KR20240079860A - Composition for Propylaxis or Treatment of Mycobacterial Infection Comprising Ginsenosides - Google Patents

Abstract

The present invention relates to a composition for preventing or treating/improving symptoms of diseases caused by non-tuberculous mycobacterial infection, and more specifically, to a composition for preventing or treating diseases caused by non-tuberculous mycobacterial infections, and more specifically, to a composition containing as an active ingredient one or more of the ginsenosides Rg6, Rh4 and Rg3. It relates to a composition for preventing or treating/improving symptoms of bacterial infectious diseases.

Description

Composition for preventing or treating mycobacterial infection containing ginsenosides {Composition for Prophylaxis or Treatment of Mycobacterial Infection Comprising Ginsenosides}

The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by non-tuberculous acid-fast bacterial infections, including Mycobacteroides abscessus .

Mycobacterium genus species are divided into two groups according to their pathogenicity: the Mycobacterium tuberculosis group, which includes the obligate pathogenic bacteria Mycobacterium tuberculosis and Mycobacterium leprosy, and the nontuberculous mycobacteria (NTM), excluding the above bacteria. In the case of tuberculosis, the cure rate is over 90% due to the development of anti-tuberculosis drugs, and the incidence and mortality rates have been continuously decreasing worldwide since the 1990s. On the other hand, non-tuberculous mycobacteria have a very high isolation rate of drug-resistant bacteria and have difficulties such as requiring long-term treatment of more than 2 years. Therefore, the incidence, prevalence, and mortality rate of infection due to non-tuberculous mycobacteria are rapidly increasing worldwide. there is.

Nontuberculous mycobacteria are widely distributed in nature, including in water and soil, and are opportunistic bacteria that infect through the respiratory tract. To date, about 150 types of non-tuberculous mycobacteria are known, and new non-tuberculous mycobacteria are discovered almost every year, but approximately 25 types are related to human diseases. There is also a large difference in the virulence that causes disease depending on the fungal species, so Mycobacterium kansasii ( M. kansasii ), Mycobacterium avium ( M. avium ) or Mycobacterium abscessus ( M. abscessus, Mabc) are relatively rare. has a high virulence, and Mycobacterium fortuitum ( M. fortuitum ) has a relatively low virulence. Diseases caused by non-tuberculous mycobacteria show four characteristic clinical symptoms: lung disease, lymphadenitis, skin, soft tissue, and bone infections, and disseminated disease. Among these, lung disease accounts for more than 90% of diseases caused by non-tuberculous mycobacteria. occupy

Non-tuberculous acid-fast bacilli are not distinguished from tuberculosis bacilli in an acid-fast bacilli smear test to detect tuberculosis bacilli. In countries where the frequency of non-tuberculous mycobacterial lung disease is relatively high, non-tuberculous mycobacteria are isolated in up to 30-50% of acid-fast bacilli smear-positive sputum, and the rate of infection due to non-tuberculous mycobacteria is reported to increase as the country becomes more advanced. It is becoming. In Korea, it is known that the frequency of non-tuberculous acid-fast bacilli disease is low, so most cases with a positive acid-fast bacillus smear are considered tuberculosis and anti-tuberculosis treatment is generally administered. However, even in Korea, more than 10% of sputum with a positive acid-fast bacillus smear contains non-tuberculosis bacteria. Mycobacterium tuberculosis is being isolated.

The most common cause of non-tuberculous mycobacterial lung disease in Korea is M. avium. am. In particular, Mabc, a fast-growing bacterium that is relatively rare in foreign countries, has the characteristic of being the second most common causative bacterium in Korea. Mabc Lung disease commonly occurs in middle-aged and older non-smoking women. Symptoms commonly include cough, fever, hemoptysis, and sputum. Mabc is particularly clinically important because it exhibits multidrug resistance and available treatments have limited efficacy. Mabc isolated from a patient who did not receive antibiotic treatment shows sensitivity to clarithromycin, amikacin, and cefoxitin in an in vitro drug susceptibility test, but is resistant to all common anti-tuberculosis drugs. Treatment of Mabc lung disease is very difficult because intravenous antibiotics must be used and a treatment period of at least 12 months is required even after successful antibacterial treatment. Patients face difficulties not only because of the symptoms of the disease, but also because of the added physical burden caused by long-term antibiotic administration. Moreover, if resistance to antibiotics develops, treatment becomes more difficult, and even with treatment including intravenous antibiotics, the cure rate is less than 10% with medical treatment alone, making it very difficult to achieve sputum cleansing, making it difficult to consider lung resection if the lesion is localized. . Mabc infection is particularly associated with immune deficiency, so the risk is further increased in patients receiving immunosuppressive treatment, elderly patients, or those with underlying pulmonary or systemic diseases. Therefore, treatment for non-tuberculous mycobacteria-related diseases, which is increasing, is required separately from the treatment for tuberculosis.

