one of the fast-growing mycobacteria, is highly pathogenic and highly drug-resistant, making it a multidrug-resistant bacterium. It commonly causes inflammatory lung infections and severe infections of the skin, joints, soft tissues, surgical sites, and disseminated infections. Antibiotic therapy is currently the preferred treatment option. However, factors such as the low permeability of the bacterium's outer membrane lipid barrier, drug-modified inactivating enzymes, efflux pump systems, and target mutations make it resistant to most clinically used antibiotics and anti-tuberculosis drugs, including rifampicin and isoniazid (Illouz, 2021). The cure rate with antibiotic treatment is only 30-50%. Therefore, it is very necessary to find and explore drugs that are more effective in treating Mycobacterium abscessus.

[0003] Fluazinam is a pyridinamine derivative developed in 1988 by the Central Research Institute of Ishihara Corporation in Japan. It boasts rapid efficacy and is less susceptible to cross-resistance. It is highly effective against potato late blight, tomato early blight, pepper anthracnose, and Chinese cabbage clubroot. Fluazinam is a potent mitochondrial oxidative phosphorylation uncoupler. Its mechanism of action primarily involves metabolic conversion at the mitochondrial level, inhibiting mitochondrial complex I activity, which can trigger apoptosis. This uncoupling effect occurs by inhibiting oxidative phosphorylation, preventing ATP from being used for ADP phosphorylation and instead dissipating it as heat energy. Research on Fluazinam domestically and internationally has primarily focused on detection methods, residue behavior, dietary risk assessment, and the impact of processing on residue levels. Studies on its antibacterial effects against nontuberculous mycobacteria have been limited. Summary of the Invention

[0004] The present invention was completed based on the discovery that Fluazinam has the activity of inhibiting Mycobacterium abscessus.

[0005] In a first aspect, the present invention provides a pharmaceutical composition comprising Fluazinam and another drug for treating Mycobacterium abscessus infection.

[0006] The pharmaceutical composition has at least one of the following effects:

[0007] a) inhibiting the activity of Mycobacterium abscessus;

[0008] b) Anti-Mycobacterium abscessus infection;

[0009] c) prevention and / or treatment of diseases caused by Mycobacterium abscessus.

[0010] Furthermore, the Mycobacterium abscessus includes a standard strain of Mycobacterium abscessus, a clinical isolate of Mycobacterium abscessus, or Mycobacterium abscessus carried by a patient infected with Mycobacterium abscessus.

[0011] Furthermore, the lowest usage concentration of the pharmaceutical composition is not lower than the minimum inhibitory concentration (MIC) of Mycobacterium abscessus that it inhibits.

[0012] Furthermore, the other anti-Mycobacterium abscessus infection drug includes one or more of antibiotics and other drugs that can help inhibit or kill Mycobacterium abscessus or provide resistance to patients.

[0013] Furthermore, the antibiotics include one or more of clofazimine, bedaquiline, fusidic acid, clarithromycin, azithromycin, cefoxitin, amikacin, tigecycline, phenelzine and fidaxomicin; and the other drugs include one or more of vitamins, amino acids, proteins or minerals.

[0014] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0015] Furthermore, the pharmaceutical composition can be prepared into various forms such as injection, tablets, powders, granules, capsules, oral solutions, ointments, creams or pharmaceutical excipients; the above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0016] Furthermore, the pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or encapsulated with other substances.

[0017] In a second aspect, the present invention provides use of Fluazinam in the preparation of a medicament for preventing and / or treating diseases caused by Mycobacterium abscessus infection.

[0018] Fluazinam works in the following ways:

[0019] 1) Fluazinam inhibits the activity of Mycobacterium abscessus;

[0020] 2) Fluazinam kills Mycobacterium abscessus;

[0021] Furthermore, the Mycobacterium abscessus includes a standard strain of Mycobacterium abscessus, a clinical isolate of Mycobacterium abscessus, or Mycobacterium abscessus carried by a patient infected with Mycobacterium abscessus.

[0022] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0023] Furthermore, the pharmaceutical composition can be prepared into various forms such as injection, tablets, powders, granules, capsules, oral solutions, ointments, creams or pharmaceutical excipients.

