Application of vorapaxar in preparation of medicine for inhibiting Mycobacterium kansasii

Abstract

According to the present invention, the vorapaxar has good antibacterial effect on Mycobacterium kansasii, the minimum inhibitory concentration of the vorapaxar on the Mycobacterium kansasii standard strain in vitro is 1.23 [mu] g / ml, and the minimum inhibitory concentration of the vorapaxar on the clinical rifampicin drug-resistant bacteria is 2 [mu] g / mL; the vorapaxar is low in cytotoxicity and good in safety, and can be used as a candidate drug to be applied to development of drugs for resisting Mycobacterium kansasii infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a small molecule compound, Vorapaxar, in the preparation of a drug for inhibiting Mycobacterium kansasii. Background Art

[0002] Nontuberculosis mycobacteria (NTM) refers to a general term for a large class of mycobacteria other than the tuberculosis complex (including tuberculosis, cattle, Africa, voles, goats, etc.) and Mycobacterium leprae. NTM is widely found in soil, water sources and livestock and poultry, with about 200 species, one-third of which can cause animal or human infection. NTM is mainly transmitted through aerosols from the environment, and in severe cases can cause host death (FEDRIZZI T, MEEHAN CJ, GROTTOLAA, et al. Genomic characterization of nontuberculous mycobacteria [J]. SciRep, 2017, 7: 45258.). Most nontuberculous mycobacteria are relatively resistant to high temperatures and have a certain degree of acid resistance. These characteristics enable them to resist the damage of heavy metals, disinfectants and antibacterial drugs to cells, have a high survival rate, and then invade the human body to cause local or systemic infection, among which lung infection is the most common. Nontuberculous mycobacteria can invade the human body through the respiratory tract, gastrointestinal tract, and skin, and then invade systemic tissues and organs. NTM infection refers to a state in which a patient is infected with NTM but does not develop related diseases. NTM disease refers to a state in which a patient is infected with NTM and causes lesions in related tissues and organs (Chinese Medical Association Tuberculosis Branch. Guidelines for the diagnosis and treatment of nontuberculous mycobacterial diseases (2020 edition) [J]. Chinese Journal of Tuberculosis and Respiratory Diseases, 2020, 43(11): 918-946). Mycobacterium kansasii is one of the NTMs with the highest clinical isolation rate at present (Weng Jianzhen, Tong Xunliang, Li Yanming. Research progress on the drug resistance mechanism of Mycobacterium kansasii [J]. Chinese Journal of Tuberculosis and Respiratory Diseases, 2019(7): 522-525.). In recent years, the incidence of nontuberculous mycobacterial lung disease has gradually increased, and the harm it brings has gradually attracted everyone's attention. However, due to the large number of strains, the classification of nontuberculous mycobacterial species has important significance for the patient's prognosis. The treatment of nontuberculous mycobacterial lung disease will change according to the different strains. Previous literature reports that the treatment of intracellular mycobacterial lung disease and kansasii mycobacterial lung disease is very different (Liu Yidian, Sha Wei. A brief discussion on the treatment of common fast-growing mycobacterial diseases [J]. Chinese Journal of Tuberculosis and Respiratory Diseases, 2020, 43(11): 914-917.). Kansasii mycobacterial disease faces multiple difficulties such as high incidence of drug resistance, long treatment cycle, and few available therapeutic drugs. It is urgent to develop new drugs, especially for rifampicin-resistant strains.

[0003] Vorapaxar is a selective, oral, potent, competitive protease-activated receptor 1 (PAR-1) antagonist that has a strong inhibitory effect on thrombin-induced platelet aggregation. It can reduce platelet activation and aggregation by blocking PAR-1 receptors, thereby reducing the risk of thrombosis. Studies have shown that vorapaxar can prevent the onset of thrombotic cardiovascular disease in patients with a history of myocardial infarction or peripheral arterial disease, and reduce the mortality of patients with myocardial infarction, stroke, and emergency coronary revascularization. On May 8, 2014, the U.S. Food and Drug Administration (FDA) approved its sulfate (trade name: Zontivity) for marketing, which can be used in combination with standard antiplatelet therapy as a secondary prevention of acute coronary syndrome, and can improve thrombotic cardiovascular events in patients with myocardial infarction. At present, there are no relevant studies on vorapaxar in the field of Mycobacterium kansasii disease, especially reports on the in vitro inhibition of Mycobacterium kansasii activity. Summary of the invention

[0004] The present invention found that Vorapaxar has good antibacterial activity against Mycobacterium kansasii in vitro, has low cytotoxicity and good safety. Based on this, the present invention was completed.

