KR101606885B1 - Pharmaceutical composition for preventing and treating allergic disease comprising adenine or pharmaceutically acceptable salt thereof as an active ingredient - Google Patents

Abstract

More particularly, the present invention relates to a pharmaceutical composition for the prevention and treatment of allergic diseases comprising adenine or a salt thereof represented by the general formula (1) and a pharmaceutical composition for preventing and treating adenine or its A composition for inhibiting mast cell degranulation, inhibiting cytokine expression, and inhibiting icosanoid formation. The composition of the present invention can be used for prevention and treatment of allergic diseases by inhibiting degranulation, inhibition of transcription of the cytokine and production of icosanoids, which are allergenic mast cells.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating an allergic disease comprising adenine or a pharmaceutically acceptable salt thereof as an active ingredient,

More particularly, the present invention relates to a pharmaceutical composition for the prevention and treatment of allergic diseases comprising adenine or a salt thereof represented by the general formula (1) and a pharmaceutical composition for preventing and treating adenine or its A composition for inhibiting mast cell degranulation, inhibiting cytokine expression, and inhibiting icosanoid formation.

Mast cells are resident cells of various tissues. When activated by various stimuli, they secrete histamine, leukotriene, prostaglandin, cytokine, chemokine, proteolytic enzyme and the like. Mast cells carry out work by the body against pathogens and parasites. However, on the other hand, it causes allergic inflammation reaction and causes atopic dermatitis, atopic asthma, allergic rhinitis, urticaria, eczema, psoriasis and hypersensitivity.

Mast cells express FcεRI, a receptor for IgE. This receptor consists of one α chain and two kinds of subunits (one β chain and a disulfide-linked homo-dimer γ chain). When FcεRI binding to antigen-specific IgE binds to receptors by multivalent antigens, signal transduction leads to degranulation and release of inflammatory eicosanoids and cytokines (Abraham, SN meat al . , Mast cell-orchestrated immunity to pathogens. Nat Rev Immunol. 10 (6): 440-52).

Accordingly, the inventors of the present invention discovered that adenine inhibits the activity of mast cells induced by antigen stimulation, and completed the present invention by developing a pharmaceutical composition for the prevention and treatment of allergic diseases including adenine.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating allergic diseases comprising adenine represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

≪ Formula 1 >

Another object of the present invention is to provide a composition for inhibiting mast cell degranulation comprising adenine represented by the general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a composition for inhibiting cytokine expression comprising adenine represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a composition for inhibiting icosanoid formation comprising adenine represented by the general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating allergic diseases comprising adenine represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

≪ Formula 1 >

In order to accomplish still another object of the present invention, there is provided a composition for inhibiting mast cell degranulation comprising adenine represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to accomplish still another object of the present invention, there is provided a composition for inhibiting cytokine expression comprising adenine represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to accomplish still another object of the present invention, there is provided a composition for inhibiting icosanoid formation comprising adenine represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of allergic diseases comprising adenine represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

≪ Formula 1 >

The adenine of the present invention and its pharmaceutically acceptable salts have the effect of preventing or treating allergic diseases.

The effects of the present invention are well illustrated in an embodiment of the present invention.

In one embodiment of the present invention, the effect of adenine on the inhibition of the synthesis of leukotriene B4 (LTB4), inhibition of degranulation and inhibition of cytokine mRNA production was examined, which is one of the isochosenoids of mast cell associated inflammation induction. As a result, it can be seen that adenine inhibits synthesis of leukotriene B4 (LTB4) in mast cells, inhibits degranulation and cytokine mRNA production, and has an effect of preventing and treating allergic diseases. In addition, adenosine has been shown to inhibit local skin hypersensitivity by measuring the hypersensitivity of localized skin hypersensitivity (PCA) in the local skin tissue of experimental animal models.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of allergic diseases comprising adenine or a pharmaceutically acceptable salt thereof.

