KR20240082793A - Composition for Propylaxis or Treatment of Mycobacterial Infection Comprising trimethoxystilbene - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by mycobacterial infection, and more specifically, to a pharmaceutical composition comprising 3,4',5-trimethoxystilbene of the following formula (1): , V46) as an active ingredient and relates to a pharmaceutical composition for the prevention or treatment of mycobacterial infectious diseases.
[Formula 1]

Description

Pharmaceutical composition for preventing or treating mycobacterial infection disease containing trimethoxy stilbene {Composition for Propylaxis or Treatment of Mycobacterial Infection Comprising trimethoxystilbene}

The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by mycobacterial infection.

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.

Mycobacterium tuberculosis proliferates very slowly using nutrients in the host's body, and although most people are cured naturally, 5-10% of infected people develop tuberculosis, which occurs when the body's immunity is weakened due to complications such as overwork, malnutrition, diabetes, etc. Mycobacterium tuberculosis develops after an incubation period for a certain period of time after infection, or develops acutely without an incubation period, causing complications such as lung inflammation and asthma, leading to the death of the infected person.

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. However, the treatment period takes a long time, 6 to 9 months, and there are many side effects. there is. In addition, in the case of simple carriers of tuberculosis bacteria, there are no subjective symptoms, so tuberculosis can be easily transmitted to others, making it difficult to prevent and treat tuberculosis.

Non-tuberculous mycobacteria are widely distributed in the natural world, including in water and soil, and are opportunistic bacteria that infect through the respiratory tract. Approximately 25 types of non-tuberculous mycobacteria are associated with 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 mycobacteria have a very high isolation rate of drug-resistant bacteria and require long-term treatment of more than 2 years. Therefore, the incidence, prevalence, and mortality rate of infection by non-tuberculous mycobacteria are rapidly increasing worldwide.

The most common causative bacterium for non-tuberculous mycobacterial lung disease in Korea is M. avium . Unusually, Mabc, a fast-growing bacterium that is relatively rare in foreign countries, is the second most common causative bacterium in Korea. Mabc is of particular clinical importance 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 due to the symptoms of the disease, but also due to 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, there is a need to develop new drugs that have preventive and therapeutic effects that protect people who are not infected with or infected with mycobacterial pathogens, such as tuberculosis bacilli or non-tuberculous mycobacteria, while being less burdensome or burdensome to take.

Although resveratrol (3,4',5-trihydoxystilbene, RSV) has various pharmacological activities, it structurally has a hydroxyl group (OH group) on the benzene ring, so it has disadvantages such as rapid metabolism and low bioavailability. Therefore, to overcome these shortcomings, 3,4',5-trimethoxystilbene (V46) was developed in which the hydroxy group in the benzene ring of resveratrol was replaced with a methoxy group. Based on the chemical structure of V46, improved metabolic stability can be predicted because all hydroxyl groups are protected by methoxylation. V46 was reported to have greater plasma exposure, longer elimination half-life, and lower clearance after oral administration. In addition, V46 is known to exhibit various activities such as anti-cancer, anti-inflammatory, anti-tumor, inhibition of osteoarthritis, and protection of vascular endothelium.

U.S. Patent No. 9539222 describes that resveratrol can be used to treat acid-fast bacteria by combining it with existing tuberculosis treatments, and Korean Patent No. 1884770 describes that resveratrol itself has anti-tuberculosis activity. However, it has not been disclosed that resveratrol derivatives are effective in preventing or treating non-tuberculous acid-fast bacteria. J. Nat. Prod. 2022, 85, 1459-1473 and Antibiotics 2020, 9, 336 state that among resveratrol derivatives, trimethoxyresveratrol does not show antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.

The present inventors confirmed that trimethoxyresveratrol has antibacterial properties against mycobacteria, unlike the above prior literature, and completed the present invention.

US Patent No. 9539222 Korean Patent No. 1884770

J. Nat. Prod. 2022, 85, 1459-1473 Antibiotics 2020, 9, 336

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating mycobacterial infectious diseases that are highly resistant to drugs and difficult to treat.

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

The present invention for achieving the above-mentioned object is a method for preventing mycobacterial infectious diseases containing 3,4',5-trimethoxystilbene (V46) of the following formula (1) as an active ingredient: Or it relates to a pharmaceutical composition for treatment. The present invention is based on the fact that V46 significantly inhibits the survival rate of mycobacteria in mycobacteria-infected macrophages and mice.

