KR102304069B1 - Composition for enhancing the effect anti-tuberculosis drugs - Google Patents

Abstract

The present invention relates to a composition for improving the therapeutic effect of an anti-tuberculosis agent and a composition for preventing or treating tuberculosis, wherein the compound according to the present invention or a pharmaceutically acceptable salt thereof inhibits drug release in M2 macrophages to reduce the amount of intracellular anti-tuberculosis agent retention By increasing it, it may contribute to enhancing the therapeutic effect of anti-tuberculosis drugs and overcoming resistance.
In addition, by using the compound or a pharmaceutically acceptable salt thereof in combination with an anti-tuberculosis agent, it is possible to effectively prevent or treat tuberculosis.

Description

Composition for enhancing the therapeutic effect of anti-tuberculosis drugs

The present invention relates to a composition for improving the therapeutic effect of an anti-tuberculosis agent and a composition for preventing or treating tuberculosis.

Macrophages play a pivotal role in tissue homeostasis, host defense, and inflammation. The cells have various phenotypes and are heterogeneous in function, but are largely divided into M1 macrophages and M2 macrophages.

The differentiation of M1 macrophages is induced by stimulation of LPS and interferon-gamma (IFN-gamma), which are the initial inflammatory inducing substances of undifferentiated macrophages due to infection and tissue damage. . M2 macrophages are differentiated by interleukin-4 (IL-4) and interleukin-13 (IL-13) and are involved in inflammation inhibition, tissue remodeling, and wound healing. Macrophages play a central role in both physiological and pathological conditions, and many studies have shown that macrophages are key therapeutic targets, especially for infectious diseases, chronic inflammatory diseases, and tumors.

Tuberculosis, caused by infection with Mycobacterium tuberculosis (Mtb), the Mycobacterium tuberculosis, is the most lethal infectious disease, causing about 1.5 million deaths each year. When first transmitted through the respiratory tract, it is usually caused in the lungs. The most difficult problem in the treatment of tuberculosis is that it is resistant to anti-tuberculosis drugs and does not respond to drug treatment. In general, the appearance and infection of drug-resistant bacteria are the main factors, but environmental factors of the lung tissue in which Mycobacterium tuberculosis survives are also important factors for drug resistance.

Mycobacterium tuberculosis (Mtb) has evolved to survive and proliferate in macrophages of lung tissue. Recently, it has been known that the characteristics and metabolic differences of macrophage subtypes are very important for the survival and proliferation of Mycobacterium tuberculosis in the cell, which is a factor in determining the presence or absence of disease. In experiments with Mycobacterium tuberculosis infection in monkeys and mice, it was found that the ratio of M1 macrophages to M2 macrophages increased with the proliferation of Mycobacterium tuberculosis in lung tissue and progression to active tuberculosis.

In addition, it is known that resistance to macrophage-targeting drugs is associated with failure to induce and maintain an intracellular titer due to overexpression of drug efflux pumps, which is more pronounced in M2 macrophages. Therefore, there is a need for the development of substances that can contribute to the increase in anti-tuberculosis drug efficacy and overcoming resistance through the regulation of drug release in M2 macrophages.

Korean Patent No. 10-0620404 (published on Sep. 13, 2006)

It is an object of the present invention to provide a composition for enhancing the therapeutic effect of an excellent anti-tuberculosis agent.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating tuberculosis using the composition for enhancing the therapeutic effect of the anti-tuberculosis agent.

In order to achieve the above object, the present invention provides a pharmaceutical composition for enhancing the therapeutic effect of an anti-tuberculosis agent containing a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

wherein X is selected from NH, CH ₂ , S or O, R ¹ and R ² may each be the same or different, (C1-C4)alkyl, (C1-C4)alkoxy, nitro, hydroxy or halogen can be selected from

In addition, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof and an anti-tuberculosis agent as active ingredients.

The compound represented by Formula 1 according to the present invention or a pharmaceutically acceptable salt thereof inhibits drug release in M2 macrophages to increase the amount of anti-tuberculosis agent in the cell, thereby enhancing the therapeutic effect of the anti-tuberculosis agent and overcoming resistance Can contribute to .

