KR100652789B1 - Pharmaceutical composition for preventing or treating of immune disease through overproduction of il-4 comprising myristicin - Google Patents

Abstract

A composition comprising myristicin is provided to be able to inhibit overproduction of interleukin-4(IL-4) and abnormal proliferation of B-cell without side effect, thereby being able to treat and prevent diseases caused by abnormal proliferation of the B-cell or allergic diseases. The pharmaceutical composition for preventing and treating immune diseases induced by overproduction of IL-4 comprises myristicin(1-allyl-5-methoxy-3,4-methylenedioxybenzene) represented by the formula(I) or a pharmaceutically acceptable salt thereof as an effective ingredient, wherein immune diseases induced by the overproduction of interleukin-4(IL-4) is diseases caused by abnormal proliferation of B-cell or allergenic diseases.

Description

Pharmaceutical composition for preventing or treating of immune disease through overproduction of IL-4 comprising myristicin}

1 is a result of measuring the degree of synthesis of IL-4 in mouse T cell line following the treatment with myristicin by ELISA.

A: Group treated with DMSO without PMA and Io (Control 1)

B: Group treated with DMSO after PMA and Io (Control 2)

C: group treated with 1 μM of myristyin after treatment with PMA and Io

D: group treated with 10 μM of myristyin after treatment with PMA and Io

E: group treated with 100 μM of myristyin after treatment with PMA and Io

F: group treated with myristyin 500μM after treatment with PMA and Io

Figure 2 shows the results of measuring the cell viability of the mouse T cell line according to the treatment with myristicin.

A: Group treated with DMSO without PMA and Io (Control 1)

B: Group treated with DMSO after PMA and Io (Control 2)

C: group treated with 1 μM of myristyin after treatment with PMA and Io

D: group treated with 10 μM of myristyin after treatment with PMA and Io

E: group treated with 100 μM of myristyin after treatment with PMA and Io

F: group treated with myristyin 500μM after treatment with PMA and Io

Figure 3 is the result of measuring the inhibition of the proliferation of B cells in mouse spleen cells by the treatment with myristicin.

A: DMSO treated without LPS (control 1)

B: Group treated with DMSO after LPS treatment (Control 2)

C: group treated with 1 μM of myristyin after LPS treatment

D: group treated with 10 μM of myristyin after LPS treatment

E: group treated with 100 μM of myristyin after LPS treatment

F: group treated with myristyin 500μM after LPS treatment

The present invention relates to a pharmaceutical composition for the prevention or treatment of immune diseases caused by overproduction of IL-4 containing myristicin as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition containing myristicin as an active ingredient and inhibiting overproduction of IL-4, thereby preventing or treating diseases caused by abnormal proliferation of B cells and allergic diseases.

Myristicin (myristicin, 1-allyl-5-methoxy-3,4-methylenedioxybenzene) is an alkenylbenzene compound that exists in nature and is the main component of nutmeg, parsley, carrots, black pepper, natural oils and spices ( O'Neil, MJ et al., The Merk Index , Merck & Co., Inc., Whitehouse Station, NJ, pp. 1134, 2001; Salzer U. J, Food Sci Nutr. 9, 345-373, 1997). Myristicin induces the synthesis of glutathione-S-transferase in the liver and small intestine (Zheng, G. et al., J. Agric. Food Chem , 40, 107-110, 1992), benzopyrene It has been reported to inhibit tumorigenicity caused by (benzopyrene) (Zheng, G et al., Carcinogenesis 13, 1921-1923, 1992).

Interleukin-4 (IL-4) is also called B cell growth factor or B cell stimulatory factor-1 because of its activity of stimulating B cell proliferation. This protein is about 20,000 daltons and is one of the most studied bioactive modulators in recent years. The IL-4 is mainly produced from activated T cells to promote growth and differentiation of B cells with antibody production function. It is also a cytokine involved in the proliferation of T cells, macrophages / monocites, mast cells and the like. In addition, the IL-4 is known to induce the expression of MHC class II antigen and IgE low affinity receptor FcεRII on the surface of B cells and monocytes.