[0005] In a first aspect, the present invention provides a use of Vorapaxar in preparing a product for inhibiting Mycobacterium kansasii, wherein the product contains Vorapaxar, and the chemical formula of Vorapaxar is as follows:

[0006]

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

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

[0009] Furthermore, the antibacterial agent may be a cleaning agent, including using Vorapaxar and its pharmaceutically acceptable substances to inhibit / eliminate Mycobacterium kansasii.

[0010] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the product.

[0011] 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.

[0012] Furthermore, the water-soluble carrier material includes, but is not limited to, one or more of polyethylene glycol, polyvinyl pyrrolidone and / or organic acid.

[0013] Furthermore, the poorly soluble carrier material includes but is not limited to one or more of ethyl cellulose and / or cholesterol stearate.

[0014] Furthermore, the enteric carrier material includes but is not limited to one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0015] 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, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0016] 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.

[0017] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0018] Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and / or aluminum silicate.

[0019] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0020] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0021] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0022] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0023] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0024] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0025] Furthermore, the pills may be prepared using a wide variety of carriers known in the art, including diluents and absorbents, binders and / or disintegrants.

[0026] Furthermore, the diluent and absorbent include but are not limited to one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin and / or talc.

[0027] Furthermore, the adhesive includes, but is not limited to, one or more of gum arabic, gum tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste and / or batter.

[0028] Furthermore, the disintegrant includes, but is not limited to, one or more of agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0029] Furthermore, the suppository may be made of a wide variety of carriers known in the art, including but not limited to one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and / or semi-synthetic glycerides.

[0030] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0031] Furthermore, the injectable preparation can use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0032] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0033] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0034] In a second aspect, the present invention provides the use of Vorapaxar in the preparation of a medicament for preventing and / or treating a disease caused by infection with Mycobacterium kansasii, wherein the medicament contains Vorapaxar, and the chemical formula of Vorapaxar is as follows:

[0035]

[0036] Furthermore, the Mycobacterium kansasii includes a clinical isolate of Mycobacterium kansasii, a standard strain of Mycobacterium kansasii, or Mycobacterium kansasii carried by a Mycobacterium kansasii infected person.

[0037] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

[0038] 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.

[0039] 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, emulsions, granules, liposomes, transdermal agents, lozenges, suppositories and / or lyophilized powder injections.

[0040] 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.

[0041] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0042] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

[0043] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0044] Beneficial Effects

[0045] The vorapaxar of the present invention has a good antibacterial effect on Mycobacterium kansasii. The minimum inhibitory concentration of vorapaxar against the standard strain of Mycobacterium kansasii in vitro is 1.23 μg / ml, and the minimum inhibitory concentration against clinical rifampicin-resistant bacteria is 2 μg / mL. Vorapaxar has low cytotoxicity and good safety, and can be used as a candidate drug for the development of drugs against Mycobacterium kansasii infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The effect of vorapaxar on the viability of A549 and THP-1 cells. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0049] The "CCK-8 method" described in this article is a cell proliferation and cytotoxicity detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt), which can be reduced to a water-soluble orange-yellow formazan dye by dehydrogenase in the mitochondria of living cells under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate, and its absorbance is measured at a wavelength of 450nm using an enzyme reader.

[0050] Example 1 Drug sensitivity test

[0051] In the present invention, the source of the standard strain of Mycobacterium kansasii is ATCC 12478. The 96-well plate microbroth dilution method was used to determine the in vitro activity of Vorapaxar against the standard strain of Mycobacterium kansasii ATCC12478, and the activity of Vorapaxar against rifampicin-resistant strains.

