KR102107659B1 - Composition for preventing or treating Mycobacterium tuberculosis or nontuberculous mycobacteria infection comprising MMAGNU1 compound - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating Mycobacterium tuberculosis or nontuberculous mycobacteria (NTM) infectious diseases comprising a MMAGNU1 compound as an effective component. Particularly, it has been confirmed that the composition of the present invention effectively inhibits Mycobacterium tuberculosis H37Rv, which is tubercular bacillus, from growing in vitro, and also inhibits Mycobacterium abscessus, which is NTM, from growing in vitro and intracellularly, so that the composition can be useful in a pharmaceutical composition and health functional food for preventing, ameliorating and treating Mycobacterium tuberculosis or NTM infectious diseases.

Description

Composition for preventing or treating Mycobacterium tuberculosis or nontuberculous mycobacteria infection comprising MMAGNU1 compound as an active ingredient MMAGNU1 compound}

The present invention relates to a composition for preventing, ameliorating or treating an infectious disease containing Mycobacterium tuberculosis or Nontuberculous mycobacteria (NTM) containing the MMAGNU1 compound as an active ingredient.

Mycobacterium tuberculosis bacteria develop after a period of incubation for a period of time after infection, or develop acutely without an incubation period, causing complications such as inflammation of the lungs and asthma, resulting in the death of the infected person. In the case of a simple carrier of tuberculosis bacteria, there is no subjective symptoms, so tuberculosis can be easily transmitted to others, and thus there is a great difficulty in preventing and treating tuberculosis. Currently, chemotherapy (an anti-tuberculosis drug) is a treatment for tuberculosis. The primary anti-tuberculosis drug for initial tuberculosis treatment is a very effective treatment method for tuberculosis that shows a treatment rate of tuberculosis of about 85% with the combination therapy of drugs (isoniazid, rifampin, pyrazinamide, and ethambutol). , It causes significant side effects, is not only expensive, but also has the problem that its efficacy is opaque to multi-drug resistant tuberculosis.

Non-tuberculous antibacterial bacteria have been known as non-pathogenic bacteria commonly found in the natural environment. However, its importance began to emerge as it was identified as an opportunistic infectious bacterium in patients with AIDS, and recently it has been known that infection can occur in patients with normal immune function. According to the national survey, nontuberculous antibacterial activity has been increasing continuously since the 1990s. Tuberculosis and non-tuberculosis are common symptoms of fatigue, cough, sputum, chest pain, etc., which is not a unique symptom seen only in patients with tuberculosis and non-tuberculosis. Moreover, since non-TB antibacterial agents show high resistance to existing anti-tuberculosis drugs, they should be treated with long-term drug combination therapy. Therefore, there is a need for an effective therapeutic agent for a non-TB anti-bacterial infection disease that continues to increase.

Korean Registered Patent No. 10-0742035 (Registration on July 16, 2007)

Accordingly, the present invention has an object to provide a composition for preventing, improving, or treating a tuberculosis bacterium or non-tuberculosis anti-bacterial infection disease containing the MMAGNU1 compound as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis or non-tuberculosis anti-bacterial infection disease containing the MMAGNU1 compound and a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving diseases of tuberculosis or non-tuberculosis anti-bacterial infections containing the MMAGNU1 compound and pharmaceutically acceptable salts thereof as an active ingredient.

The present invention relates to a composition for preventing, improving or treating an infectious disease of Mycobacterium tuberculosis or nontuberculous mycobacteria (NTM) containing the MMAGNU1 compound as an active ingredient, and in particular, Mycobacterium tuberculosis , Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv was effectively inhibited from growth in vitro, and Mycobacterium abscessus , a non-tuberculosis antibacterial, was tested in vitro and intracellularly. It can be used in pharmaceutical compositions and health functional foods useful for the prevention, improvement and treatment of tuberculosis and non-tuberculosis antimicrobial infections by confirming growth inhibition in intracellular).