The normal function of IL-4 in vivo is to maintain homeostasis of B cells and to regulate the differentiation of helper T cells and the class switching of immunoglobulins. On the other hand, when the IL-4 is excessively produced, abnormal proliferation of B cells occurs. Aberrant proliferation of the B cells is known to cause diseases such as mantle cell lymphoma, follicular lymphoma, Burkitt's lymphoma, and large B cell lymphoma ( Kluin, PM et al., Malignant lymphomas stem from different B cell population.In : Ferrarini, M. and Caligaris-Cappio, F. (eds.) Human B cell population.Farger, Basel, Switzerland, 1997, pp70-84). .

In addition, when IL-4 is overproduced, helper T cells are differentiated into TH2 cells, which increases the number of TH2 cells, resulting in antigen-antibody reactions and eoxinophilic inflammatory reactions involving IgE and IgG1. It is known that the function is suppressed, leading to atopic and allergic diseases (Song, Z. et al. J. Immunol . 156, 424-429, 1996; Hopkin, JM Curr. Opin. Immunol . 9, 788-792, 1997 Rosenwasser, L. J. Int. Arch. Allergy Immunol. 118, 268-270, 1999).

In addition, airway hyperreactivity, allergic conjunctivities of the eye, and mild B cell hyperplasia have been reported in mice transfected with T cells with IL-4. (Tepper, RI Res. Immunol. 144, 633-637, 1994). Many studies have reported the association between IL-4 and allergic diseases in humans. For example, increased IL-4 gene expression due to the development of single nucleotide polymorphism in the promoter region of the IL-4 gene (Noguchi, E. et al. Clin.Exp. Allergy 28, 449-453). , 1998) and allergic diseases such as atopic dermatitis (Kawashima, T. et al. J. Med. Genet. 35, 502-504, 1998) have been reported (Song, Z. et al. J. Immunol. 156, 424-429, 1996; Rosenwasser, LJ Int. Arch. Allergy Immunol. 118, 268-270, 1999).

Accordingly, for the prevention or treatment of the above diseases, a drug that can suppress the expression of IL-4 has been developed. The inhibitors of IL-4 developed so far are known as cyclosporin A (SO) and suppressor of cytokine signaling (SOCS). However, the IL-4 inhibitors have the disadvantage of showing severe side effects.

Therefore, there is a need for the development of a safe and effective new IL-4 inhibitor without side effects.

Therefore, the inventors of the present invention, while studying to develop a pharmaceutical composition that does not have side effects that can prevent and treat immune diseases caused by the overproduction of IL-4, myristicin not only inhibits the overproduction of IL-4, but also B The present invention was completed by confirming the inhibition of proliferation of cells.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of immune diseases caused by overproduction of IL-4 containing myristicin as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of immune diseases caused by IL-4 overproduction containing myristicin.

Hereinafter, the present invention will be described in detail.

The pharmaceutical composition containing myristicin according to the present invention as an active ingredient has an activity of inhibiting overproduction of IL-4. In addition, the myristicin does not affect the survival rate of the cell does not exhibit side effects, and because it is a natural substance derived from edible plants, there is a general safe feature.

In one embodiment of the present invention, the myriticin was treated in mouse T cell line to investigate whether myristyin has an activity of inhibiting overproduction of IL-4, and the degree of synthesis of IL-4 was analyzed by ELISA (Example 1). As a result, it was confirmed that the synthesis of IL-4 at the protein level was inhibited by the treatment of myristicin (see FIG. 1). Furthermore, the effect of the treatment with myristicin on the survival of T cells was examined by trypan blue staining. As a result, it was confirmed that myristicin does not affect the survival of T cells (see FIG. 2).

In this regard, the present inventors examined whether the myristicin also inhibits the proliferation of B cells involved in IL-4.

That is, in another embodiment of the present invention, in order to examine whether myristicin has an activity of inhibiting the proliferation of B cells, the myelocytes isolated from the mouse were treated with myristicin at different concentrations and the effects on B cell proliferation were investigated. (See Example 2). As a result, it was found that myristyin inhibited the proliferation of B cells in a concentration-dependent manner (see FIG. 3).

From the above experimental results, the inventors have found that myristicin according to the present invention is very useful for preventing or treating diseases caused by abnormal proliferation of B cells or allergic diseases by inhibiting overproduction of IL-4.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of immune diseases caused by overproduction of IL-4 containing myristicin as an active ingredient.

In the present invention, an immune disease caused by overproduction of IL-4 may include all diseases that occur abnormally in the immune system, and include, for example, diseases caused by abnormal proliferation of B cells and allergic diseases.

