KR20180130340A - Pharmaceutical composition for prevention or treatment of Mycobacterium Tuberculosis or nontuberculous Mycobacterium infection - Google Patents

Abstract

The present invention relates to a pharmaceutical composition capable of inhibiting proliferation of tuberculosis or non-tuberculosis mycobacteria and preventing or treating infectious disease. Specifically, the present invention relates to a pharmaceutical composition for inhibiting the proliferation of tuberculosis or non-tuberculosis mycobacteria or preventing or treating infectious disease thereof containing pifithrin-α as an active ingredient among p53 inhibitors.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating tuberculosis or nontuberculous Mycobacterium infection,

The present invention relates to a pharmaceutical composition capable of inhibiting the proliferation of tuberculosis or nontuberculous autoantibacteria and preventing or treating infectious diseases.

The Mycobacterium genus includes not only species that cause serious diseases in humans and animals, such as tuberculosis, bovine tuberculosis, and leprosy, but also microorganisms called opportunistic infections Species, and saprophytic species, which can be found in natural environments. To date, about 72 species have been known, of which 25 are known to be related to human diseases. Such a Mycobacterium genus is not easily stained with a commonly used stain solution but once it is stained, it is also called an auto acid bacteria because it is not easily discolored even when it is treated with alcohol or hydrochloric acid.

Among mycobacterial strains, tuberculosis is infectious to about one third of the world's population, including Korea, about 200 million, and 5 to 10% of them can be converted into active TB for a lifetime. After being infected through the respiratory tract, the tubercle bacillus enters the incubation period for a certain period of time depending on the immune status of the person, and then undergoes active tuberculosis or long-term latent infection process.

Mycobacterium abscessus, a non - tuberculous mycobacteria other than tuberculosis, has been reported to be a major problem in infectious diseases due to the increased incidence of infection in Korea and its resistance to most antibiotics. M. abscessus, like M. tuberculosis, differs from intracellular parasitic bacteria or M. tuberculosis in that it is widely found in soil such as soil and water. It is the only antibiotic resistant to all currently used antimicrotubule agents have. In the past, it was recognized as an opportunistic infectious disease causing illness in people with impaired immune function. However, recently, it has been recognized as a true pathogen that causes serious diseases to human body regardless of whether immune function is normal or not. As a feature, the genome size of M. abscessus ATCC19977T is about 5 Mb, and unlike other mycobacteria, it does not have a characteristic insertion sequence but has a full-length prophage of 81 kb in length . Mycobacterium infosus can be classified into a rough type and a smooth type depending on the shape of the colonies growing on the solid medium. Generally, coarse type strains are highly pathogenic.

Treatment of tuberculosis and nontuberculous mycobacteria is basically the first-line treatment of isoniazid, rifampicin, pyrazinamide, and ethambutol. Amikacin, kanamycin and moxifloxacin are used as secondary drugs. These anti-tuberculosis drugs have been developed for decades, and resistant strains have become a big problem. Drug-resistant tuberculosis (MDR-TB) is resistant to isoniazid and rifampicin. Drug-resistant tuberculosis is referred to as a broad-spectrum drug resistance (XDR-TB) strain when resistant to secondary drugs.

Mycobacterium abscessus is known to be resistant to most anti-tuberculosis agents and antibiotics, and the standard treatment period is 2 years, which means that the cure rate is low despite treatment for a very long period of time. Therefore, for the treatment of tuberculosis and nontuberculous mycobacterial infections, development of a new antibiotic to inhibit the growth of the microorganism is the primary goal. Recently, the microorganism suppresses the growth of the microorganism by controlling the signal transduction mechanism and immune regulation of the infected host. Host-directed therapies have been actively studied.

The present invention is intended to provide a pharmaceutical composition capable of effectively preventing or treating the infectious disease by inhibiting the proliferation of tuberculosis or non-tuberculous aerobic bacteria.

The inventors of the present invention firstly confirmed the ability of pifithrin-alpha to inhibit anti-acid bacteria by activating p53-dependent transcriptional regulation and cell death blocker through a large predation cell infection model and a mouse infection model Leading to the present invention.