Ginseng is completely harmless to the human body even when taken over a long period of time. It is known to have various effects such as fatigue recovery, anti-aging, tonic, and anti-cancer as a medicinal herb that can regulate the body's condition and prevent aging by taking it. . Ginsenoside is the main saponin component that exhibits the pharmacological effects of ginseng, and has a unique structure and characteristics that are different from saponins found in other plants such as onion, bellflower root, and garlic. Ginsenosides are expressed in the form of RX, such as Rb1, Rg3, and Rd, depending on the distance traveled when chromatography is performed, and more than 40 types of ginsenosides have been isolated and confirmed from ginseng or processed ginseng. However, the efficacy of these ginsenosides against non-tuberculous mycobacteria has not yet been reported.

Registered Patent No. 10-1751930 Registered Patent No. 10-2365657

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating non-tuberculous mycobacterial infectious diseases that are highly resistant to anti-tuberculosis drugs and are difficult to treat, or a health functional food composition for preventing or improving symptoms.

Another object of the present invention is to provide a composition for inhibiting the proliferation or growth of non-tuberculous mycobacteria.

The present invention for achieving the above-described object relates to a pharmaceutical composition for the prevention or treatment of non-tuberculous mycobacterial infectious diseases containing one or more of ginsenosides Rg6, Rh4 and Rg3 as active ingredients.

In macrophages infected with non-tuberculous mycobacteria, ginsenosides Rg6, Rh4, or Rg3 significantly inhibited the survival rate of non-tuberculous mycobacteria, confirming their usefulness as a pharmaceutical composition for the prevention or treatment of non-tuberculous mycobacterial infection diseases. When applied directly to non-tuberculous mycobacteria, the IC ₅₀ value of Rg6, Rh4 or Rg3 was not considered to be superior to that of other ginsenoside compositions, but in non-tuberculous mycobacteria-infected macrophages, the growth of non-tuberculous mycobacteria was inhibited by other ginsenoside compositions. It was significantly inhibited compared to senosides. This is understood to be because Rg6, Rh4, or Rg3 do not act directly on non-tuberculous mycobacteria to exhibit antibacterial activity, but rather exhibit antibacterial activity through other mechanisms within the cell.

Ginsenoside is an active ingredient in ginseng or processed ginseng such as red ginseng or black ginseng, and has been taken for a long time as an alternative medicine, health functional food, or health supplement. Therefore, it can be used in different applications even without a separate toxicity test or safety test. In addition, it is known not only for its activity against non-tuberculous mycobacteria but also for its beneficial effects on the living body, so it has the advantage of obtaining useful effects including anti-cancer efficacy and antioxidant efficacy in addition to the prevention or treatment of non-tuberculous mycobacterial infection diseases.

The non-tuberculous acid-fast bacteria may be M. kansasii , M. avium , or Mabc. In particular, it may be Mabc, a fast-growing bacterium that is commonly found in infection cases in Korea and has limited efficacy with conventional drugs, and can be more useful for Mabc, which is resistant to all conventional anti-tuberculosis drugs.

In the present invention, non-tuberculous mycobacterial infection disease includes all clinical symptoms caused by infection with non-tuberculous mycobacteria, and the disease includes lung disease, lymphadenitis, skin, soft tissue, bone infection, or disseminated disease. .

The composition of the present invention, which consists of one or more of ginsenosides Rg6, Rh4, and Rg, can be used not only for therapeutic purposes after infection with non-tuberculous mycobacteria, but also for preventive purposes in groups at high risk of infection with non-tuberculous mycobacteria. there is. In other words, since the ginsenoside inhibits the proliferation and growth of non-tuberculous mycobacteria, it can also be used for preventive purposes in cases where there is a high risk of non-tuberculous mycobacterial infection, such as in patients with a family history or a persistently unstable immune system such as AIDS patients. It is natural that it exists.