[Formula 1]

The mycobacteria may be M. tuberculosis, Mycobacterium kansasii , M. avium , or Mycobacterium absesus (Mabc). In particular, it may be Mycobacterium absesus, 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 against Mycobacterium acesus, which is resistant to all conventional anti-tuberculosis drugs. there is.

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

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

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

The pharmaceutical composition for preventing or treating mycobacterial infectious diseases according to the present invention is powdered 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 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 that is sensitive to mycobacteria. 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 mycobacterial infectious diseases containing V46 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.

The present invention also relates to a composition for inhibiting the proliferation or growth of mycobacteria containing V46 as an active ingredient. The composition of the present invention can be used as a quasi-drug composition that inhibits proliferation or growth by directly acting on mycobacteria existing in nature before mycobacterial infection.

As described above, according to the present invention, by utilizing the newly discovered efficacy of V46, it can be usefully used to prevent or treat/improve symptoms of mycobacterial infectious diseases without spending a long time and money on separate safety tests.

Figure 1 is a diagram showing the effect of V46 treatment on Mabc-S infected mouse macrophages.
Figure 2 is a diagram showing the effect of V46 treatment on Mabc-R infected mouse macrophages.
Figure 3 is a diagram showing the effect of V46 treatment on Mtb-infected mouse macrophages.
Figure 4 is a diagram showing the effect of V46 treatment on Mabc-R infected mice.

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]

1. Preparation of samples, etc.

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

Macrophages were collected from the bone marrow of 7-week-old wild-type C57BL/6 female mice and cultured in DMEM (Lonza) containing 10% fetal bovine serum (Lonza), penicillin (100 IU/ml), and streptomycin (100 μg/ml). ) were 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 for 3 to 5 days to induce macrophage differentiation, 37°C, 5% CO ₂ , cultured under humidified conditions.

(2) Preparation of mycobacteria

Mabc is divided into smooth-type strains (with) and rough-type strains (without) depending on the presence or absence of glycoprotein lipids in the cell membrane. When the glycoprotein lipid is deleted from the Mabc-S strain, it is converted to Mabc-R. For this study, Mabc-S (ATCC 19977) was used according to the method used in previous reports, and Mabc-R strain obtained by subculturing Mabc-S and deleting glycoprotein lipids. Mtb H37Rv was used as tuberculosis bacteria. did. The culture methods for the three strains are the same as below. Mabc-S and Mabc-R were inoculated into Middlebrook 7H9 (Difco, 271310) liquid medium supplemented with 0.2% glycerol, 0.05% Tween 80 (Sigma-Aldrich) and albumin-dextrose catalase, respectively, at 140 rpm and 37°C. Cultured until mid-logarithmic phase (OD600=0.4~0.5). The bacterial culture was collected by centrifugation at 3000 rpm for 20 minutes, and after adding 0.05% Tween 80 to PBS (phosphate-buffered saline), the pellet was released with this reagent and centrifuged under the same conditions as above. After centrifugation, release the pellet once more and centrifuge to remove the supernatant. In this process, before centrifugation, the bacteria are released into single bacteria by reacting three times for 30 seconds each in an ultrasonic shaker. After resuspending in PBS, the cells were divided into 1.5 ml vials, and viable colony forming units (cfu) were measured in Middlebrook 7H9 medium from several vials at random.

2. Assaying the effect of V46 on the growth of mycobacteria

(1) Mycobacterial infection and treatment of V46

① In case of Mabc-S infection

Mouse macrophages (BMDM) 1x10 ⁵ cells/well (500 μl each) were dispensed and cultured for 4 days at 37°C, 5% CO ₂ , and humidified conditions. Each cultured well was treated with Mabc-S for 2 hours so that the average number of infected bacteria per macrophage (multiplicity of infection, MOI) was 1, and then washed with PBS three times to remove bacteria present outside the cells. It was washed and treated with gentamycin, and 500 μl of DMEM (Lonza) medium was added.

As shown in Table 1 below, four test groups were cultured at 37°C, 5% CO ₂ , and humidified conditions (triple experiment). The volume of culture medium in the test groups was equalized. In the table, RSV is resveratrol and V46 is 3,4',5-trimethoxystilbene.

After culturing, macrophages were burst with distilled water to release intracellular bacteria, and distilled water from each well was collected. 0dpi (test group 1) was 1/5, 1/5 ² , 1/5 ³ , and 1/5 ⁴ . The rest were serially diluted 1/5 ³ , 1/5 ⁴ , 1/5 ⁵ , and 1/5 ⁶ .