1 shows the chemical structure of MMAGNU1.
Figure 2 shows the results of in vitro activity of fluorescence based MMAGNU1 against Mycobacterium tuberculosis H37Rv.
Figure 3 shows the results of in vitro activity based on the fluorescence of MMAGNU1 against M. abscessus .
Figure 4 shows the cytotoxicity results of MMAGNU1 on mouse Raw 264.7 macrophages, human colon cancer cells (HCT116) and embryonic kidney cells (HEK 293). Data were expressed as mean ± sd by repeating 3 times.
Figure 5 shows the result of quantifying the intracellular M. abscessus 3 days after MMAGNU1 treatment based on luminescence.

The present invention provides a pharmaceutical composition for the prevention or treatment of tuberculosis bacteria or non-tuberculosis anti-bacterial infection disease containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Preferably, the non-tuberculosis antibiotics may be Mycobacterium abscessus , but is not limited thereto.

Preferably, the Mycobacterium tuberculosis or non-TB antimicrobial infection disease includes all clinical symptoms caused by the infection of Mycobacterium tuberculosis or non-TB tuberculosis, specifically, tuberculosis, lung disease, lymphadenitis, skin, soft tissue, bone infection, or It may be a disseminated disease, but is not limited thereto.

The pharmaceutically acceptable salt is the pharmaceutically acceptable salt is hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartrate, maleate, gluconate, succinate, formate, trifluor Loacetate, oxalate, fumarate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, camphorsulfonate, sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt, It is not limited to this.

In the present specification, the compound represented by Chemical Formula 1 was named MMAGNU1.

When the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition may be prepared by using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and as an adjuvant, disintegrant, sweetener, binder, coating agent , Solubilizers such as expanders, lubricants, lubricants or flavoring agents. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include, as sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

Pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of active compounds, etc. You can. The pharmaceutical composition of the present invention is a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of diseases. Thus, the type of disease, the severity of the disease, the type and content of active and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health status, gender and diet, time of administration, route of administration and composition It can be adjusted according to various factors, including the rate of secretion, the duration of treatment, and drugs used simultaneously. Without being limited thereto, for example, in the case of adults, once to several times a day, the composition of the present invention is administered once to several times a day, in the case of a compound, 0.1 ng / kg to 10 g / kg dose You can.

In addition, the present invention provides a health functional food composition for preventing or improving a tuberculosis bacterium or non-tuberculosis antibacterial infection disease containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Preferably, the non-tuberculosis antibiotics may be Mycobacterium abscessus , but is not limited thereto.

Preferably, the Mycobacterium tuberculosis or non-TB antimicrobial infection disease includes all clinical symptoms caused by the infection of Mycobacterium tuberculosis or non-TB tuberculosis, specifically, tuberculosis, lung disease, lymphadenitis, skin, soft tissue, bone infection, or It may be a disseminated disease, but is not limited thereto.

When the composition of the present invention is a health functional food composition, the health functional food composition may be provided in the form of a powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is other food or food additives other than the active ingredient It can be used in conjunction with, and can be suitably used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to its purpose of use, for example, prophylactic, health or therapeutic treatment.

The effective dose of the active ingredient contained in the dietary supplement composition can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for health and hygiene purposes or for health control purposes, the effective dose is below the above range. It can be, it is clear that the active ingredient can be used in an amount above the range because there is no problem in terms of safety.

There are no particular restrictions on the type of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Experimental Example>

The following experimental examples are intended to provide an experimental example commonly applied to each embodiment according to the present invention.

1. Strains and culture

M. abscessus ATCC 19977 was used as a wild type strain. Clinical strains were obtained from Korea Mycobacterium Resource Center (KMRC). M. abscessus AMK-R, CFX-R and CLA-R mutations were produced in previous studies (Antimicrob Agents Chemother 61: e02752-16, 2017). M. abscessus strains include Middlebrook 7H9 broth with 10% albumin-dextrose-catalase (ADC) and Middlebrook 7H10 plates with 10% oleic acid-ADC (ODC). Incubated at 37 ° C. MMAGNU1 was purchased from Sigma-Aldrich. Mycobacterium tuberculosis H37Rv was used as Mycobacterium tuberculosis .