Additionally, the pharmaceutical composition can be used to treat diseases not only in humans but also in other animals/plants infected by non-tuberculous mycobacteria.

The pharmaceutical composition for preventing or treating non-tuberculous mycobacterial infection diseases according to the present invention can be prepared by itself or mixed with a pharmaceutically acceptable carrier, forming agent, diluent, etc., by a method known in the pharmaceutical field. It can be manufactured and used in dosage forms such as powder, granules, tablets, capsules, or injections.

The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment. The effective dose level is determined by factors including the type of patient's disease, severity, age, gender, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. It can be determined depending on factors.

The composition of the present invention can be administered alone or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. The pharmaceutical composition of the present invention may further include, for example, an antibiotic. The antibiotic may include, but is not limited to, clarithromycin, amikacin, cefoxitin, or a combination thereof to which non-tuberculous mycobacteria are sensitive. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art. In general, the above compound can be administered to adults once or several times in an amount of 0.1 to 100 mg per 1 kg of body weight per day.

Another aspect of the present invention relates to a health functional food composition for preventing or alleviating symptoms of non-tuberculous mycobacterial infection disease, which contains one or more of ginsenosides Rg6, Rh4 and Rg3 as an active ingredient.

The effective dose of the health functional food composition of the present invention can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range. , the active ingredient can be used in amounts exceeding the above range because there is no problem in terms of safety. The health functional food of the present invention includes the form of tablets, capsules, pills, or liquid, and foods to which the composition of the present invention can be added include, for example, various foods, beverages, gum, tea, snacks, There are alcoholic beverages, chocolate, vitamin complexes, health functional foods, etc.

As described above, according to the present invention, a new application of ginsenoside, a major pharmacological ingredient of ginseng or processed ginseng, which has been used for a long time in the field of alternative medicine and health functional foods and has proven safety, is proposed, and a separate safety test is conducted over a long period of time. It can be usefully used to prevent or treat/improve symptoms of non-tuberculous mycobacterial infection without spending any money.

Figure 1 is a graph showing the direct effect of ginsenosides on the growth of non-tuberculous mycobacteria.
Figure 2 is a graph showing the effect of ginsenosides on the growth of non-tuberculous mycobacteria in macrophages infected with non-tuberculous mycobacteria.

The present invention will be described in more detail below with reference to the attached drawings and examples. However, these drawings and examples are merely examples for easily explaining the content and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed thereby. Based on these examples, it will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention.

[Example]

Example 1: Preparation of non-tuberculous mycobacteria

Mabc (ATCC 19977) was cultured at 37°C at 140 rpm using Middlebrook 7H9 broth (Difco, 271310) supplemented with 0.2% glycerol, 0.05% Tween 80 (Sigma-Aldrich), and albumin-dextrose catalase. The bacterial culture was placed in an Erlenmeyer flask containing new medium so that the initial optical density (OD600) of the medium was 0.01, and cultured at 140 rpm in a 37°C incubator to reach the logarithmic growth phase (mid-logarithmic phase, OD600=0.4~). Cultured until 0.5). After mixing 100% sterilized glycerol with the culture medium to a final concentration of 20%, 1 ml was dispensed into cryovials and stored at -80°C before use. After dissolving the vial before use, viable colony forming units (CFU) were measured in Middlebrook 7H10 medium.

Example 2: Assay for the direct effect of ginsenosides on the growth of non-tuberculous mycobacteria

Using the non-tuberculous acid-fast bacteria and ginsenosides prepared in Example 1, the effect of each ginsenoside on the growth of non-tuberculous acid-fast bacteria was tested by measuring absorbance according to bacterial growth.

Specifically, 7H9 medium was uniformly dispensed into a 96-well microplate, and then the ginsenoside composition listed in Table 1 was serially diluted with a concentration gradient in the medium using a two-fold dilution method. When the ginsenoside composition in Table 1 consists of a mixture of ginsenosides, each ginsenoside was mixed in the same weight ratio. The Mabc culture medium cultured as in Example 1 was centrifuged at 3000 rpm for 10 minutes to obtain a pellet, and after adding 0.05% Tween 80 to PBS (phosphate-buffered saline), the pellet was released with this reagent and the same conditions as above were obtained. Centrifuge. After centrifugation, release the pellet once more and centrifuge to remove the supernatant. After adding the above medium to the pellet and adding glass beads (3 mm), the bacterial culture was vortexed (3 times for 5 seconds each). Afterwards, centrifuge for 3 minutes at 250 Afterwards, Mabc was added to each well at a final OD of ₆₀₀ = 0.01, and cultured at 37°C for 3 days. As a control group, DMSO (Dimethyl sulfoxide), which was used as a solvent instead of ginsenoside, was treated. Figure 1 shows a graph of absorbance measurement results according to concentration, and the IC ₅₀ values calculated therefrom are listed in Table 1.