[0024] Furthermore, the pharmaceutical composition can be introduced into the body through physical or chemical methods, such as into muscle, intradermal, subcutaneous, intravenous or mucosal tissues, or can be mixed or encapsulated with other substances and then introduced into the body.

[0025] Beneficial effects

[0026] Fluazinam in the present invention has a significant antibacterial effect compared to conventional anti-Mycobacterium abscessus drugs, and the MIC against Mycobacterium abscessus can reach 0.0313 μg / mL;

[0027] The invention broadens the application field of Fluazinam and provides a new method for treating Mycobacterium abscessus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 4 shows the distribution of MIC values ​​of Fluazinam against 37 clinical isolates of Mycobacterium abscessus.

[0029] Figure 2 The effect of Fluazinam on THP-1 cell cytotoxicity.

[0030] Figure 3 These are the CFU counts and histopathological results of Mab-infected mice treated with Fluazinam. DETAILED DESCRIPTION

[0031] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The present invention is further described in detail below in conjunction with the specific embodiments. The examples provided below can serve as a guide for further improvement by those skilled in the art and do not constitute a limitation of the present invention in any way. The experimental methods in the examples, unless otherwise specified, are conventional methods and conditions. The experimental materials used in the examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all repeated 3-5 times, and the results were averaged.

[0032] Definition and Description

[0033] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. The absence of a specific definition for a particular term or phrase should not be construed as ambiguous or unclear, but rather should be understood according to its ordinary meaning.

[0034] Multidrug-resistant organisms (MDROs) refer to bacteria that are resistant to three or more types of clinically used antimicrobial drugs, mainly including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, extended-spectrum β-lactamase-producing bacteria, carbapenem-resistant Acinetobacter baumannii, etc.

[0035] Efflux pumps refer to some transport membrane proteins on the bacterial cell membrane. These membrane proteins change their own conformation by actively consuming energy, selectively or non-selectively pumping drugs out of the bacterial cell, significantly reducing the concentration of antimicrobial drugs reaching the target site and leading to bacterial resistance.

[0036] Cross-resistance refers to the phenomenon that after developing resistance to a certain herbicide, resistance to other herbicides with the same mechanism of action is also developed. Cross-resistance to ALS herbicides has been found in resistant weeds such as bluegrass, foxtail millet and cleaver.

[0037] Cardiac QT interval prolongation: In a 12-lead resting electrocardiogram, the QT interval is the time from the start of the QRS complex to the end of the T wave, representing the duration of ventricular depolarization and repolarization. The measured QT interval must be corrected for heart rate. The corrected QT interval, known as QTc, can be used for comparison between individuals. The normal reference value for QTc is 350-430 ms for healthy men and 350-450 ms for women. If the QTc exceeds the upper limit of the normal reference range, QTc prolongation is diagnosed.

[0038] Fluazinam: purchased from MCE, product number: HY-B1839. Fluazinam molecular formula: C 13 H4C l2 F6N4O4, CAS No.: 79622-59-6.

[0039] The structural formula is as follows:

[0040]

[0041] Mycobacterium abscessus standard strain: ATCC 19977.

[0042] Example 1. Detection of Fluazinam's antibacterial activity against Mycobacterium abscessus standard strains

[0043] Detected substance: Fluazinam

[0044] 1. Add 100 μL of Mueller Hinton (MH) medium to each well of a 96-well plate.

[0045] 2. After completing step 1, take the 96-well plate and add 100 μL of a 4 μg / mL test drug solution (prepared in DMSO) to column 2. Mix thoroughly, aspirate 100 μL, and add it to column 3. After serial dilution to column 11, remove 100 μL and discard. Columns 1 and 12 contain no drug and serve as negative and positive control wells, respectively. Set up three replicate wells for each concentration.

[0046] 3. After completing step 2, take the 96-well plate and add 100 uL of the Mycobacterium abscessus standard strain bacterial suspension to each well so that the final volume in each well is 200 uL; the final drug concentration in each well is shown in Table 1. Preparation method of the Mycobacterium abscessus standard strain bacterial suspension: The Mycobacterium abscessus standard strain is inoculated into a neutral Roche medium and cultured in an incubator at 37°C for about 3 days. After scraping the strain in the logarithmic growth phase on the neutral Roche medium, the bacteria are ground and turbidized, and then diluted with Mueller Hinton (MH) medium.