2. REMA  Used MIC  Measure

MICs of MMAGNU1 were measured using a resazurin microtiter assay (REMA). Briefly, 100 μl of 7H9 medium with 10% ADC added was added to each well of a 96-well microtiter plate, and antibiotics diluted twice in a row were added directly to each well. The plate was covered and incubated for 3 days at 37 ° C. Resazurin (0.025% [wt / vol]) was added to each well and the plates were incubated overnight. Fluorescence was measured using a SpectraMax ^® M3 Multi-Mode Microplate Reader (Molecular Devices, CA, USA) (ex. 560 / em. 590 nm). MIC values were calculated using Prism 6 (GraphPad Software, Inc., La Jolla, CA).

3. Cell Viability  analysis

RAW 264.7 murine macrophages (KCLB No. 40071), THP-1 human monocytic cell line (KCLB 40202), HEK293 human embryonic kidney cells (KCLB No. 21573) and HCT116 human colon cancer cell line (KCLB No. 10247) are Korea Cell Line Bank (KCLB). The cells were cultured in 5% CO ₂ -wet atmospheric conditions, 37 ° C. in Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS and antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin) added. THP-1 monocytes (KCLB No. 40202) were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate solution and 2 mM GlutaGro supplement (Welgen). Cell viability was tested for human cell lines HEK293 and HCT116 using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) (Sigma M2128) viability assay. Briefly, cells were distributed in 96-well plates (4.0 × 10 ⁴ cells / well) and cultured at 37 ° C. for 24 hours. Thereafter, cells were treated with various concentrations of MMAGNU1. After 24 hours of further incubation, 100 μL MTT (40 μg / mL in PBS) was added to each well, and culture was continued for 2 hours. Then, the medium was removed and 100 μl DMSO was added. The final color was analyzed at 570 nm using a SpectraMax ^® M3 Multi-Mode Microplate Reader (Molecular Devices, CA, USA).

4. Macrophage analysis

Intracellular activity for the drug (intracellular activity) was performed in RAW 264.7 cells (KCLB No. 40071). RAW 264.7 cells were maintained in DMEM with 10% heat-inactivated FBS. Cells were seeded in 96-well white plates at a concentration of 5 x 10 ⁴ cells per well and allowed to attach overnight. For infection, log phase M. abcessuess -LuxG13 was washed twice with 7H9 + ADS + 0.05% Tween 80. Bacterial aggregation was destroyed 20-30 times through a 26G 1 ml syringe, and the bacterial suspension was sonicated twice for 30 seconds in a bath sonicator (BRANSON 3800). Thereafter, Raw 264.7 cells were infected with a bacterial suspension at a ratio of 1 MOI 1: 1 per cell and incubated for 2 hours. After 2 hours of infection at 37 ° C. with 5% CO ₂ conditions, macrophages were treated with 50 μg / mL gentamicin for 30 minutes and washed three times with DMEM to remove extracellular bacteria. Finally, 200 μL DMEM complete medium was added to each well with or without clarithromycin and MMAGNU1. Each drug concentration was tested in triplicate. Data were expressed as percentages (e.g., RLUs of drug treated infected cells / RLUs of untreated drug treated cells x100) and plotted according to drug concentration.

5. MMAGNU1  Preparation of bone marrow-derived macrophages to measure intracellular activity

C57BL / 6 mouse is KOATECH (Gyeonggi-do, Korea), bone marrow-derived macrophages (BMDMs) were isolated from 7-week-old mice, and macrophage colony promoting factor (M-CSF; JW CreaGene JW-M001-0250) It was differentiated in a medium containing 0.5% CO ₂ incubator at 37 ° C. for 4 days. Thereafter, MOI 1:10 bacteria were added diluted in DMEM containing 10% FBS. After 2 days, intracellular bacteria were extracted and lysate was diluted 10-fold with PBS. Each diluted bacterium was inoculated on 7H10 agar plates containing 50 μg / ml kanamycin, and cultured at 37 ° C. for 3 days in a 0.5% CO ₂ incubator.