According to one embodiment of the present invention, a pharmaceutical composition for inhibiting the proliferation of tuberculosis or non-tuberculous acid-fast bacteria containing, as an effective ingredient, pifithrin-alpha among p53 inhibitors.

According to another embodiment of the present invention, there is provided a pharmaceutical composition capable of preventing or treating infectious diseases of tuberculosis or nontuberculous autoantibacteria including pifithrin-alpha as an active ingredient, among p53 inhibitors.

In the present invention, the use of the above-mentioned pipitin-alpha inhibits the proliferation of tuberculosis or non-tuberculous mycobacteria by host-directed therapy, and furthermore, the infectious disease of the above-mentioned bacteria can be treated.

More specifically, the peptitrin-alpha of the present invention has antitumor antibiotic resistance against Mycobacterium abscessus, BCG (Bacillus Calmette-Guerin) and Mycobacterium tuberculosis (standard strain, Korean strain, multidrug-resistant bacteria) Lt; / RTI >

In addition, in the present invention, the peptitrin-alpha can be used not only for Mycobacterium abscessus showing a smooth type of colonies but also for displaying a rough type colony, Mycobacterium abscessus has the effect of inhibiting proliferation and infectious diseases, but more preferably, it has an excellent proliferation inhibitory effect against Mycobacterium abscessus showing particularly rough type colonies.

In addition, in the present invention, the tuberculosis or non-tuberculous mycobacterial infection includes all the clinical symptoms caused by infection of the Mycobacterium tuberculosis or non-tuberculous mycobacterium, and the infectious disease includes lung disease, lymphadenitis, skin, soft tissue, Disseminated disease, and the like.

In the present invention, "prevention" may include, without limitation, any action that uses the pharmaceutical composition of the present invention to block the symptoms of an infectious disease, or to inhibit or delay the symptoms of an infectious disease.

In the present invention, "treatment" may include, without limitation, any action that improves or alleviates symptoms of an infectious disease using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may further comprise a commonly used therapeutic agent for infectious diseases of tuberculosis and non-tuberculous aerobic bacteria. Examples thereof include, but are not limited to, at least one selected from the group consisting of isoniazid, rifampicin, pyrazinamide, ethambutol, amikacin, kanamycin and moxifloxacin.

In the present invention, the pharmaceutical composition may be in the form of a capsule, a tablet, a granule, an injection, an ointment, a powder or a drink, and the pharmaceutical composition may be a human.

The pharmaceutical composition of the present invention may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories and sterilized injection solutions according to a conventional method, . The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other, solutions, suspensions, tablets, capsules, sustained-release preparations and the like.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The route of administration of the pharmaceutical composition according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, , Sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention may be administered orally or parenterally depending on various factors including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the mode of administration, the route of administration and the period of time, and is preferably from 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical compositions according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

The present invention can inhibit the proliferation of tuberculosis and non-tuberculous acid-fast bacteria and further effectively prevent or treat the infection.

FIG. 1 (a) is a graph showing cytotoxicity of pipitrin-alpha to large predominant cells.
Fig. 1 (b) and Fig. 1 (c) show the results obtained by infecting macrophages with rough and smooth types of microbacterium, respectively, And the number of bacteria was measured after 3 days.
Fig. 2 (a) and Fig. 2 (b) respectively show that BCG (Bacillus Calmette-Guerin) of Mycobacterium bovis and H37Rv strain, a standard strain of Mycobacterium tuberculosis, Was treated with an amount of 5 uM and the number of bacteria was measured after 3 days.
Fig. 3 (a) and Fig. 3 (b) show the results obtained by infecting large predominant cells with Mtbk and MDR-tb strains of Korean tubercle bacilli, respectively, followed by treatment with 5 μM of phytatein- Respectively.
FIG. 4 is a schematic view showing a whole experimental method for an animal treatment experiment using mouse infection and phytolin-alpha of Mtbk, a Korean highly pathogenic strain.
FIG. 5 (a) is a graph showing the number of bacteria in the lungs and spleen after 4 weeks of infecting the mouse with Mtbk, a highly pathogenic Korean strain, and injecting with Pipitorin-Alpha.
FIG. 5 (b) is a photograph showing the lesion of the lung at the 4th week after infecting the mouse with the high-pathogenic strain Mtbk and injecting with phyptrelin-alpha.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