The above-mentioned " allergic disease " refers to a disease caused by hypersensitivity to a substance in the human body, that is, an excessive reaction of the body's immune system against a substance introduced from the outside. Examples of the allergic diseases include, but are not limited to, bronchial asthma, hay fever, allergic rhinitis, urticaria, anaphylaxis, digestive tract allergy, drug allergy, allergic conjunctivitis and atopic dermatitis.

The present invention provides a composition for inhibiting mast cell degranulation comprising adenine represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The adenine of the present invention and its pharmaceutically acceptable salts have an effect of inhibiting degranulation of mast cells.

In one embodiment of the present invention, the amount of β-hexosaminidase enzyme in the intracellular granules released into the extracellular buffer solution by degranulation was measured in activated RBL-2H3 cells. Adenine dose-dependently inhibited the release of β-hexosaminidase in RBL-2H3 cells. The maximal inhibitory dose was 5 mM.

The present invention provides a composition for inhibiting cytokine expression comprising adenine represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The adenine of the present invention and its pharmaceutically acceptable salts have a cytokine inhibitory effect.

In one embodiment of the present invention, the effect of adenine in the transcript production of IL-3 and MCP-1 released by activated mast cells was measured by Real-Time PCR. Adenine completely blocked the transcription of cytokines such as IL-3 and MCP-1.

The present invention provides a composition for inhibiting icosanoid formation comprising adenine represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The adenine of the present invention and its pharmaceutically acceptable salt have an effect of inhibiting icosanoid formation of mast cells.

In one embodiment of the present invention, the extent of activation of RBL-2H3 cells by leukotriene B4, which is a kind of icosanoid, is released from the cells. The amount of leukotriene B4 released from the cells after stimulation of the cells for 30 minutes was measured using an LTB4 ELISA kit. Adenine effectively inhibited leukotriene B4 release at a dose of 1 mM.

Eicosanoids are derived from polyunsaturated fatty acids such as arachidonic acid and linolenic acid, and refer to any type of compound associated with cellular activity. Refers to an unsaturated fatty acid metabolite having 20 carbon atoms such as eicosatrienoic acid, eicosatetraenoic acid, and eicosapentaenoic acid. Hydroxyeicosatetraenoic acid or hydroperoxyeicosatetraenoic acid, prostaglandin, thromboxane, leukotriene, lipoxin, and the like. Of the arachidonic acid metabolites or analogs thereof and all of the chemically modified derivatives thereof.

The composition of the present invention as described above may be a pharmaceutical composition.

The composition or pharmaceutical composition of the present invention is characterized by comprising adenine or a pharmaceutically acceptable salt thereof.

The adenine of the present invention has a structure represented by the following general formula (1), and can be isolated and purified from nature, commercially purchased, or can be produced by a chemical synthesis method known in the art.

≪ Formula 1 >

Preferably, the adenine can be isolated / purified from the nucleic acid of the cell. The adenine according to the present invention can be extracted by a known method commonly used in the art such as organic solvent extraction.

 The adenine according to the present invention may be used as such or in the form of a pharmaceutically acceptable salt thereof. As used herein, the term " pharmaceutically acceptable " means physiologically acceptable and does not normally cause an allergic reaction or similar reaction when administered to humans, and the salt includes a pharmaceutically acceptable free acid ) Are preferred. The free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The pharmaceutical composition according to the present invention may contain adenine or a pharmaceutically acceptable salt thereof singly or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition for preventing or treating allergic diseases of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI > Preferably, the pharmaceutical composition of the present invention can be transdermally administered. The term 'transdermal administration' as used herein refers to the administration of the pharmaceutical composition of the present invention to cells or skin to allow the active ingredient contained in the pharmaceutical composition for prevention or treatment of allergic diseases to be delivered into the skin. For example, the pharmaceutical composition of the present invention can be administered in a scanning type formulation, pricking the skin lightly with a 30 gauge needle, or directly applying it to the skin.