[0047] Table 1 Final drug concentrations in each column of wells

[0048]

[0049] 4. After completing step 3, place the 96-well plate in an incubator for culture.

[0050] 5. After completing step 4, take the 96-well plate, add 20uL of Lamar blue and 50uL of 5% Tween 80 to each well, and then continue to culture in an incubator.

[0051] 6. After completing step 5, take the 96-well plate, read the minimum inhibitory concentration (MIC) and calculate the inhibition rate.

[0052] Minimum Inhibitory Concentration (MIC) Reading Method: The minimum inhibitory concentration (MIC) is the drug concentration that can inhibit 90% bacterial growth. The minimum drug concentration that inhibits >90% of reduced Alamarblue production is measured by fluorescence detection (E× / Em, 530nm / 600nm).

[0053] Inhibition rate % = 100% - (fluorescence value of the detection well - background fluorescence value) / (fluorescence value of the growth control well - background fluorescence value) × 100%.

[0054] 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.

[0055] The negative control was a culture medium without drugs and bacterial solution, and the positive control was a culture medium containing bacteria without drugs.

[0056] The results showed that the MIC of Fluazinam against M. abscessus standard strain ATCC 19977 was 0.0313 μg / mL.

[0057] Example 2, Detection of the bacteriostatic activity of Fluazinam against clinical isolates of M. abscessus

[0058] Clinical isolates: 37 strains isolated and cultured from sputum samples of patients infected with M. abscessus, identified as M. abscessus by 16S rRNA, hsp65, rpoB, 16-23S rRNA intergenic region sequencing.

[0059] Tested drug: Fluazinam

[0060] According to the method in Example 1, the bacteriostatic activity of the tested drug against 37 clinical strains of M. abscessus was detected. The MIC results are shown in Table 2. The statistical results of the MIC concentration distribution are shown in Table 3 and Figure 3. Figure 1

[0061]

[0062]

[0063]

[0064]

[0065] Table 3 is the statistical results of the MIC concentration distribution of Fluazinam against clinical isolates of M. abscessus

[0066]

[0067] The results showed that Fluazinam had good bacteriostatic activity against clinical isolates of M. abscessus. No more potent drugs / compounds against M. abscessus in vitro have been reported in the literature, and Fluazinam is expected to find new uses in the treatment of M. abscessus infection diseases.

[0068] Example 3, Determination of the minimum bactericidal concentration (MBC) of Fluazinam against M. abscessus standard strain

[0069] Tested substance: Fluazinam

[0070] 1. According to the CLSI guidelines, MBC refers to the concentration of the drug that can achieve 99.9% kill rate against the final inoculum.

[0071] According to Example 1, the MIC value was determined. ​

[0072] 2. According to the MIC results, find the corresponding wells on the MBC plate and mark the wells as 1×, 2×, 4×, 8×, 16×, 32×, 64×, and 128×MIC. Aspirate the culture medium from the different MIC wells and record them as 1×100 respectively, and then perform serial dilutions.

[0073] 3. Spot the dilutions of different concentrations on Mueller Hinton (MH) solid culture plates.

[0074] 4. After 3-4 days, count the CFU of the colonies on the culture plate.

[0075] 5. According to CLSI guidelines, the MBC is defined as the minimum effective drug concentration in CFU that is at least 3 log10 lower than the initial CFU. When the MBC / MIC ratio is ≤4, the antibiotic is considered bactericidal; otherwise, it is considered bacteriostatic.

[0076] The results showed that the minimum bactericidal concentration of Fluazinam against the standard strain of Mycobacterium abscessus was 0.0625 μg / mL. Therefore, the MBC / MIC ratio of Fluazinam against the standard strain of Mycobacterium abscessus was 2, indicating that the drug had a bactericidal effect against Mycobacterium abscessus.

[0077] Example 4: Detection of Fluazinam Cytotoxicity to THP-1 Cells by CCK-8 Method

[0078] 1. Cell culture

[0079] THP-1 cells in the logarithmic growth phase (purchased from the cell bank of the Chinese Academy of Sciences) were seeded into 96-well plates (10,000 cells / well), and 100 ng / L LPMA (purchased from Sigma, catalog number P1585-1MG) was added and incubated for 48 h to induce differentiation into macrophages.