1.1 Of large predominant cells  Separation and induction

Femur bone marrow was collected from C57BL / 6 mice using injection for bone marrow harvesting. The collected bone marrow was washed and the red blood cells were removed with ammonium chloride. Separated cells were cultured in DMEM (Fetal bovine serum, 2 mM L-glutamine, 100 U / ml penicillin / streptomycin, 10 ng / ml M-CSF) Lt; / RTI >

1.2 Myco bacteria  culture

(M. abscessus ATCC19977T) was purchased from the American Type Culture Collection, Manassas, Va., And cultured in 7H9 broth (Difco Laboratories, Detroit, Mich.) Medium supplemented with 10% vol / vol) oleic acid-albumin-dextrose-catalase (OADC, Becton Dickinson, Spark, MD) for 7 days. At this time, M. corbiculata abcissus shows a smooth type of colonies. To transform the strain into a rough form, C57BL / 6J mice were infected and treated with a macrolide antibiotic. Subsequently, Maoobacterium infosus was obtained from the mouse lungs in a coarse form, and the change of the genotype was confirmed through the VNTR technique. The cells were cultured in 7H9 broth containing OADC, centrifuged at 10,000 xg for 20 minutes, and washed three times with PBS. The centrifuged pellet was incubated in a homogenizer for 1 minute and then passed through an 8 μm pore size filter to form a single cell. It was stored at -80 ° C and dissolved immediately after infection.

On the other hand, M. bovis BCG, H37Rv, MtbK and MDR-tb strains were cultured in 7H9-OADC broth for 15 days after being distributed from US ATCC and Korea International Tuberculosis Research Institute. The cultured strains were then harvested and disrupted by vortexing with 6 mm glass beads. After the cell aggregates were submerged, the supernatant was collected and stored at -70 ° C. After thawing, live Mycobacterium tuberculosis was counted by gradual dilution plate culture in Middlebrook 7H11 agar (Difco, Detroit, MI, USA). To inoculate the mice with the Mycobacterium tuberculosis bacteria, the above-described Mycobacterium bacterium suspension was sonicated lightly in a sonic bath and diluted with PBS (phosphate buffered saline) at pH 7.2 to obtain the desired number of bacteria.

1.3. Antibiotic acid  Animal Infection Model

C57BL / 6 (Japan SLC, Inc, Shijuoka), which had no specific pathogen for 5 to 6 weeks after birth, was housed in the restricted space of the BL-3 biohazard animal room in Yonsei University Medical Research Center. Mice were randomly given sterile, commercial mouse food and water. Animal tuberculosis infection was induced by inhalation of 200 to 250 Mycobacterium tuberculosis bacteria per mouse using a Glas-Col inhalation device (Terre Haute, IN).

2. In large predominant cells  Mycobacterium Abseusu  After infection Pipitrin - Measurement of bacterial changes by alpha (pifithrin-α) treatment

To confirm the antimicrobial activity of pepticline-alpha against M. bovis, first we determined the concentrations that had no effect on the survival of large predominant cells. At this time, CCK-8 was used for cell viability assay. Pipitrine-Alpha up to 20 uM was not toxic until 48 hours. However, treatment with 20 uM pipitrin-alpha for 72 hours was slightly cytotoxic (Fig. 1 (a)). Thus, in this patent, a suitable concentration of 5 μM of pipitrin-alpha was determined and then the experiment was conducted. Maocobacterium adepisus rough and smooth molds were infected with large proliferating cells at MOI (multiplicity of infection) = 0.5, respectively. Then, the pipitrin-alpha was left for 72 hours after 5 uM treatment. As a result, the pepticline-alpha significantly inhibited the growth rate of the Maccobacterium adenoside rough type and the smooth type in the large phagocytic cells (Fig. 1 (b) and Fig. 1 (c)).