In addition, the pharmaceutical composition may be formulated into oral or parenteral formulations according to the route of administration as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective amount of the adenine of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the preferred total dose of the adenine of the present invention may be from about 0.01 to 1,000 mg, and most preferably from 0.1 to 100 mg, per kg body weight of the patient per day. However, the dose of adenine is determined based on various factors such as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate as well as administration route and frequency of treatment of the pharmaceutical composition, The person skilled in the art will be able to determine the appropriate effective dose according to the specific use of the adenine as a preventive or therapeutic agent for allergic diseases. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of allergic diseases comprising adenine or a pharmaceutically acceptable salt thereof as an active ingredient. The composition of the present invention can be used for the prevention and treatment of allergic diseases by inhibiting the synthesis of LTB4, inhibiting degranulation and cytokine mRNA production in mast cells which are involved in inflammation induction.

1 shows the effect of inhibiting degranulation by adenine (Ag-: DNP-HSA untreated, Ag +: DNP-HSA treated).
FIG. 2 shows the effect of adenine hemisulfate (Ag-: DNP-HSA untreated, Ag +: DNP-HSA treated) against adenine hemisulfate.
FIG. 3 shows the inhibitory effect of adenine on the production of inflammatory molecule, leukotriene B4 (LTB4).
Figure 4 shows the effect of adenine at the transcription level of cytokine (IL-3) mRNA in RBL-2H3 cells (CN: control (using PBS)).
Figure 5 shows the effect of adenine at the transcription level of RBL-2H3 cytokine (MCP-1) mRNA (CN: control (using PBS)).
FIG. 6 shows the effect of suppressing the local skin hypersensitivity when mice are administered with adenine peritoneal cavity. (Ad: adenine treatment, Control: physiological saline treatment)

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Cell culture

RBL-2H3 cells (American Type Culture Collection, Rockville, Md., USA) were inoculated with EMEM containing 15% FBS (v / v) (PAA Lab. Canada), 100 U / ml penicillin, 100 μg / ml streptomycin Lonza). The culture was maintained at 37 ° C in a 5% CO ₂ incubator. The cells were separated by trypsin EDTA solution. After washing, the cells were resuspended in fresh medium and used in the next experiment.

≪ Example 2 >

Degranulation Assay (β- hexosaminidase  Release Assay ) And leukotriene  B4 ( LTB4 ) Measure

RBL-2H3 cells (1 × 10 ⁵ / ml) were sensitized with 100 ng / ml of DNP specific IgE (Sigma, St. Louis, Mo., USA) overnight. After washing three times in Tyroide buffer, cells were exposed for 30 minutes at 37 ° C by changing the dose of adenine (Sigma-Aldrich) and adenine hemisulfate (Sigma-Aldrich). After incubation, cells were placed in DNP-HSA (Sigma, St. Louis, MO, USA) at 100 ng / ml for 20 min at 37 ° C. The supernatant was collected and the released β-hexosaminidase and LTB4 were measured. Cells were resuspended in 0.5% triton X-100 for 5 min for the remaining β-hexosaminidase in the cells. 10 μl of supernatant and lysate were mixed with 50 μl of a substrate solution ( p- nitrophenyl- N- acetyl-β-D-glucosaminide, 4 mM in citrate 0.1 M, pH 4.5) And cultured for 1 hour. 150 μl of 0.2 M glycine (pH 10.7) was added to complete the reaction and the absorbance was measured at 405 nM using a microplate reader. For LTB4 measurement, 100 μl of the supernatant was measured using an LTB4 ELISA kit (Enzo Life Science, USA). LTB4 in the sample and LTB4 bound to alkaline phosphatase were competitively bound to the anti-LTB4 antibody, then developed with alkaline phosphatase substrate and absorbance was measured at 404 nm.

As the degranulation index of RBL-2H3 cells, the degree of release of β-hexosaminidase in the cells into the buffer solution was measured. As shown in FIG. 1 and FIG. 2, adenine and adenine hemisulfate inhibited the release of β-hexosaminidase in RBL-2H3 cells in a dose-dependent manner at a maximal inhibitory dose of 5 mM.