In addition, by using the compound or a pharmaceutically acceptable salt thereof having such an effect in combination with an anti-tuberculosis agent, it is possible to more effectively prevent or treat tuberculosis.

1 is a graph comparing the drug excretion ability of M1 macrophages and M2 macrophages according to an embodiment of the present invention.
2 is a graph showing the effect of inhibiting drug release activity of M2 macrophages treated with T0070907 according to an embodiment of the present invention.
Figure 3 is a graph showing the change in the concentration of the anti-tuberculosis agent pyrazinamide (Pyrazinamide) in M2 macrophages treated with T0070907 according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors have completed the present invention by identifying a substance that effectively inhibits drug release in M2 macrophages to increase the intracellular concentration of an anti-tuberculosis agent.

The present invention provides a pharmaceutical composition for enhancing the therapeutic effect of an anti-tuberculosis agent containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the above formula, X _{may be selected from NH, CH 2} , S or O, R ¹ and R ² may each be the same or different, (C1~C4)alkyl, (C1~C4)alkoxy, nitro, hydroxy or halogen.

Preferably, in the above formula, X may be NH, R ¹ and R ² may each be the same or different, and may be selected from nitro or halogen.

More preferably, it may be a compound of Formula 2 or a pharmaceutically acceptable salt thereof.

[Formula 2]

The compound of Formula 2 is 2-chloro-5-nitro-N-4-pyridinyl-benzamide (2-chloro-5-nitro-N-4-pyridinyl-benzamide), PPAR-gamma (peroxisome proliferator-activated) It is a selective antagonist of receptor gamma), and may be named "T0070907".

In the pharmaceutical composition according to the present invention, the compound may be used in the form of a pharmaceutically acceptable salt within the range having the same effect. The "pharmaceutically acceptable" means that the composition is not toxic to cells or humans exposed to the composition. That is, the salt may be a salt that does not cause serious irritation to the administered organism and does not impair the biological activity and properties of the compound.

The salt may be used in the form of any one of a pharmaceutically acceptable basic salt or an acid salt. The basic salt may be used in the form of any one of an organic base salt, an inorganic base salt, sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethyl It may be selected from the group consisting of an aminium salt and a pyridinium salt, but is not limited thereto.

As the acid salt, an acid addition salt formed by a free acid is useful. The free acid may include an inorganic acid and an organic acid, and as the inorganic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, double phosphoric acid, nitric acid, etc. may be used. As the organic acid, citric acid, acetic acid, maleic acid, malic acid, and puma Lyric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid , glutamic acid, citric acid, aspartan acid, stearic acid, and the like can be used.

In addition, the compound according to the present invention may include all salts, hydrates and solvates that can be prepared by conventional methods as well as pharmaceutically acceptable salts. The addition salt can be prepared by a conventional method, and the compound is dissolved in a water-miscible organic solvent, for example, acetone, methanol, ethanol, or acetonitrile, and an excess of an organic base is added or an aqueous base solution of an inorganic base is added, followed by precipitation. It can be prepared by crystallization or crystallization. Alternatively, an addition salt may be obtained by evaporating the solvent or excess base from the mixture and drying, or it may be prepared by suction filtration of the precipitated salt.

In the pharmaceutical composition according to the present invention, the compound or a pharmaceutically acceptable salt thereof controls the drug excretion ability of M2 macrophages to control the macrophage-targeting drug, ATP-binding cassette (ATP-Binding Casette, ABC) transporter It is possible to increase the intracellular concentration of the target drug.

Preferably, by increasing the intracellular concentration of the anti-tuberculosis agent, the therapeutic effect of the anti-tuberculosis agent can be enhanced.

The M2 macrophages are cells present in tuberculosis granuloma, 'granuloma' refers to tuberculosis granuloma, tuberculosis granuloma, and tuberculosis tuberculosis granuloma.

The anti-tuberculosis agent is pyrazinamide, isoniazid, streptomycin, para-aminosalicylic acid, neomycin, biomycin, kanamycin and rifampin ( rifampin) may be one or more selected from the group consisting of, but is not limited thereto.