[0080] 2. Verification of Fluazinam's cytotoxicity against THP-1 cells

[0081] 2.1. THP-1 cells were treated with Fluazinam solutions at concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.16 μM, and 0.08 μM, respectively. Control cells were treated with 1640 cell culture medium containing 0.1% DMSO (200 μL / well). Incubation was performed for 24 and 48 hours, respectively. Five replicates were performed for each concentration.

[0082] 2.2. Aspirate the culture medium (to remove the influence of the original drug), add 100 μL of incomplete culture medium and 10 μL of CCK-8 solution (purchased from Solebow, catalog number: CA1210) to each well, incubate in a 37°C incubator for 2 hours, and measure the absorbance at 450 nm using a microplate reader.

[0083] 3. Viability Calculation: The cell viability of THP-1 cells cultured at different Fluazinam concentrations was calculated according to the following formula.

[0084] Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100

[0085] 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 2. Figure 2 shown.

[0086] from Figure 2 It can be seen that when fluazinam ≤ 5 μM, the cell survival rate was 90-100% after incubation with THP-1 for 24 hours, indicating that it has low cytotoxicity; when fluazinam was 10 μM, the cell survival rate was 80.36% after 24 hours of incubation and 62.85% after 48 hours of incubation.

[0087] Example 5: Safety evaluation of high-dose fluazinam in mice

[0088] 20g female BALB / C mice were gavaged with 1000mg / kg / d for more than one week. No significant changes were observed in the activity status of the mice during daily observation.

[0089] Ten days later, the mice were sacrificed, and the size and weight of their vital organs were observed visually, revealing no significant differences compared to the untreated group. Pathological examination of the lungs, spleen, and kidneys revealed no significant changes (data not shown), indicating that the mice tolerated the high dose of fluazinam well.

[0090] Example 6: The killing effect of Fluazinam on Mycobacterium abscessus in mice

[0091] 1. The standard strain of Mycobacterium abscessus (ATCC 19977) was cultured at 1x10 7 Nude mice were infected by intravenous injection of CFU / mouse;

[0092] 2. On the second day after infection, mice were treated with Fluazinam at a concentration of 100 mg / kg by gavage. After 5 consecutive days of treatment, CFU counts were performed on different organs of the mice. The results were as follows: Figure 3shown.

[0093] The results showed that compared with the non-treatment group, the CFU counts in the lungs, spleen and kidneys were significantly reduced, and pathological examination results showed that the degree of organ inflammation was significantly improved.

Claims

1. A pharmaceutical composition comprising Fluazinam and another drug for treating Mycobacterium abscessus infection, wherein the Fluazinam has the following structural formula:

2. The pharmaceutical composition according to claim 1, wherein the Mycobacterium abscessus comprises a standard strain of Mycobacterium abscessus, a clinical isolate of Mycobacterium abscessus, or Mycobacterium abscessus carried by a patient infected with Mycobacterium abscessus.

3. The pharmaceutical composition according to claim 1 or 2, wherein the another anti-Mycobacterium abscessus infection drug comprises one or more of antibiotics and other drugs that can help inhibit or kill Mycobacterium abscessus or provide patients with resistance.

4. The pharmaceutical composition according to claim 3, wherein the antibiotic comprises one or more of clofazimine, bedaquiline, fusidic acid, clarithromycin, azithromycin, cefoxitin, amikacin, tigecycline, phenelzine and fidaxomicin.

5. The pharmaceutical composition according to claim 3, wherein the other drugs comprise one or more of vitamins, amino acids, proteins or minerals.

6. Use of Fluazinam in the preparation of a medicament for preventing and / or treating diseases caused by Mycobacterium abscessus infection, wherein: Fluazinam works by: Fluazinam inhibits the activity of Mycobacterium abscessus; Fluazinam kills Mycobacterium abscessus.

7. The use according to claim 6, wherein the Mycobacterium abscessus comprises a standard strain of Mycobacterium abscessus, a clinical isolate of Mycobacterium abscessus, or Mycobacterium abscessus carried by a patient infected with Mycobacterium abscessus.

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