The "disease caused by abnormal proliferation of B cells" refers to a disease caused when B cells proliferate to higher levels than normal levels. For example, but not limited to, mantle cell lymphoma, follicular lymphoma, Burkitt's lymphoma, and Large B cell lymphoma.

The "allergic disease" refers to a disease caused by an excessive response of the body immune system to a substance coming from the outside, and in the present invention, particularly, hypersensitivity of the body immune system caused by overproduction of IL-4. Examples of the allergic disease may include allergic rhinitis, allergic asthma, allergic otitis media, allergic conjunctivitis, anaphylatic shock and allergic skin diseases.

Myristicine (1-allyl-5-methoxy-3,4-methylenedioxybenzene) which is an active ingredient of the pharmaceutical composition according to the present invention is represented by the following formula (I).

Formula I

In addition, myristicin according to the invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. “Pharmaceutically acceptable salts” means salts suitable for use in contact with human and lower animal tissues without causing excessive toxicity, irritation, allergic reactions, etc. within the scope of medical judgment. Kinds of such pharmaceutically acceptable salts are well known in the art. For example, the pharmaceutically acceptable salts are described in detail in the literature (SM Berge et al., J. Pharmaceutical Sciences , 1977, 66: 1). Pharmaceutically acceptable salts can be broadly divided into acid addition salts and basic addition salts. Acid addition salts of myristicin according to the invention can be prepared in situ during the final separation and purification of the compounds of the invention or by reacting the free base functional groups with suitable organic acids in a separate reaction system. Representative acid addition salts include but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerol Lophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isothionate), lactate, maleate, methanesulfonate , Nicotinate, 2-naphthalenesulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate , Phosphate, glutamate, bicarbonate, p-toluenesulfonate and udecanoate It is included. Examples of acids that can be used to form acid addition salts include inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid.

Basic addition salts may be used to convert carboxylic acid moieties in situ during the final separation and purification of the compounds of the invention into suitable bases (e.g., hydroxides, carbonates or bicarbonates of pharmaceutically acceptable metal cations). Nate), ammonia, organic primary amines, secondary amines or tertiary amines. Pharmaceutically acceptable basic addition salts include, but are not limited to, alkali metal or alkaline earth metal cations such as lithium, sodium, potassium, calcium, magnesium and aluminum salts, nontoxic quaternary ammonia and amine cations (ammonium, tetramethyl Ammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium and ethylammonium). Other representative organic amines useful for the formation of base addition salts include ethyleneamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

The myristyin can be isolated from nature, chemically synthesized or purchased commercially using known methods.

As a method of separating the myristicin from nature, the myristicin may be separated and purified from a plant or part of a plant such as nutmeg, parsley, carrot, black pepper.

In addition, myristicine of the present invention may be purchased commercially from Sigma-Aldrich, or may be chemically prepared using known synthetic methods.

In the pharmaceutical compositions of the present invention, myristicin or a pharmaceutically acceptable salt thereof of the present invention may be used alone or formulated in combination with one or more pharmaceutically acceptable carriers depending on the route of administration chosen.

The route of administration of the pharmaceutical composition of the present invention may be oral or parenteral. Parenteral routes of administration may include, but are not limited to, subcutaneous, intravenous, intramuscular, or intraperitoneal administration.

For oral administration of the pharmaceutical compositions of the present invention may be formulated in solid forms such as capsules, tablets, pills, granules and powders or in liquid form such as elixis syrups and suspensions. When formulated in solid form for oral administration, fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like known in the art can be used as suitable carriers. For example, to formulate the pharmaceutical composition of the present invention into capsules, carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid can be used. In addition, it can be prepared in the form of compressed tablets using a diluent and both the tablets and capsules can be prepared in a sustained release according to methods known in the art to allow the continuous release of the drug over several hours. Compressed tablets can be sugar coated or film coated. When formulated in liquid form for oral administration, suspending agents, emulsions, syrups, diluents, wetting agents, sweetening agents, fragrances, preservatives and the like known in the art can be used as suitable carriers.

For parenteral administration of the pharmaceutical compositions of the invention, the form of injectables in ampoules or vials is preferred. Suitable carriers when formulated as injectables may include stabilizers, buffers, preservatives and the like known in the art. Suitable stabilizers include sodium bisulfite, sodium sulfite and ascorbic acid, and the like, and preservatives include benzalkonium chloride, methyl or propyl-paraben and chlorobutanol.