Preferably it may be pyrazinamide (pyrazinamide), isoniazid (isoniazid), streptomycin (streptomycin).

In the pharmaceutical composition according to the present invention, the tuberculosis is pyrazinamide-resistant tuberculosis (PZAr-TB), isoniazid-resistant tuberculosis (INHr-TB) and streptomycin-resistant tuberculosis. tuberculosis, STRr-TB) may be one or more resistant tuberculosis selected from the group consisting of, but is not limited thereto. By increasing the intracellular concentration of the anti-tuberculosis agent by controlling the drug excretion ability of the M2 macrophages, it is possible to overcome resistance to the anti-tuberculosis agent and increase the therapeutic effect.

In the pharmaceutical composition according to the present invention, the tuberculosis is selected from the group consisting of ocular tuberculosis, skin tuberculosis, renal tuberculosis, lymph gland tuberculosis, laryngeal tuberculosis, intestinal tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, throat tuberculosis, breast tuberculosis and spinal tuberculosis. It may be one or more, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof and an anti-tuberculosis agent as active ingredients.

[Formula 1]

In the above formula, X _{may be selected from NH, CH 2} , S or O, R ¹ and R ² may each be the same or different, (C1~C4)alkyl, (C1~C4)alkoxy, nitro, hydroxy or halogen.

Preferably, in the above formula, X may be NH, R ¹ and R ² may each be the same or different, and may be selected from nitro or halogen.

More preferably, it may be a compound of Formula 2 or a pharmaceutically acceptable salt thereof.

[Formula 2]

The compound of Formula 2 is 2-chloro-5-nitro-N-4-pyridinyl-benzamide (2-chloro-5-nitro-N-4-pyridinyl-benzamide), PPAR-gamma (peroxisome proliferator-activated) It is a selective antagonist of receptor gamma), and may be named "T0070907".

The anti-tuberculosis agent is pyrazinamide, isoniazid, streptomycin, para-aminosalicylic acid, neomycin, biomycin, kanamycin and rifampin ( rifampin) may be one or more selected from the group consisting of, but is not limited thereto.

Preferably it may be pyrazinamide (pyrazinamide), isoniazid (isoniazid), streptomycin (streptomycin).

In the pharmaceutical composition according to the present invention, the tuberculosis is pyrazinamide-resistant tuberculosis (PZAr-TB), isoniazid-resistant tuberculosis (INHr-TB) and streptomycin-resistant tuberculosis. tuberculosis, STRr-TB) may be one or more resistant tuberculosis selected from the group consisting of, but is not limited thereto. By increasing the intracellular concentration of the anti-tuberculosis agent by controlling the drug excretion ability of the M2 macrophages, it is possible to overcome resistance to the anti-tuberculosis agent and treat tuberculosis.

In the pharmaceutical composition according to the present invention, the tuberculosis is selected from the group consisting of ocular tuberculosis, skin tuberculosis, renal tuberculosis, lymph gland tuberculosis, laryngeal tuberculosis, intestinal tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, throat tuberculosis, breast tuberculosis and spinal tuberculosis. It may be one or more, but is not limited thereto.

The compound or a pharmaceutically acceptable salt thereof may increase the intracellular concentration of the anti-tuberculosis agent by controlling the drug excretion ability of M2 macrophages, thereby enhancing the therapeutic effect of the anti-tuberculosis agent.

According to an embodiment of the present invention, it was confirmed that when M2 macrophages were treated with the compound T0070907 of Formula 2, the intracellular concentration of pyrazinamide was increased. Accordingly, by using the compound or a pharmaceutically acceptable salt thereof and the anti-tuberculosis drug in combination, the anti-tuberculosis agent therapeutic efficacy can be improved to more effectively prevent or treat tuberculosis.

The "prevention" means any action of suppressing the onset or delaying the onset of tuberculosis or at least one or more symptoms of the disease by administration of the pharmaceutical composition according to the present invention. It may also include treating a subject in remission of the disease to prevent or prevent recurrence.