Dispense 10 μl of the diluted sample onto the medium agar plate in 1(2) above without overlapping, culture in a 37°C, 5% CO ₂ incubator for 3 to 4 days, and measure CFU to check intracellular bacterial survival rate (triple) experiment) and the results are shown in Figure 1. The p-value in the figure was derived by Tukey's post hoc test of one-way ANOVA.

As can be seen in the figure, when 20 μM of V46 was treated with BMDM infected with Mabc-S, the number of surviving bacterial colonies in the cells was significantly reduced. In addition, compared to conditions treated with the same concentration of RSV, which is known to have an antibacterial effect against Mabc-S, the number of viable bacterial colonies in cells was significantly reduced when treated with V46. As a result, it was confirmed that V46 has an antibacterial effect against the corresponding bacteria. The p-value in the figure was derived by Tukey's post hoc test of one-way ANOVA (same as below).

② In case of Mabc-R infection

In the case of Mabc-S infection, the test was performed in the same manner as ① above, except that Mabc-R was infected instead and cultured under the conditions shown in Table 2 below, and the results are shown in Figure 2.

As can be seen in the figure, when V46 was treated with Mabc-R-infected BMDM, the number of surviving bacterial colonies in the cells was significantly reduced, and the reduction effect increased as the treatment concentration of V46 increased. In addition, compared to the condition in which RSV with the same parent nucleus structure was treated at the same concentration (20 μM), the number of viable bacterial colonies in the cells when treated with V46 was significantly reduced. This confirmed that V46 has an antibacterial effect against Mabc-R.

③ In case of tuberculosis infection

In the case of Mabc-S infection, Mtb was infected instead, cultured under the conditions in Table 3 below, and the test was performed in the same manner as ① above, except that the bacterial colonies were cultured in an incubator for more than 2 weeks, and the results are shown in Figure 3. did.

As can be seen in the figure, when V46 was treated with Mtb-infected BMDM, the number of surviving bacterial colonies in the cells was rapidly reduced, and the reduction effect increased as the treatment concentration of V46 increased. This confirmed that V46 has an antibacterial effect against Mtb.

3. Evaluation of anti-tuberculosis activity in Mabc-R infected mice

Wild-type C57BL/6 female mice were intranasally infected with 1×10 ⁶ CFU of Mabc-R. Five or 10 days after infection, V46 (50 mg/kg) dissolved in PBS buffer was administered intraperitoneally. As a control group, the same volume of PBS buffer was administered instead of drug.

Mice were sacrificed 5 days (5 dpi) and 10 days (10 dpi) after infection, and then lung tissue was collected, finely ground using a homogenizer to destroy lung tissue cells, and diluted 10 times with PBS. The tissue lysate was dispensed onto a 7H10 agar plate and cultured at 37°C in a 5% CO ₂ incubator for about 3 days. Colony forming units (CFU) were measured, and the results are shown in Figure 4. The p-value in the figure was derived using the unpaired Student's t test.

As confirmed in the figure, it can be seen that V46 shows antibacterial activity against mycobacteria by reducing the survival rate of Mabc-R in vivo . These results show that the antibacterial effect of V46 further increases over time.

The results of the above examples show that V46 (3,4',5-trimethoxystilbene) acts directly on mycobacteria and inhibits their growth, and also inhibits the growth of mycobacteria in vivo, so V46 is used to prevent mycobacterial infection. It shows that it can be useful in preventing or treating diseases caused by

Claims (7)

A pharmaceutical composition for preventing or treating mycobacterial infectious diseases containing 3,4',5-trimethoxystilbene of the following formula (1) as an active ingredient.
[Formula 1]

In claim 1,
Mycobacterial infection, characterized in that the mycobacteria are M. tuberculosis, Mycobacterium kansasii , Mycobacterium avium ( M. avium ), or Mycobacterium absesus (Mabc) Pharmaceutical composition for preventing or treating diseases.
In claim 1 or claim 2,
A pharmaceutical composition for the prevention or treatment of mycobacterial infectious diseases, wherein the 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 preventing or treating mycobacterial infectious diseases, 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 mycobacterial infectious diseases containing 3,4',5-trimethoxystilbene as an active ingredient.
In claim 5,
A composition for inhibiting the proliferation or growth of mycobacteria, characterized in that the composition is in the form of a tablet, capsule, pill, or liquid.
A composition for inhibiting the proliferation or growth of mycobacteria containing 3,4',5-trimethoxystilbene as an active ingredient.