The dosage of the pharmaceutical composition of the present invention can be determined by a doctor under medical judgment and includes the age, health, weight, excretion, diet, disease severity, activity of the compound used, sex, time of administration, route of administration, Of course, it can be determined according to the treatment period and the number of treatments. Preferably, the effective dose of the pharmaceutical composition of the present invention is 0.000001 to 1 mg / kg / day, more preferably 0.0001 to 0.1 mg / kg / day can be administered 1 to 3 times a day .

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

<Example 1>

Investigation of the effects of myristicin treatment on IL-4 synthesis and cell viability

It was investigated whether the treatment of myristicin inhibited the production of IL-4. To this end, EL4.IL-2 cells (ATCC TIB-181, Manassas, VA, USA), which are mouse T cell lines, were activated with phorbol-12-myristate-13-acetate (PMA) and ionomycin (Io). Synthesis was induced and analyzed by ELISA to see if myristyin inhibited the synthesis of IL-4. The EL4.IL-2 cells are T cell lines, but show little IL-4 synthesis when inactivated, but IL-4 synthesis is induced when activated with PMA and Io.

First, EL4.IL-2 cells were 5 × 10 ⁵ cells / ml using a medium containing 100 units / ml of penicillin, 100 μg / ml of streptomycin, and 5% fetal bovine serum in RPMI 1640 medium. Suspended at a concentration of and put in a culture dish and incubated in a condition of 37 ℃ in 5% CO ₂ incubator. At this time, treated with myristyin (Sigma-Aldrich, St. Louis, MO, USA) dissolved in DMSO at a concentration of 1, 10, 100, 500 μM and treated with PMA 80 nM and Io 1 μM to activate the cells after 1 hour. Then incubated for 24 hours. As a control group, an inactive group treated with only dimethyl sulfoxide (DMSO) without treatment with PMA and Io (control group 1) and a group treated with DMSO and then treated with DMSO (control group 2) were used. The concentration of DMSO treated in the experimental and control groups was 0.1%, which is lower than 1%, a concentration known to be harmless, to exclude the effect of the solvent. When the culture was completed, the culture solution was recovered and used for analysis of IL-4 protein, and the cells were recovered and used for measuring cell viability. Protein levels of IL-4 were analyzed using known ELISA methods using anti-mouse IL-4 antibodies (BD Bioscience Pharmingen, San Diego, Calif., USA) (Ouyang, YL et al., Toxicology, 104: 187). -202, 1995). Cell viability was expressed as a ratio of the total number of cells treated with trypan blue and the number of viable cells not stained by trypan blue under a microscope. The experiment was performed three times independently and the results are expressed as mean ± standard deviation. Significance was verified by Dunnett's two-tailed t-test.

As a result, EL4.IL-2 cells were activated by treatment of PMA and Io, and it was confirmed that IL-4 was synthesized. The synthesized IL-4 was shown to be inhibited by the treatment of myristicin. In particular, it was shown that the synthesis of IL-4 is significantly inhibited when the myristyin is treated at a concentration of 100 μM or more (FIG. 1).

On the other hand, the viability of EL4.IL-2 cells did not change by treatment of PMA and Io and treatment of myristicin (FIG. 2).

Therefore, it was confirmed that the myristyin has an activity that significantly inhibits the synthesis of IL-4 without affecting the survival rate of T cells.

<Example 2>

Investigation of the effects of myristicin treatment on the proliferation of B cells

In Example 1, it was confirmed that the inhibition of the synthesis of IL-4 by myristyin was investigated whether the myristicin was directly involved in the proliferation of IL-related B cells and inhibited the proliferation of B cells. To this end, splenocytes of mice were isolated and treated with myristicin, and then the proliferation of B cells was measured.