The "treatment" means any action that improves or beneficially changes the symptoms, such as alleviating, reducing, or disappearing at least one symptom of tuberculosis or the disease by administration of the pharmaceutical composition according to the present invention.

The present invention may provide a pharmaceutical composition for enhancing the therapeutic effect of an anti-tuberculosis agent containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutical composition for preventing or treating tuberculosis comprising the anti-tuberculosis agent as an active ingredient. Corresponding features may be substituted for the above-mentioned parts.

The pharmaceutical composition according to the present invention may be prepared according to a conventional method in the pharmaceutical field.

The pharmaceutical composition may be combined with a suitable pharmaceutically acceptable carrier according to the formulation, and if necessary, an excipient, diluent, dispersant, emulsifier, buffer, stabilizer, binder, disintegrant, solvent, etc. to be prepared. can The appropriate carrier and the like do not inhibit the activity and properties of the compound according to the present invention, or a pharmaceutically acceptable salt thereof, or an anti-tuberculosis agent, and may be selected differently depending on the dosage form and formulation.

The pharmaceutical composition according to the present invention may be applied in any dosage form, and more specifically, it may be formulated and used in parenteral dosage forms of oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods.

The solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may be included. In addition, the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Among the oral dosage forms, liquid formulations include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.

The effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered to any animal capable of developing tuberculosis, and the animal may include, for example, not only humans and primates, but also livestock such as cattle, pigs, horses, and dogs.

The pharmaceutical composition may be administered by an appropriate administration route depending on the form of the formulation, and may be administered via oral or parenteral various routes as long as it can reach the target tissue. The administration method is not particularly limited, and for example, oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. .

The pharmaceutical composition according to the present invention may be used alone for preventing or treating tuberculosis, or may be used in combination with surgery or other drug treatment.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. 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 completely explain the present invention to those of ordinary skill in the art.

<Example 1> Comparative analysis of drug excretion ability of M1 macrophages and M2 macrophages

To compare and analyze the drug excretion ability of M1 macrophages and M2 macrophages, Hoechst 33342 fluorescent dye was used.

THP-1 cells are human monocyte cells, which are widely used as models to study monocyte and macrophage biology and function. THP-1 cells were 100U/ml penicillin, 100μg/ml streptomycin (HyClone™) and 10% FBS (HyClone™) and 55μM 2-mercaptoethanol (2-mercaptoethanol, Gibco) added RPMI. It was suspended in 1640 medium and cultured in an incubator at _{37° C., 5% CO 2 .} Cells were divided into group 1 M0 macrophages, group 2 M1 macrophages, and group 3 M2 macrophages, and the amount of Hoechst 33342 retained in the cells was confirmed.

The drug treatment method is as follows. Cells in culture were treated with phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) at 200 ng/ml to induce differentiation into M0 macrophages. After 6 hours, 100ng/ml LPS (Sigma-Aldrich) and 20ng/ml recombinant human IFN-γ (R&D System) were treated to differentiate into M1 macrophages, and recombinantly to differentiate into M2 macrophages. Human IL-4 (recombinant human IL-4) and recombinant human IL-13 (recombinant human IL-13, Peprotech) were each treated at 20 ng/ml. After inducing differentiation into M1 and M2 macrophages, after 96 hours, the cells were washed with phosphate buffered saline (PBS), and in a medium containing 5 μg/ml of Hoechst 33342 (Sigma-Aldrich) at 37 ° C, 5% CO ₂ was cultured in an incubator. After 1 hour, the cells were washed with PBS and cultured for 30 minutes in a medium not containing Hoechst 33342. After incubation, intracellular Hoechst 33342 was measured using a spectrofluorometer (SpectraMax M2e; 350 excitation and 461 emission). Intracellular Hoechst 33342 concentration was analyzed based on 100% M0 macrophages.

1 is a graph comparing the drug excretion ability of M1 macrophages and M2 macrophages according to an embodiment of the present invention. Referring to this, the retention of Hoechst 33342 in M1 macrophages is lower than that of M2 macrophages. It was confirmed that the drug excretion activity of M1 macrophages was higher than that of M1 macrophages.

