Use of pentamidine in the treatment of Mycobacterium abscessus infection

Abstract

The present invention belongs to the field of biomedical technology, and particularly relates to the application of aromatic diamidines in the treatment of Mycobacterium abscessus infection. Pentamidine in the present invention has good antibacterial effects on both standard strains and clinical isolates of Mycobacterium abscessus; the MIC of pentamidine against standard strains of Mycobacterium abscessus can reach 8 μg / mL, and the best MIC against clinical isolates of Mycobacterium abscessus can reach 2 μg / mL, which broadens the application field of pentamidine and provides a new method for the treatment of Mycobacterium abscessus infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of aromatic diamidine drugs in the treatment of Mycobacterium abscessus infections. Background Art

[0002] Nontuberculous mycobacteria (NTM) refer to all mycobacteria other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. The incidence and related mortality of NTM pulmonary infections are increasing globally (Non-tuberculous mycobacterial pulmonary disease[J]. Eur Respir J, 2019, 54(1): 1900250.). NTM are divided into rapidly growing mycobacteria and slowly growing mycobacteria. Among them, Mycobacterium abscessum (MAB) is the most common human pathogenic rapidly growing mycobacterium. MAB is one of the most difficult-to-treat non-tuberculous mycobacteria, being naturally resistant to most antibiotics. The treatment drugs recommended by the American Thoracic Society, the Chinese Medical Association's Tuberculosis Branch, and the Chinese Anti-Tuberculosis Association are only a limited number of antibiotics, and most of them are injectable drugs (Prevalence and speciation of non-tuberculous mycobacteria among pulmonary and extrapulmonary tuberculosis suspects in South India[J]. J Infect Public Health, 2021, 14(3): 320 - 323.). However, despite long-course and expensive treatment, most patients still experience treatment failure, with a cure rate of less than 30% and a high recurrence rate (tidrug-resistant Mycobacterium abscessus threatens patients with cystic fibrosis[J]. Lancet Respir Med, 2017, 5(1): 15.). This is related to MAB's natural resistance to most antibiotics, the high incidence of adverse reactions of existing effective drugs, and poor patient compliance due to the long treatment course and mostly intravenous use. There is an urgent need to develop drugs with effective treatment, low incidence of adverse reactions, and good compliance for the treatment of MAB. Mycobacterium abscessum has a special drug resistance mechanism, making its drug resistance spectrum wider than that of other microorganisms. Drug resistance can be divided into two categories: natural resistance and acquired resistance. Among them, acquired resistance is mostly caused by gene mutations due to exposure to an antibiotic environment. Fully exploring the anti-Mycobacterium abscessum activity of existing compounds or antibiotics is also an efficient way to develop anti-Mycobacterium abscessum drugs.

[0003] Pentamidine is an anti-parasitic drug, a chemically synthesized aromatic diamidine drug with a half-life of 0.5 - 1 h. Its commonly used clinical form is the hydroxyethyl sulfonate (pentamidine isethionate). Pentamidine isethionate has a direct killing effect on Leishmania protozoa and is an effective drug for the treatment of kala-azar. It can also be used for the early treatment of African trypanosomiasis and the treatment of pulmonary inflammation caused by Pneumocystis carinii (Zhao Zhao, Yang Yihong, Zhang Heng, et al. Improvement of the synthesis process of pentamidine [J]. Chinese Journal of Synthetic Chemistry, 2009, 17(5): 642 - 644.). In addition, studies have found that pentamidine has anti-tumor effects. Pentamidine can inhibit the proliferation of various tumor cells, including malignant melanoma, prostate cancer, rectal cancer, and breast cancer, in vitro (Wu Yanyin, Zhang Mingyue, Ding Hong, et al. Study on the inhibitory effect of pentamidine on human ovarian cancer CAOV3 cells [J]. Military Medical Journal of Southeast China, 2012, 14(1): 27 - 28.). Currently, there are no reports on the inhibition of Mycobacterium abscessus by pentamidine. Summary of the Invention

[0004] The present invention discovers that pentamidine has the effect of inhibiting the activity of Mycobacterium abscessus, and thus completes the present invention.

[0005] In a first aspect, the present invention provides an application of pentamidine in the preparation of a product for inhibiting Mycobacterium abscessus.

[0006] Further, the Mycobacterium abscessus includes a standard strain of Mycobacterium abscessus, a clinically isolated strain of Mycobacterium abscessus, or Mycobacterium abscessus carried by a patient infected with Mycobacterium abscessus.

[0007] Further, the product for inhibiting Mycobacterium abscessus includes a medical product or a non-medical product.

[0008] Even further, the medical product refers to a medical drug, and the non-medical product refers to an experimental reagent or a bacteriostatic agent.

