Application of pentamidine in resisting mycobacterium abscessus infection

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an aromatic diamidine medicine in resisting mycobacterium abscessus infection. The pentamidine disclosed by the invention has a relatively good antibacterial effect on standard strains and clinical isolates of mycobacterium abscessus; the MIC of the pentamidine can reach 8 mu g / mL for a standard strain of the mycobacterium abscessus, and the MIC of a clinical isolated strain of the mycobacterium abscessus can reach 2 mu g / mL preferably, so that the application field of the pentamidine is widened, and a new method is provided for treating mycobacterium abscessus infection.

Description

Technical Field

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

[0002] Nontuberculous mycobacteria (NTM) refers to all mycobacteria except Mycobacterium tuberculosis complex and Mycobacterium leprae. The incidence and related mortality of NTM lung infection are increasing worldwide (Non-tuberculous mycobacterial pulmonary disease [J]. Eur Respir J, 2019, 54 (1): 1900-250.). NTM is divided into fast-growing mycobacteria and slow-growing mycobacteria, among which Mycobacterium abscessum (MAB) is the most common human pathogenic fast-growing mycobacteria. MAB is one of the most difficult non-tuberculous mycobacteria to treat and is naturally resistant to most antibiotics. The treatment drugs recommended by the American Thoracic Society, the Tuberculosis Branch of the Chinese Medical Association, and the Chinese Anti-Tuberculosis Association are only a limited number of antibiotics, most of which 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 and expensive treatment, most patients still fail to receive treatment, 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 the natural resistance of MAB to most antibiotics, the high incidence of adverse reactions to existing effective drugs, and the poor patient compliance due to the long course of treatment and the fact that most of the drugs are intravenous. There is an urgent need to develop effective drugs with low incidence of adverse reactions and good compliance for the treatment of MAB. Mycobacterium abscessus has a special resistance mechanism, which makes its resistance spectrum wider than other microorganisms. Resistance can be divided into two categories: natural resistance and acquired resistance, of which acquired resistance is mostly caused by gene mutations caused by exposure to antibiotic environments. Fully exploring the anti-Mycobacterium abscessus activity of existing compounds or antibiotics is also an efficient way to develop anti-Mycobacterium abscessus drugs.

[0003] Pentamidine is an antiparasitic drug, a chemically synthesized aromatic diamidine drug with a half-life of 0.5-1h. Its isethionate salt (pentamidine isethionate) is commonly used in clinical practice. Pentamidine isethionate has a direct killing effect on Leishmania 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 lung inflammation caused by Pneumocystis carinii (Zhao Zhao, Yang Yihong, Zhang Heng, et al. Improvement of the synthesis process of pentamidine [J]. 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, colorectal 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]. Southeast National Defense Medicine, 2012, 14 (1): 27-28.). At present, there are no reports on the inhibition of Mycobacterium abscessus by pentamidine. Summary of the invention

[0004] The present invention is completed based on the discovery that pentamidine has the effect of inhibiting the activity of Mycobacterium abscessus.

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

[0006] 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.

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

[0008] Furthermore, the medical product refers to medical drugs, and the non-medical product refers to experimental reagents or antibacterial agents.

[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, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

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

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0020] 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.

[0021] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0022] 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.

[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, comprising pentamidine and another drug for preventing Mycobacterium abscessus infection, wherein 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) prevention and / or treatment of 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 another anti-Mycobacterium abscessus infection drug includes one or more of antibiotics and other drugs that can help inhibit or kill Mycobacterium abscessus or improve the patient's resistance.

[0030] Furthermore, 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 may be added to the pharmaceutical composition.

[0032] Furthermore, the pharmaceutical composition can be prepared into various forms such as injection, tablet, powder, granule, capsule, oral solution or pharmaceutical excipients; the above various dosage forms of drugs can be prepared according to 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 tissue by injection, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or encapsulated with other substances.