The results in Figure 1 overall show that low-concentration ginsenoside treatment has insufficient activity to directly inhibit the growth of non-tuberculous mycobacteria. In the case of G1 or G2, it showed a killing effect on Mabc when treated at high concentration (1mM), but the remaining ginsenoside compounds (G3~11) showed no killing effect or no change in growth even when treated at high concentration. . From these results, the growth inhibition effect of ginsenosides on Mabc is significantly higher than the IC ₅₀ of many existing drugs, and since many of them did not show a killing effect even when treated at high concentrations, it is possible that they may have a direct growth inhibition effect. This suggests that the possibility is low.

Example 3: Testing the effect of ginsenosides on the growth of non-tuberculous mycobacteria in immune cells infected with non-tuberculous mycobacteria

1) Collection and culture of mouse bone marrow-derived macrophages (BMDMs)

Macrophages were collected from the bone marrow of 8-week-old wild-type C57BL/6 mice and cultured in DMEM (Lonza) containing 10% fetal bovine serum (Lonza), penicillin (100 IU/ml), and streptomycin (100 μg/ml). Cultured in medium. Macrophage colony-stimulating factor (M-CSF, R&D Systems) was added to the medium at a concentration of 25 ng/ml, and cultured and differentiated for 3 to 5 days at 37°C, 5% CO ₂ , and humidified conditions. .

2) Treatment of non-tuberculous mycobacterial infection and ginsenosides

After treating the non-tuberculous acid-fast bacterium Mabc in the mouse BMDMs medium prepared in 1) for 2 hours so that the average number of infected bacteria per cultured cell (multiplicity of infection, MOI) was 3, bacteria existing outside the cells were treated. To remove it, it was washed twice with PBS. Afterwards, the new medium was treated with gentamycin to kill external bacteria, and ginsenosides from Table 1 dissolved in DMSO were treated at a concentration of 20 μM and cultured at 37°C for 2 days. As a control group, only DMSO without ginsenoside was treated.

After culturing, macrophages were burst with distilled water to release intracellular bacteria, diluted 5-fold with PBS, and then serially diluted. It was dispensed into 7H10 medium and cultured at 37°C for 3 to 4 days. Colony forming units (CFU) were measured to determine intracellular bacterial survival. The results are shown in Figure 2.

Figure 2 shows that ginsenosides Rg6, Rh4, and Rg3 exhibit a significant Mabc growth inhibition effect compared to other ginsenosides.

Combining the results of Example 1, ginsenosides may act directly on non-tuberculous mycobacteria to inhibit their growth, but the effect is minimal, and instead, they inhibit the growth of non-tuberculous mycobacteria through a different mechanism within the cell. It is thought to inhibit growth, and this shows that it can be usefully used in the prevention or treatment of non-tuberculous mycobacteria.

Claims (5)

A pharmaceutical composition for the prevention or treatment of non-tuberculous mycobacterial infectious diseases containing as an active ingredient one or more of ginsenosides Rg6, Rh4 and Rg3.
In claim 1,
A pharmaceutical composition for preventing or treating non-tuberculous mycobacterial infectious diseases, wherein the non-tuberculous mycobacterium is Mycobacterium abscessus ( M. abscessus ).
In claim 1 or claim 2,
A pharmaceutical composition for the prevention or treatment of non-tuberculous mycobacterial infectious diseases, wherein the non-tuberculous mycobacterial infectious diseases are lung diseases, lymphadenitis, skin, soft tissue, bone infections, or disseminated diseases.
In claim 1 or claim 2,
A pharmaceutical composition for the prevention or treatment of non-tuberculous mycobacterial infection disease, characterized in that it is administered in combination with clarithromycin, amikacin, cefoxitin, or a combination thereof.
A health functional food composition for preventing or alleviating symptoms of non-tuberculous mycobacterial infection disease, containing one or more of ginsenosides Rg6, Rh4 and Rg3 as active ingredients.