< Example >

MMAGNU1 the growth of the M. tuberculosis H37Rv and M. abscessus - in order to make sure that indicates the inhibitory effect, the inventors have carried out the drug sensitivity test for M. tuberculosis H37Rv and M. abscessus ATCC 19977 by the resazurin microtiter assay. Drug sensitivity tests for M. tuberculosis H37Rv and M. abscessus were performed in 7H9 broth medium.

As shown in Figure 2, MMAGNU1 significantly reduced the resazurin fluorescence for M. tuberculosis H37Rv in a concentration-dependent manner.

As shown in Figure 3, MMAGNU1 significantly reduced the resazurin fluorescence for M. abscessus in a concentration-dependent manner. MMAGNU1 showed a low MIC value of 1.0 μM in 7H9 medium, showing a strong inhibitory effect.

Next, the present inventors confirmed the antibacterial activity of MMAGNU1 against M. abscessus in macrophages. Previously, the cytotoxic effect of MMAGNU1 was tested by MTT analysis on HCT116 (human colorectal cancer cells), Raw 264.7 and HEK293 (human embryonic kidney cells). As shown in Figure 4, MMAGNU1 treatment showed 90% survival in all treated cells. Like Mycobacterium tuberculosis , M. abscessus is infected and multiplied inside human macrophages, indicating its ability to evade antibacterial reactions in macrophages. Therefore, in order to inhibit the intracellular growth of M. abscessus , it is important to confirm the activity of MMAGNU1. The present inventors quantified intracellular bacterial growth after treatment with MMAGNU1 using a luminescent M. abscessus - lux strain. Bacterial growth in cells was measured by M. abscessus strain with bacterial lux operon using pMV306hsp + LuxG13 in RAW 264.7 mouse macrophages. For quantitation of M. abscessus - lux in RAW 264.7 cells, the optimal time point for growth measurement was 3 days after infection, at which time M. abscessus growth in cells was homogenized, and macrophage death was hardly observed. As shown in Fig. 5, after 3 days of exposure, MMAGNU1 significantly reduced the luminescence expressed from M. abscessus - lux in cells. This was comparable to the decrease in luminescence observed after clarithromycin treatment.

Since the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that this specific technique is only a preferred embodiment, whereby the scope of the present invention is not limited. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

A pharmaceutical composition for the prevention or treatment of a tuberculosis bacterium or non-tuberculosis anti-bacterial infection disease containing a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

According to claim 1, wherein the non-tuberculosis antibiotics Mycobacterium abscessus ( Mycobacterium abscessus ) characterized in that the Mycobacterium tuberculosis or non-TB antimicrobial infection disease prevention or treatment pharmaceutical composition. According to claim 1, wherein the Mycobacterium tuberculosis or non-tuberculosis anti-bacterial infection disease is tuberculosis, lung disease, lymphadenitis, skin, soft tissue, bone infection or disseminated disease, characterized in that the tuberculosis or non-tuberculous antibacterial infection disease prevention or treatment Pharmaceutical composition. A health functional food composition for preventing or improving a tuberculosis bacterium or non-tuberculosis anti-bacterial infection disease containing a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

The method of claim 4, wherein the non-tuberculosis antibacterial bacteria Mycobacterium abscessus ( Mycobacterium abscessus ) Mycobacterium tuberculosis or non-tuberculosis antibacterial infection health functional food composition for preventing or improving disease. According to claim 4, wherein the Mycobacterium tuberculosis or non-tuberculosis antibacterial infection disease is tuberculosis, lung disease, lymphadenitis, skin, soft tissue, bone infection or disseminated disease, characterized in that the tuberculosis or non-tuberculous antibacterial infection disease prevention or improvement Health functional food composition.

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