<Example 2> Confirmation of the inhibitory effect of M2 macrophage drug release activity of T0070907

In order to confirm the drug excretion activity according to T0070907 treatment in M2 macrophages, it was evaluated using Hoechst 33342 fluorescent dye, a substrate of the ABC transporter.

Cells in culture were divided into group 1 M0 macrophages, group 2 M2 macrophages treated with solvent control, and group 3 M2 macrophages treated with T0070907 (Sigma-Aldrich), and the intracellular Hoechst 33342 retention was confirmed.

The drug treatment method is as follows. Cells in culture were treated with PMA (Sigma-Aldrich) at 200 ng/ml to induce differentiation into M0 macrophages. After 6 hours, differentiation into M2 macrophages was induced by treatment with 20 ng/ml of IL-4 and IL-13 (Peprotech), and T0070907 was treated with IL-4 and IL-13 at 10 μM 1 hour before treatment. After 96 hours, the cells were washed with PBS and cultured in a medium containing 5 μg/ml of Hoechst 33342 (Sigma-Aldrich) at 37° C., 5% CO _{2 in an} incubator. After 1 hour, the cells were washed with PBS and cultured for 30 minutes in a medium not containing Hoechst 33342. After incubation, intracellular Hoechst 33342 was measured using a spectrofluorometer (SpectraMax M2e; 350 excitation and 461 emission). Intracellular Hoechst 33342 concentration was analyzed based on 100% M0 macrophages.

2 is a graph showing the drug release activity inhibitory effect of M2 macrophages treated with T0070907 according to an embodiment of the present invention. Referring to FIG. 2 , M2 macrophages treated with T0070907 have intracellular Hoechst 33342 retention It was confirmed that the ability was recovered and the drug retention ability was improved.

<Example 3> Confirmation of the effect of increasing the concentration of the anti-tuberculosis agent pyrazinamide in M2 macrophages of T0070907

In order to determine whether the drug concentration of the anti-tuberculous drug pyrazinamide (Pyrazinamide, PZA) was maintained in the T0070907-treated M2 macrophages according to the present invention, the following experiment was performed.

THP-1 cells in culture were differentiated into M2 macrophages in the same manner as in Example 2, and then divided into M2 macrophages treated with group 1 solvent control and M2 macrophages treated with group 2 T0070907. After 96 hours, the cells were washed with PBS and cultured in a medium containing 50 μM pyrazinamide at 37° C., 5% CO _{2 in an} incubator. After 20 minutes, the cells were washed with cold PBS, the cells were recovered in 70% acetonitrile buffer, and the intracellular drug concentration was measured using HPLC-MS/MS analysis.

3 is a graph showing the change in the concentration of the anti-tuberculosis agent pyrazinamide in M2 macrophages treated with T0070907 according to an embodiment of the present invention. Referring to FIG. 3, M2 macrophages treated with T0070907 are intracellular It was confirmed that the drug retention capacity was improved and the pyrazinamide drug concentration increased.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (9)

A reagent composition for inhibiting the release of an anti-tuberculosis agent from M2 macrophages, containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

delete The method of claim 1,
The M2 macrophages are
Reagent composition, characterized in that the cells present in tuberculosis granuloma. The method of claim 1,
The anti-tuberculosis agent,
With pyrazinamide, isoniazid, streptomycin, para-aminosalicylic acid, neomycin, biomycin, kanamycin and rifampin A reagent composition, characterized in that at least one selected from the group consisting of. The method of claim 1,
The tuberculosis is
One selected from the group consisting of pyrazinamide-resistant tuberculosis (PZAr-TB), isoniazid-resistant tuberculosis (INHr-TB) and streptomycin-resistant tuberculosis (STRr-TB) Reagent composition, characterized in that the above-resistant tuberculosis. The method of claim 1,
The tuberculosis is
Eye tuberculosis, skin tuberculosis, renal tuberculosis, lymph gland tuberculosis, laryngeal tuberculosis, intestinal tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, throat tuberculosis, breast tuberculosis and spinal tuberculosis, characterized in that at least one selected from the group consisting of, a reagent composition. delete delete delete

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