Also, as shown in Fig. 3, adenine inhibited LTB4 production. Thus, adenine inhibits the degranulation of mast cells and the production of icosanoid LTB4.

≪ Example 3 >

Measurement of cytokines (IL-3 and MCP-1) mRNA

RBL-2H3 cells were sensitized and treated with adenine as described in the degranulation assay of Example 2. And stimulated with DNP-HSA (100 ng / ml) for 3 hours. After washing the cells with PBS, RNA was extracted with the easy BLUE RNA extraction kit. One μg of total RNA was used to synthesize cDNA according to the method indicated by Reverse Transcriptase Master premix (5X). The cDNA synthesized to measure cytokine mRNA for cytokine IL-3 and MCP-1 was used for real-time PCR with primers and SYBR Green reagent (Applied Biosystem).

Sequence name order SEQ ID NO: IL-3 (forward) 5'-TCCTGATGCTCTTCCACC-3 ' One IL-3 (reverse) 5'-TCGCAGCTGCAGGAATACAACACT-3 ' 2 MCP-1 (forward) 5'-TGGCAAGATGATCCCAAT-3 ' 3 MCP-1 (reverse) 5'-AGGTGGTTGTGGAAAAG-3 ' 4

The effect of adenine on the production of IL-3, MCP-1 transcripts was measured by Real-Time PCR. As shown in [Figure 4] and [Figure 5], the treatment of adenine at the cytokine transcription level of IL-3 and MCP-1 showed complete blocking.

< Example  4>

Local skin sensitization ( passive cutaneous anaphylaxis : PCA )

Anti-DNP IgE, an antibody specifically binding to dinitrophenyl (DNP), was subcutaneously injected 250 ng into each ear of mice (mouse strain DBA / 1J), and after 24 hours, adenine was weighed 3.4 kg per kg of body weight 6.8 mg were intraperitoneally injected. As a control experiment, PBS was intraperitoneally injected instead of adenine. After 1 hour, 200 μl of a 10: 1 mixture of DNP-HSA (DNP-conjugated to dinitrophenyl-human serum albumin, DNP conjugated to human serum albumin) and 4% Evans blue solution was added to the tail Intravenously. Evans blue solution is released from the blood vessels to the site where the vascular permeability is increased by the allergic reaction. After 30 minutes, the mouse's ears are cut into several pieces and placed in 500 μl of formamide solution. When left overnight at 63 ° C, evans blue that has flowed out through the ear vein comes out as a solution. Measure with Evans blue absorbance at 620 nm.

As shown in FIG. 6, when hypersensitivity was measured in the local skin tissue of the experimental animal model with PCA as a local skin immune response, Evans blue leakage amount was decreased in a dose dependent manner when adenine was injected. Thus, adenine was shown to inhibit local skin sensitization.

As described above, the present invention provides a pharmaceutical composition for the prevention and treatment of allergic diseases comprising adenine or a pharmaceutically acceptable salt thereof as an active ingredient. The composition of the present invention can be used for the prevention and treatment of allergic diseases because it inhibits the inhibition of the synthesis of leukotriene B4 (LTB4) and the inhibition of degranulation and cytokine mRNA production of mast cells that cause allergies.

Claims (5)

A pharmaceutical composition for the prevention and treatment of allergic diseases comprising adenine represented by the general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
&Lt; Formula 1 >

The composition according to claim 1, wherein the allergic disease is selected from the group consisting of bronchial asthma, hay fever, allergic rhinitis, urticaria, anaphylaxis, digestive tract allergy, drug allergy, allergic conjunctivitis and atopic dermatitis.
A pharmaceutical composition for preventing and treating allergic diseases caused by inhibition of mast cell activation comprising adenine represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
&Lt; Formula 1 >

A pharmaceutical composition for preventing and treating allergic diseases by inhibiting cytokine expression comprising adenine represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
&Lt; Formula 1 >

A pharmaceutical composition for preventing and treating allergic diseases caused by inhibition of icosanoid formation comprising adenine represented by the general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
&Lt; Formula 1 >

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