KR102132712B1 - 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 the proliferation of tuberculosis or non-tuberculous antibacterial and preventing or treating infectious diseases. Specifically, the present invention is a pharmaceutical composition for inhibiting or preventing the proliferation of tuberculosis or non-tuberculosis antimicrobial bacteria, which contains pipithrin-α as an active ingredient among p53 inhibitors. It is about.

Description

Pharmaceutical composition for prevention or treatment of Mycobacterium Tuberculosis or nontuberculous Mycobacterium infection

The present invention relates to a pharmaceutical composition capable of inhibiting the proliferation of tuberculosis or non-tuberculous antibacterial and preventing or treating infectious diseases.

The Mycobacterium genus is known as opportunistic infectious bacteria, as well as species that cause serious illness to humans and animals, such as tuberculosis, right tuberculosis, and leprosy. About 72 species have been known to date, including species and saprophytic species found in the natural environment, of which 25 are known to be related to human disease. The genus Mycobacterium is also called an antibacterial because it is not easily dyed with a commonly used dyeing solution, but once stained, it is not easily discolored even when treated with alcohol or hydrochloric acid.

Among antibacterial bacteria, tuberculosis is latently infected by about one-third of the world's population, including Korea, that is, more than 200 million people, and 5-10% of them can be transferred to active tuberculosis throughout their lives. The tuberculosis bacteria are infected through the respiratory tract and then go through an active tuberculosis or long-term latent infection process after a period of incubation for a period of time depending on a person's immune status.

Mycobacterium abscessus among non-tuberculosis antibacterial bacteria, except tuberculosis, has been significantly increased in Korea and has been pointed out as a major problem in infectious diseases because it has resistance to most antibiotics. M. abscessus, like tuberculosis bacteria, is a bacterium widely present in nature, such as soil and water, unlike parasitic bacteria and tuberculosis bacteria in cells, and is the only antibacterial agent that is resistant to all currently used anti-tuberculosis drugs. have. In the past, it was recognized as an opportunistic infectious bacterium that causes disease in people with reduced immune function. Recently, it has been recognized as a true pathogen that causes serious disease in the human body regardless of whether or not the immune function is normal. As a feature, the genome size of M. abscessus ATCC19977T is about 5 Mb and, unlike other mycobacteria, does not have a characteristic insertion sequence, but has a full-length prophage of 81 kb. . Mycobacterium Absesus can be divided into a rough type and a smooth type depending on the shape of a colony growing on a solid medium. Generally, a strain of a rough type is highly pathogenic.

The primary therapeutic agents for isoniazid, rifampicin, pyrazineamide and ethambutol are primarily used for the treatment of these tuberculosis and non-tuberculosis antibacterial agents. Amikacin, kanamycin and moxifloxacin are used as secondary drugs. These anti-tuberculosis drugs have been developed for decades, and resistant strains have emerged as a major problem. Drug-resistant tuberculosis is a multi-drug resistant tuberculosis strain (MDR-TB) that is resistant to isoniazid and rifampicin, and is called a broad spectrum drug-resistant (XDR-TB) strain when it is resistant to secondary drugs.

Mycobacterium Absesus is known to be resistant to most anti-tuberculosis drugs and antibiotics, and has a low rate of cure despite treatment for a very long period with a standard treatment period of 2 years. Therefore, for the treatment of these tuberculosis and non-tuberculosis antibacterial infections, the development of new antibiotics that inhibit the growth of the bacteria itself is the primary goal, but recently, the growth of the bacteria is suppressed by regulating the signaling mechanism and immune regulation of the infected host. Research into host-directed therapy has been actively conducted.
(Patent Document 1) KR10-2016-0139074 A

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

The inventors of the present invention first confirmed the anti-antibacterial activity of pifithrin-α, known as a cell death blocker, by activating p53-dependent transcription regulation through a macrophage infection model and a mouse infection model. It came to this invention.

According to an embodiment of the present invention, among p53 inhibitors, the present invention relates to a pharmaceutical composition for inhibiting the proliferation of tuberculosis or non-tuberculosis antibacterial bacteria, which particularly includes pifithrin-α as an active ingredient.

According to another embodiment of the present invention, among p53 inhibitors, the present invention relates to a pharmaceutical composition capable of preventing or treating infectious diseases of tuberculosis or non-tuberculosis antimicrobial acid, particularly comprising pifithrin-α as an active ingredient.

In the present invention, the use of the pitrine-alpha can inhibit the proliferation of tuberculosis or non-tuberculosis antibacterial bacteria by a host-directed therapy method and further treat the infectious disease of the bacteria.

More specifically, the phythrin-alpha of the present invention has anti-antibacterial activity against Mycobacterium abscessus (BCG), Bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis (standard strain, Korean strain, multi-drug resistant bacteria). Can have

In addition, in the present invention, the pitrine-alpha is not only Mycobacterium abscessus showing a smooth type colony, but also modified to show a rough type colony or so. It shows the effect of inhibiting proliferation and treating infectious diseases in all of Mycobacterium abscessus, but more preferably, it has an excellent effect of inhibiting proliferation against Mycobacterium abscessus, which shows a particularly rough colony.