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

[0010] Even further, the carrier materials include, but are not limited to, one or more of water-soluble carrier materials, poorly soluble carrier materials, and / or enteric-soluble carrier materials.

[0011] Further, the water-soluble carrier materials include, but are not limited to, one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acids.

[0012] Further, the poorly soluble carrier materials include, but are not limited to, one or more of ethyl cellulose and / or cholesterol stearate.

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

[0014] Further, the product can be made into various dosage forms, including, but not limited to, one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, buccal tablets, suppositories, and / or lyophilized powder injections.

[0015] In a second aspect, the present invention provides an application of pentamidine in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection, and the pentamidine exerts its effect by inhibiting the activity of Mycobacterium abscessus.

[0016] Further, 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.

[0017] Further, one or more pharmaceutically acceptable carriers or excipients can also be added to the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection.

[0018] Furthermore, the carrier material includes, but is not limited to, one or more of water-soluble carrier materials, poorly water-soluble carrier materials, and / or enteric-coated carrier materials.

[0019] Further, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be made into various dosage forms, including, but not limited to, one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, buccal tablets, suppositories, and / or lyophilized powder injections.

[0020] Furthermore, the preparation can be one or more of an ordinary preparation, a sustained-release preparation, a controlled-release preparation, and / or various particulate drug delivery systems.

[0021] Further, if necessary, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, or other materials can also be added to the pharmaceutical preparation.

[0022] Further, the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection can be administered by injection, intravaginal administration, respiratory administration, or mucosal administration.

[0023] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection, etc.

[0024] In a third aspect, the present invention provides a pharmaceutical composition, the pharmaceutical composition contains pentamidine and another drug for anti-Mycobacterium abscessus infection, and the pharmaceutical composition has at least one of the following effects:

[0025] a) Inhibiting the activity of Mycobacterium abscessus;

[0026] b) Anti-Mycobacterium abscessus infection;

[0027] c) Preventing and / or treating diseases caused by Mycobacterium abscessus.

[0028] 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.

[0029] 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 enhance the resistance of patients.

[0030] Even further, the antibiotics include one or more of clofazimine, bedaquiline, fusidic acid, clarithromycin, azithromycin, cefoxitin, amikacin, tigecycline, phenelzine, and fidaxomicin; the other drugs include one or more of vitamins, amino acids, proteins, or minerals.

[0031] Furthermore, one or more pharmaceutically acceptable carriers can be added to the pharmaceutical composition.

[0032] Furthermore, the pharmaceutical composition can be made into various forms such as injection solutions, tablets, powders, granules, capsules, oral liquids, or pharmaceutical excipients, etc.; the drugs in the above various dosage forms can all be prepared according to the conventional methods in the pharmaceutical field.

[0033] Furthermore, the pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, or mucosal tissues by injection, penetration, absorption, physical or chemical mediated methods; or it can be introduced into the body after being mixed or encapsulated with other substances.

[0034] Beneficial effects

[0035] Pentamidine in the present invention has good antibacterial effects on both the standard strain and clinical isolates of Mycobacterium abscessus; the MIC for the standard strain of Mycobacterium abscessus can reach 8 μg / mL, and the best MIC for the clinical isolates of Mycobacterium abscessus can reach 2 μg / mL.

[0036] The present invention broadens the application field of pentamidine and provides a new method for the treatment of Mycobacterium abscessus infection. Specific embodiments

[0037] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not limit the present invention. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0038] In the following experimental methods of the examples, unless otherwise specified, they are all conventional methods. Unless otherwise specified, the test materials used in the following examples can all be obtained through conventional commercial channels.

[0039] Material description

[0040] 1. McFarland turbidity standard tubes

[0041] McFarland turbidity standard tubes are standard turbidity tubes with different turbidities for microbial turbidity measurement invented by McFarland. They are prepared according to the ratio of sulfuric acid and barium chloride, specifically as follows:

[0042]

[0043] 2. Test strains and drugs

[0044] Mycobacterium abscessus standard strain: ATCC 19977.

[0045] Pentamidine: Molecular formula: C 19 H 24 N4O2, and the structural formula is as follows:

[0046]

[0047] Pentamidine isethionate has a molecular formula of C 23 H 36 N4O 10 S2, and the structural formula is as follows:

[0048]

[0049] 3. Test drug conversion

[0050] Pentamidine isethionate has a molecular weight of 592.68 g / mol, and the molecular weight of isethionic acid is 125.12 g / mol. After calculation, the potency of pentamidine in the compound pentamidine isethionate is 79.06%. According to the calculated potency, weigh 16.19 mg of pentamidine isethionate (pentamidine content is 12.8 mg) and add it to 1 ml of DMSO to prepare a pentamidine drug stock solution with a final concentration of 12.8 mg / ml. Dilute it at a ratio of 1:50 to a working solution with a concentration of 256 μg / ml.