[0052] 1. Experimental Materials

[0053] 1.1 Test bacteria: standard strain of Mycobacterium kansasii ATCC12478, 3 strains of clinical rifampicin-sensitive bacteria (No. 1, 2, and 3), and 2 strains of rifampicin-resistant bacteria (No. 4 and 5).

[0054] 1.2 Antibacterial drugs: Vorapaxar.

[0055] 1.3 Culture medium: Cation-Adjusted Mueller-Hinton Broth (CAMHB).

[0056] 1.4 Color indicator: Resazurin.

[0057] 2. Experimental methods

[0058] 2.1 MIC detection of standard strains of Mycobacterium kansasii

[0059] Vorapaxa was diluted with CAMHB medium containing 5% OADC and then added to a 96-well plate containing 100 μl of 5% OADC CAMHB broth and serially diluted. 100 μl of medium without drug was added to the negative control wells and the positive control wells, as shown in Table 1.

[0060] Table 1 Distribution of various concentrations of Vorapasa in 96-well plates of standard strains of Mycobacterium kansasii

[0061] 1 2 3 4 5 6 7 8 9 10 11 12 PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS 20μM 10μM 10μM 2.5μM 1.25μM 0.625μM 0.312μM 0.156μM PBS PBS PBS PBS 20μM 10μM 5μM 2.5μM 1.25μM 0.625μM 0.312μM 0.156μM PBS PBS PBS PBS Positive Positive Positive Positive Positive Positive Positive Positive PBS PBS PBS PBS Negative Negative Negative Negative Negative Negative Negative Negative PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

[0062] Note: Drug concentration is in μM

[0063] Pick a colony of the standard strain of Mycobacterium kansasii ATCC12478 in the logarithmic growth phase, grind the bacteria and measure the turbidity to 0.5 McFarland units, then dilute 100 times, and pipette 100 μl of bacterial solution into the drug well and positive control well of the aforementioned 96-well plate. The bacterial inoculation amount is about 5×10^5 CFU / mL, and the final concentration range of Vorapax is 20 μM to 0.156 μM. Add 100 ul of bacterial solution to the positive control well and 100 ul of culture medium to the negative control well. Seal the plate with a sealing film and incubate.

[0064] 2.2 MIC detection of rifampicin-sensitive and rifampicin-resistant clinical strains of Mycobacterium kanssa

[0065] Vorapaxar (final concentration range 4 μg / ml to 0.125 μg / ml) was serially diluted using CAMHB broth containing 5% OADC according to the above method, and 100 ul of medium without drug was added to the negative control wells and the positive control wells, as shown in Table 2.

[0066] Table 2 Distribution of various concentrations of Vorapasa in 96-well plates of clinical strains of Mycobacterium kansasii

[0067] 1 2 3 4 5 6 7 8 9 10 11 12 A PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS B PBS Negative Positive 4 4 PBS PBS PBS PBS PBS PBS PBS C PBS Negative Positive 2 2 PBS PBS PBS PBS PBS PBS PBS D PBS Negative Positive 1 1 PBS PBS PBS PBS PBS PBS PBS E PBS Negative Positive 0.5 0.5 PBS PBS PBS PBS PBS PBS PBS F PBS Negative Positive 0.25 0.25 PBS PBS PBS PBS PBS PBS PBS G PBS Negative Positive 0.125 0.125 PBS PBS PBS PBS PBS PBS PBS H PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

[0068] Note: Drug concentration is in μg / ml

[0069] Select 5 well-growing clinical strains of Mycobacterium kansasii (No. 1, 2, and 3 are rifampicin-sensitive strains, and No. 4 and 5 are rifampicin-resistant strains) and colonies of the standard strain ATCC12478, prepare a bacterial suspension concentration of 0.5 McFarland units, and then dilute it 100 times. Pipette 100 μl of bacterial solution into the aforementioned 96-well plate to ensure that the bacterial inoculation concentration is about 5×10^5 CFU / mL, add 100 ul of bacterial solution to the negative control well, and add 100 ul of culture medium to the negative control well. Seal the plate with a sealing film and incubate.

[0070] 2.3 Adding color indicator

[0071] The 96-well culture plate was taken out, and resazurin indicator was added to each well, and the culture was continued to be incubated.