In addition, in the present invention, the tuberculosis or non-tuberculosis antibacterial infections include all clinical symptoms caused by infection of the tuberculosis bacteria or non-tuberculosis antibiotics, and the infections include lung disease, lymphadenitis, skin, soft tissue, and bone infection. And disseminated diseases.

In the present invention, "prevention" may include, without limitation, any action that blocks or suppresses or delays the symptoms of an infectious disease using the pharmaceutical composition of the present invention.

In the present invention, "treatment" may include, without limitation, any action that improves or benefits the symptoms of infection using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may further include a therapeutic agent for infectious diseases of commonly used tuberculosis and non-tuberculosis antibacterial agents. Examples of this may be at least one selected from the group consisting of isoniazid, rifampicin, pyrazineamide, ethambutol, amikacin, kanamycin and moxifloxacin, but is not limited thereto.

In the present invention, the pharmaceutical composition may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized as targeting humans.

The pharmaceutical composition of the present invention is not limited to these, but may be used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc., when administered orally, and in the case of injections, buffers, preservatives, painless Agents, solubilizers, isotonic agents, stabilizers, etc. can be used in combination, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. can be prepared in the form of injections, and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. have. Others can be formulated as solutions, suspensions, tablets, capsules, and sustained release preparations.

On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

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

In the present invention, “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, epidural, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention depends on a number of factors, including the activity, age, weight, general health, sex, diet, administration time, route of administration, release rate, drug combination and severity of the particular disease to be prevented or treated, of the specific compound used. It may vary, and the dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, dosage form, administration route and duration, but can be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions.

The present invention suppresses the proliferation of tuberculosis and non-tuberculosis antibacterial bacteria, and furthermore, can effectively prevent or treat the infectious disease.

Figure 1 (a) shows a graph confirming the cytotoxicity of phythrin-alpha for macrophages.
1 (b) and 1 (c) are each infected with Mycobacterium Absesus rough type and smooth type in large macrophages, followed by the amount of 5 uM of phytrine-alpha. It was shown as a graph by measuring the number of bacteria 3 days after treatment with.
Figure 2 (a) and Figure 2 (b), respectively, the macrophage cells Mycobacterium bovis (Mycobacterium bovis) of BCG (Bacillus Calmette-Guerin) and the tuberculosis standard strain H37Rv strain, respectively, and then infected with phypitrin-alpha It was treated with an amount of 5 uM, and after 3 days, the number of bacteria was measured and graphed.
3(a) and 3(b) are each infected with Korean tuberculosis strains Mtbk and MDR-tb strains in macrophages, respectively, and then treated with piprine-alpha in an amount of 5 uM, and the number of bacteria measured after 3 days. It is shown graphically.
4 is a schematic diagram showing the entire experimental method for the animal treatment experiments using mouse infection and pitrine-alpha of the Korean high pathogenic strain Mtbk.
FIG. 5(a) is a graph showing the number of bacteria in the lungs and spleen at 4 weeks of infection after injecting phytrine-alpha after infection of Mtbk, a Korean pathogenic strain, in mice.
Figure 5 (b) shows a picture confirming the lesions of the lungs 4 weeks after infection, after injecting pitrine-alpha after infection with the Korean high-pathogenic strain Mtbk in mice.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

1.1 Macrophage Separation and induction

Femoral bone marrow was collected from a C57BL/6 mouse using a bone marrow injection. After washing the collected bone marrow, red blood cells were removed using ammonium chloride. The separated cells were added to DMEM (10% FBS (Fetal bovine serum), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 10 ng/ml M-CSF) on a 100 mm plate for 6 days. Cultured for a while.

1.2 Mycobacteria culture

Mycobacterium Absesus, a standard tuberculosis strain (M. abscessus ATCC19977T), was pre-sold from the American Type Culture Collection, Manassas, VA (ATCC), and then 10% (7%) in 7H9 broth (Difco Laboratories, Detroit, MI) medium. vol/vol) Oleic acid-albumin-dextrose-catalase (OADC, Becton Dickinson, Spark, MD) was maintained at 37° C. and cultured for 7 days. At this time, Mycobacterium Absesus shows a smooth colony. In order to convert the bacteria into a rough form, C57BL/6J mice were infected with macrolide antibiotics. Then, a mycobacterium Absesus in the form of switching from the mouse lung to a rough form was obtained to confirm the change in the genotype through the VNTR technique. After culturing it in 7H9 broth containing OADC, it was centrifuged at 10,000 xg for 20 minutes and washed three times with PBS. The centrifuged pellet was subjected to a homogenizer for 1 minute and then passed through an 8 μm pore size filter to make a single cell. It was stored at -80°C and then dissolved immediately after infection.