3. In large predominant cells  After TB infection Pipitrin - Alpha processing Number of bacteria  Change measurement

M. bovis BCG and H37Rv standard strains were used to confirm the antimicrobial activity of pepticline-alpha against M. tuberculosis infection. In other words, M. bovis BCG and H37Rv were infected with large proliferating cells with MOI (multiplicity of infection) = 0.5, respectively. Then, the pipitrin-alpha was treated in an amount of 5 uM and left for 72 hours. As a result, peptitrin-alpha significantly inhibited the growth rate of M. bovis BCG and H37Rv in large proliferating cells (Fig. 2 (a) and Fig. 2 (b)).

Likewise, phyptrelin-alpha was also effective in Korean high-pathogenic MtbK and MDR strains resistant to isoniazid and rifampicin (Fig. 3 (a) and Fig. 3 (b)).

4. In vitro modeling of tuberculosis infection in mice in vivo Pipitrin - Alpha Antituberculous ability  Confirm

In order to confirm the antitumor activity of peptitrin - alpha in the mouse tuberculosis infection model, 300 Korean high - pathogenic MtbK strains were infected with C57BL / 6 mice per mouse. At this time, the infection of the M. tuberculosis using the air infectious method. After infecting the Mycobacterium tuberculosis, Pifitrin-Alpha was injected into the mouse tail vein twice weekly for 3 weeks at 2.2 mg / kg (FIG. 4). Bacterial counts were measured in the lungs and spleen of mice at 4 weeks after infection. As a result, it was confirmed that the tuberculosis bacillus was reduced in the experimental group injected with the phyptyricin-α in the lung and spleen. In addition, it was confirmed that the degree of histopathological and macroscopic inflammation of the lung was greatly reduced (Figs. 5 (a) and 5 (b)).

Claims (10)

A pharmaceutical composition for inhibiting the proliferation of tuberculosis or non-tuberculous mycobacteria comprising a p53 inhibitor as an active ingredient. The method according to claim 1,
Wherein the p53 inhibitor is pifithrin-alpha, a composition for inhibiting the proliferation of tuberculosis or non-tuberculous mycobacteria. The method according to claim 1,
Wherein the tuberculosis or non-tuberculous mycobacteria are at least one selected from the group consisting of Mycobacterium abscessus, BCG (Bacillus Calmette-Guerin) and Mycobacterium tuberculosis, Lt; / RTI > The method according to claim 1,
The non-tuberculous mycobacteria are Mycobacterium abscessus, which shows a rough type or a smooth type of colony, and a pharmaceutical composition for inhibiting proliferation of tuberculosis or non-tuberculous mycobacteria Gt; 5. The method of claim 4,
The pharmaceutical composition for inhibiting proliferation of tuberculosis or non-tuberculous mycobacteria, wherein the non-tuberculous mycobacteria are Mycobacterium abscessus showing a rough type of colonies. A pharmaceutical composition for the prophylaxis or treatment of an infectious disease of tuberculosis or nontuberculous autoantibacteria comprising a p53 inhibitor as an active ingredient. The method according to claim 6,
Wherein said p53 inhibitor is pifithrin-alpha, a pharmaceutical composition for the prophylaxis or treatment of infectious diseases of tuberculosis or non-tuberculous aerobic bacteria. The method according to claim 6,
Wherein the tuberculosis or non-tuberculous mycobacteria are at least one selected from the group consisting of Mycobacterium abscessus, BCG (Bacillus Calmette-Guerin) and Mycobacterium tuberculosis, ≪ / RTI > The method according to claim 6,
The pharmaceutical composition for preventing or treating infectious disease of tuberculosis or non-tuberculous autoantibody is selected from the group consisting of lung disease, lymphadenitis, skin, soft tissue, bone infectious disease and disseminated disease. The method according to claim 6,
Wherein said pharmaceutical composition further comprises at least one member selected from the group consisting of isoniazid, rifampicin, pyrazinamide, ethambutol, amikacin, kanamycin and moxifloxacin for the prevention or treatment of infectious diseases of tuberculosis or non- A pharmaceutical composition.

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