[0034] Beneficial Effects

[0035] The pentamidine in the present invention has good antibacterial effect on both the standard strain and clinical isolate of Mycobacterium abscessus; the MIC of the standard strain of Mycobacterium abscessus can reach 8 μg / mL, and the MIC of the clinical isolate of Mycobacterium abscessus can reach 2 μg / mL at best.

[0036] The invention broadens the application field of pentamidine and provides a new method for treating mycobacterium abscessus infection. DETAILED DESCRIPTION

[0037] 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.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0039] Material Description

[0040] 1. McFarland turbidimetric tube

[0041] McFarland turbidimetric tube is a standard turbidity tube of different turbidity invented by McFarland for microbial turbidity measurement. It is prepared according to the ratio of sulfuric acid and barium chloride, 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, the structural formula is as follows:

[0046]

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

[0048]

[0049] 3. Conversion of experimental drugs

[0050] Pentamidine isethionate has a molecular weight of 592.68 g / mol, of which the molecular weight of isethionate is 125.12 g / mol. The potency of pentamidine in pentamidine isethionate is calculated to be 79.06%. According to the calculated potency, 16.19 mg of pentamidine isethionate (pentamidine content is 12.8 mg) is weighed and added 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 standard strain suspension of Mycobacterium abscessus

[0053] Take the standard strain of Mycobacterium abscessus (ATCC19977) and inoculate it into the culture medium. When it grows to the logarithmic phase, scrape the colony and place it in a grinding bottle. Measure the turbidity to 0.5 McFarland turbidimetric tube turbidity, dilute it 1:200 and set it aside.

[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 serially diluted drugs to columns 1 to 11;

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

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

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

[0060] Table 1 Drug concentration 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] MIC reading method: MIC is the drug concentration that can inhibit 90% of the colony growth. Blue wells are sterile growth, pink wells are bacterial growth, and 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 a culture medium containing bacteria without drugs, i.e. column 12; another 96-well plate is added with a culture medium without drugs and bacteria, i.e. the negative control. When the positive control is pink and the negative control is blue, the MIC data of the batch is valid.

[0064] 1.3 Test results

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

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

[0067] 2.1 Test samples and methods

[0068] Clinical isolates: 34 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.

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

[0070] 2.2 Test results

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

[0072] Table 2 MIC of pentamidine against clinical isolates of Mycobacterium abscessus

[0073]

[0074]

[0075] Table 3 MIC concentration distribution statistical results

[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 clinical isolates of Mycobacterium abscessus, with the MIC distribution range of 2-16μg / mL, and the best value was 2μg / mL.

Claims

1. Use of pentamidine in the preparation of a product for inhibiting Mycobacterium abscessus, wherein the pentamidine molecular formula is: C 19 H 24 N4O2, the structural formula is:

2. The use 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 use according to any one of claims 1 or 2, wherein the product for inhibiting Mycobacterium abscessus comprises a medical product or a non-medical product; the medical product refers to a medical drug, and the non-medical product refers to an experimental reagent or an antibacterial agent.

4. Use of pentamidine in the preparation of a medicament for preventing and / or treating diseases caused by Mycobacterium abscessus infection, wherein the pentamidine exerts its effect by inhibiting the activity of Mycobacterium abscessus.

5. The use according to claim 4, 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.

6. The use according to any one of claims 4 or 5, wherein 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, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

7. The use according to any one of claims 4 to 6, wherein the drug for preventing and / or treating diseases caused by Mycobacterium abscessus infection can be administered by injection, cavity administration, respiratory tract administration or mucosal administration.

8. A pharmaceutical composition comprising pentamidine and another drug for treating Mycobacterium abscessus infection, wherein the pharmaceutical composition has at least one of the following effects: a) inhibiting the activity of Mycobacterium abscessus; b) Anti-Mycobacterium abscessus infection; c) prevention and / or treatment of diseases caused by Mycobacterium abscessus.

9. The pharmaceutical composition according to claim 8, 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 improve the patient's resistance.

10. The pharmaceutical composition according to claim 9, wherein 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.

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