[0072] 2.4 Record the color of each well and determine the MIC

[0073] After the incubation, the 96-well culture plate was removed, and the color changes of each well in the 96-well microplate were observed and recorded (blue indicates no bacterial growth, while pink indicates bacterial growth), and the MIC (minimum inhibitory concentration is defined as the lowest drug concentration that inhibits visible bacterial growth) was determined. The MIC value is defined as the lowest drug concentration at which the color of the culture plate changes from blue to pink after adding the resazurin indicator 24-48 hours after the 96-well plate was cultured in a 37°C constant temperature incubator for 10 days, and the change to pink indicates that the bacteria have begun to grow.

[0074] 3. Experimental results

[0075] The MIC of vorapaxar against the standard strain of Mycobacterium kansasii ATCC12478 was 1.23 μg / ml, and the MIC against two clinical rifampicin-resistant strains was 2 μg / ml.

[0076] Table 3 Minimum inhibitory concentration of vorapaxar against clinical strains of Mycobacterium kansasii

[0077]

[0078]

[0079] The experimental results show that Vorapaxar has a good inhibitory effect on Mycobacterium kansasii in vitro and is worthy of further research.

[0080] Example 2 Cytotoxicity Experiment

[0081] 1. Experimental Methods

[0082] The cytotoxicity of Vorapaxar was evaluated against human non-small cell lung cancer cells (A549) and a human monocytic leukemia cell line (THP-1):

[0083] (1) Dilute the A549 cell content to 2.5×10^5 cells / ml, pipette 100 μl into a 96-well plate, and culture until it adheres to the wall and grows;

[0084] (2) Dilute THP1 cells to a density of 5 × 10^5 cells / ml and add 50 nM sodium dodecyl sulfate (PMA). Take 100 μl and transfer to a 96-well plate for culture to induce differentiation into macrophages and adhere to the wall for growth.

[0085] (3) After the two cell lines were completely attached, the culture medium was replaced with DMEM or RPMI medium containing vorapaxa (20 μM, 10 μM, 5 μM). 10 μl of CCK8 was added 24 h after the addition of the drug, and the absorbance at 450 nm was measured.

[0086] 2. Experimental results

[0087] After A549 cells were co-cultured with 5μM, 10μM, and 20μM vorapaxat for 24 hours, the cell viabilities were 100%, 107%, and 109%, respectively. There was no statistical difference between each concentration and the control group.

[0088] After THP-1 was co-cultured with 5μM and 10μM Vorapax for 24 hours, the cell viability was 99% and 94%, respectively, and there was no statistical difference in the cell viability between these two concentrations and the control group. After THP-1 was co-cultured with 20μM Vorapax for 24 hours, the cell viability was 85%, slightly lower than 99% of the control group, and there was a statistical difference between the two groups (P=0.02).

Claims

1. Use of Vorapaxar in preparing a product for inhibiting Mycobacterium kansasii, wherein the product contains Vorapaxar, and the chemical formula of Vorapaxar is as follows:

2. The product for inhibiting Mycobacterium kansasii as claimed in claim 1 is an experimental reagent or an antibacterial agent.

3. The antibacterial agent as claimed in claim 1 can be a cleaning agent, comprising using Vorapaxar and its pharmaceutically acceptable substances to inhibit / eliminate Mycobacterium tuberculosis.

4. Use of Vorapaxar in the preparation of a drug for preventing and / or treating a disease caused by Mycobacterium kansasii infection, wherein the drug contains Vorapaxar, and the chemical formula of Vorapaxar is as follows:

5. The Mycobacterium kansasii according to claim 1 or 4 comprises a clinical isolate of Mycobacterium kansasii, a standard strain of Mycobacterium kansasii or Mycobacterium kansasii carried by a Mycobacterium kansasii infected person.

6. The use as claimed in claim 4, wherein one or more pharmaceutically acceptable carriers or excipients may be added to the medicine.

7. The use according to claim 6, wherein 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.

8. The use as claimed in claim 4, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, transdermal preparations, suppositories and / or lyophilized powder injections.

9. The use according to claim 8, wherein 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.

10. The use according to claim 4, wherein the drug can be administered by injection, cavity administration, respiratory tract administration or mucosal administration.

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