[0051] Example 1 Detection of the antibacterial activity of pentamidine against standard strains of Mycobacterium abscessus

[0052] 1.1 Preparation of a suspension of standard strains of Mycobacterium abscessus

[0053] Inoculate the standard strain of Mycobacterium abscessus (ATCC19977) into the culture medium. When it grows to the logarithmic phase, scrape the colonies and place them in a grinding bottle. Adjust the turbidity to 0.5 McFarland turbidity tube, and dilute it 1:200 for later use.

[0054] 1.2 Minimum inhibitory concentration (MIC)

[0055] (1) Add 100 μL of Mueller Hinton (MH) medium to a 96-well plate;

[0056] (2) Add sequentially diluted drugs to columns 1-11;

[0057] (3) Add the diluted suspension of the standard strain of Mycobacterium abscessus to columns 1-11 so that the final concentration of the bacterial solution is 4×10 5 CFU / mL; The specific final drug concentrations in each well are shown in Table 1, and then incubate;

[0058] (4) Add 30 μL of resazurin chromogenic solution, and observe the color change of the well plate after incubation;

[0059] (5) Read the minimum inhibitory concentration (Minimal inhibitory concentration, MIC).

[0060] Table 1 Drug concentrations of pentamidine

[0061] Number of columns 1 2 3 4 5 6 7 8 9 10 11 12 Drug concentration (μg / mL) 64 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 0

[0062] Method for reading MIC: MIC is the drug concentration that can inhibit the growth of 90% of the colonies. The blue wells indicate no bacterial growth, and the pink wells indicate bacterial growth. The lowest drug concentration that prevents the color from changing from blue to pink is recorded as the MIC that can inhibit bacterial growth.

[0063] The positive control is the bacteria-containing culture medium without the addition of drugs, that is, column 12; Take another 96-well plate and add the culture medium without both drugs and bacterial solution as the negative control. When the positive control is pink and the negative control is blue, the MIC data measured in this batch are valid.

[0064] 1.3 Test results

[0065] The MIC of pentamidine against the standard strain of Mycobacterium abscessus is 8 μg / mL.

[0066] Example 2 Detection of the antibacterial activity of pentamidine against clinically isolated strains of Mycobacterium abscessus

[0067] 2.1 Test Samples and Methods

[0068] Clinically isolated strains: 34 strains isolated and cultured from sputum specimens of patients infected with Mycobacterium abscessus, and identified as Mycobacterium abscessus by sequencing of 16S rRNA, hsp65, rpoB, and the 16-23S rRNA intergenic region.

[0069] According to the method in Example 1, the in vitro antibacterial activity of pentamidine against 34 clinically isolated strains of Mycobacterium abscessus was detected.

[0070] 2.2 Test Results

[0071] The MIC results of pentamidine against clinically isolated strains of Mycobacterium abscessus are shown in Table 2, and the statistical results of the MIC concentration distribution are shown in Table 3.

[0072] Table 2 MIC of Pentamidine against Clinically Isolated Strains of Mycobacterium abscessus

[0073]

[0074]

[0075] Table 3 Statistical Results of MIC Concentration Distribution

[0076] Drug concentration (μg / mL) 0.0625 0.125 0.25 0.5 1 2 4 8 16 32 Number of strains (strains) 0 0 0 0 0 2 0 8 24 0

[0077] The results showed that pentamidine had good antibacterial activity against clinically isolated strains of Mycobacterium abscessus, with the MIC distribution range of 2 - 16 μg / mL, and the best could reach 2 μg / mL.

Claims

1. Use of pentamidine in the preparation of a product for inhibiting Mycobacterium abscessus, wherein the pentamidine is the only active ingredient in the product for inhibiting Mycobacterium abscessus; the molecular formula of the pentamidine is: C 19 H 24 N4O2, and the structural formula is: 。 2. The application according to claim 1, wherein the Mycobacterium abscessus includes a standard strain of Mycobacterium abscessus, a clinically isolated strain of Mycobacterium abscessus, or Mycobacterium abscessus carried by a patient infected with Mycobacterium abscessus.

3. An application of pentamidine in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection, wherein the pentamidine is the sole active ingredient in the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection; the pentamidine exerts its effect by inhibiting the activity of Mycobacterium abscessus.

4. The application according to claim 3, wherein the Mycobacterium abscessus includes a standard strain of Mycobacterium abscessus, a clinically isolated strain of Mycobacterium abscessus, or Mycobacterium abscessus carried by a patient infected with Mycobacterium abscessus.

5. The application according to any one of claims 3 or 4, wherein the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, and / or suppositories.

6. The application according to any one of claims 3-5, wherein the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection is administered by injection, via the respiratory tract, or mucosally.

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