Six-week-old female B6C3F1 mice (Korean Biolink, Chungbuk Negative, Korea) were bred for at least one week in a controlled environment with a temperature of 21-24 ° C, a relative humidity of 40-60%, and a 12-hour light / dark cycle. When the weight of the mouse is 17 to 20g, the cervical spine is degenerated and lethal. Then, spleens were removed from the sterile workplace to separate splenocytes. The medium was mixed with RPMI1640 medium using penicillin 100 Unit / ml, streptomycin 100 ㎍ / ml, and fetal bovine serum 5%. Suspension to a concentration of 10 ⁶ cells / ml. This was placed in a culture dish and incubated at 37 ° C. in a 5% CO ₂ incubator. At this time, the spleen cells were treated with myristyin dissolved in DMSO at a concentration of 1, 10, 100, 1000 μM, and after 1 hour, 1 μg / ml of lipopolysaccharide (LPS) was further incubated for 48 hours. As a control group, an inactive group treated with DMSO only (control 1) without LPS and a group treated with LSO and then treated with DMSO (control 2) were used. The concentration of DMSO treated in both the experimental and control groups was 0.1%. Then thymidine radiolabeled with tritium 5 μCi / ml treatment was further incubated for 16 hours. After culturing, the cells were recovered and the amount of thymidine absorbed when the cells proliferated was determined by radiometric measurement. The experiment was performed three times independently and the measured values were expressed as mean ± standard deviation. Statistical significance was also verified (Dunnett's two-tailied t-test, Dunnett, CW J. Amer Stat. Assoc ., 50, 1096-1121, 1995).

As a result, the treatment of B cells (about 50%) and T cells (about 50%), the constituents of the spleen, was performed when LPS, a mitogen that proliferated only B cells, occurred. The increased proliferation of B cells was inhibited concentration dependently by treating myristicin. In particular, the degree of inhibition of proliferation of B cells was found to be statistically significant when the concentration of myristicin was 500 μM or more (FIG. 3).

<Manufacture example 1>

Preparation of a medicament containing myristicin as an active ingredient

<1-1> Preparation of Tablet

25 mg of myristicin with U-type mixer together with 26 mg of lactose for excipients and 3.5 mg of Avicel (microcrystalline cellulose), 1.5 mg of sodium starch glyconide as a disintegration aid and 8 mg of L-HPC (low-hydrosyprophylcellulose) for binders And mixed for 20 minutes. After mixing was completed, 1 mg of magnesium stearate was further added as a lubricant and mixed for 3 minutes. Tablets were prepared by tableting and film coating after a quantitative test and a humidity test.

<1-2> Preparation of Syrup

2 g of acid addition salt of myristicin, 0.8 g of saccharin and 25.4 g of sugar of the present invention were dissolved in 80 g of warm water. After cooling the solution, 8.0 g of glycerin, 0.04 g of flavor, 4.0 g of ethanol, 0.4 g of sorbic acid, and an appropriate amount of distilled water were mixed. Water was added to the mixture to 100 ml.

<1-3> Preparation of Capsule

A capsule was prepared by mixing 50 mg of myristicin, 50 mg of lactose, 46.5 mg of starch, 1 mg of talc and 1 mg of magnesium stearate and filling it into hard gelatin capsules.

<1-4> Preparation of Injection

1 g of hydrochloric acid salt of myristicin, 0.6 g of sodium chloride and 0.1 g of ascorbic acid of the present invention were dissolved in distilled water to obtain a final volume of 100 ml. The solution was filled in ampoules and heat sterilized.

As described above through the above examples, the pharmaceutical composition containing myristicin according to the present invention as an active ingredient has an effect of inhibiting overproduction of IL-4 and proliferation of abnormal B cells. Therefore, the pharmaceutical composition of the present invention is very useful for preventing or treating diseases or allergic diseases caused by abnormal proliferation of B cells caused by overproduction of IL-4.

Claims (4)

To overproduce IL-4 (Interleukin-4) containing myristicin (1-allyl-5-methoxy-3,4-methylenedioxybenzene) represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical composition for the prevention or treatment of immune diseases caused by. Formula I

According to claim 1, wherein the immune disease caused by the overproduction of IL-4 is a disease caused by abnormal proliferation of B cells or Pharmaceutical composition, characterized in that the allergic disease. According to claim 2, wherein the disease caused by aberrant cell proliferation (Mantle cell lymphoma, follicular lymphoma (Follicular lymphoma), Burkitt's lymphoma and Large B cell lymphoma (Large B cell lymphoma) Pharmaceutical composition, characterized in that selected from the group consisting of. The method of claim 2, wherein the allergic disease is allergic rhinitis, allergic asthma, allergic otitis media, anaphylactic A pharmaceutical composition, characterized in that selected from the group consisting of shock (anaphylatic shock), allergic conjunctivitis and allergic skin diseases.

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