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

1.3. Antibacterial Animal infection model

As a test animal, C57BL/6 (Japan SLC, Inc, Shijuoka) without specific pathogens 5 to 6 weeks of age was bred in a limited space of the BL-3 biohazard animal room in the Yonsei University Medical Research Center. Mice were provided with sterile commercial mouse food and water at random. Animal tuberculosis infection was infected by inhaling the MtbK strain with 200 to 250 tuberculosis bacteria per mouse using a Glas-Col inhalation device (Terre Haute, IN).

2. In macrophages Mycobacterium Absesus After infection Pitrine -Measurement of changes in the number of bacteria according to alpha (pifithrin-α) treatment

In order to confirm the antimicrobial ability of phypitrin-alpha against Mycobacterium Absesus, a concentration that does not affect the survival of macrophages was first determined. In this case, CCK-8 was used for the cell viability assay. There was no toxicity up to 48 hours even when the piptrin-alpha was treated up to 20 uM. However, when treated with 20 uM pipitrine-alpha for 72 hours, it was slightly cytotoxic (FIG. 1(a)). Thus, in this patent, 5 µM of pitrine-alpha was determined as a suitable concentration, and the experiment was conducted thereafter. Mycobacterium Absesus coarse and smooth were respectively infected with macrophages of multiplicity of infection (MOI)=0.5. Then, pitrine-alpha was left for 72 hours after 5 uM treatment. As a result, the growth rate of the coarse and smooth forms of Mycobacterium Absesus in macrophage cells was significantly inhibited by phythrin-alpha (FIGS. 1(b) and 1(c)).

3. In macrophages After tuberculosis infection Pitrine -Alpha treatment Bacteria Change measurement

M. bovis BCG and H37Rv standard strains were used to confirm the antimicrobial ability of phytrine-alpha against tuberculosis infection. That is, M. bovis BCG and H37Rv were infected with macrophages of multiplicity of infection (MOI)=0.5, respectively. Then, pitrine-alpha was treated with an amount of 5 uM and then left for 72 hours. As a result, phythrin-alpha significantly inhibited the growth rate of M. bovis BCG and H37Rv in macrophages (Fig. 2(a) and Fig. 2(b)).

Likewise, pitrine-alpha also showed efficacy in Korean high pathogenic MtbK, isoniazid, and rifampicin-resistant MDR strains (Figures 3(a) and 3(b)).

4. in using the mouse tuberculosis infection model in vivo Pitrine -Alpha Anti-tuberculosis ability Confirm

In order to confirm the anti-tuberculosis activity of phytrine-alpha in the mouse tuberculosis infection model, C57BL/6 mice were infected with 300 Korean pathogenic MtbK strains per mouse. At this time, the infection of tuberculosis bacteria was used in the air infection method. After infecting Mycobacterium tuberculosis, pipitrin-alpha was injected into the mouse tail vein twice a week for 3 weeks at 2.2 mg/kg (Fig. 4). Four weeks after infection, the number of bacteria was measured in the lungs and spleen of the mouse. As a result, it was confirmed that the tuberculosis bacteria were reduced in both the experimental group injected with pitrine-α in the lung and spleen. In addition, it was confirmed that the degree of histopathological and gross inflammation of the lung was significantly reduced (FIG. 5(a) and FIG. 5(b)).

Claims (10)

A pharmaceutical composition for inhibiting the proliferation of tuberculosis or non-tuberculosis antibacterial bacteria, comprising pifithrin-α as an active ingredient. delete According to claim 1,
The Mycobacterium abscessus (Mycobacterium abscessus), BCG (Bacillus Calmette-Guerin) and Mycobacterium tuberculosis (Mycobacterium tuberculosis) is one or more selected from the group consisting of tuberculosis or non-TB tuberculosis. Pharmaceutical composition for inhibition. According to claim 1,
The non-tuberculosis antibacterial bacteria is Mycobacterium abscessus, which shows a colony of a rough type or a smooth type, and is a pharmaceutical for inhibiting the growth of tuberculosis or non-tuberculous acid bacteria Ever composition. According to claim 4,
The non-tuberculosis antibiotics are Mycobacterium abscessus, which shows a rough type colony, and a pharmaceutical composition for inhibiting the proliferation of tuberculosis or non-tuberculosis antibiotics. A pharmaceutical composition for the prevention or treatment of infectious diseases of tuberculosis or non-tuberculosis antibacterial bacteria, comprising pifithrin-α as an active ingredient,
The tuberculosis or non-tuberculosis antibacterial infection is selected from the group consisting of pulmonary disease, lymphadenitis, skin, soft tissue, bone infection and disseminated disease, pharmaceutical composition for the prevention or treatment of infection of tuberculosis or non-tuberculosis antibacterial bacteria. delete The method of claim 6,
The tuberculosis or non-tuberculosis antimicrobial infection is at least one selected from the group consisting of Mycobacterium abscessus (BCG), Bacillus Calmette-Guerin (BCG), and Mycobacterium tuberculosis, an infectious disease of tuberculosis or non-tuberculosis antibiotics Pharmaceutical composition for the prevention or treatment of. delete delete

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