KR20190074649A - Pharmaceutical composition for preventing or treating allergic inflammatory response comprising coumarin derivative - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating allergic inflammatory diseases containing 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate. A composition containing 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate of the present invention can be usefully used as the pharmaceutical composition for preventing or treating allergic inflammatory diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating an allergic inflammatory disease comprising a coumarin derivative,

The present invention relates to a pharmaceutical composition for preventing or treating an allergic inflammatory disease, and more particularly to a pharmaceutical composition for preventing or treating an allergic inflammatory disease comprising a coumarin derivative having a specific chemical structure.

Allergies are hypersensitivity reactions of the immune system caused by foreign antigens (allergens) in a harmless environment. Symptoms such as itching, fever and pain, which are major symptoms of asthma, atopic dermatitis and allergic rhinitis, are caused by allergic reactions, which are caused by excessive activation of mast cells.

Mast cells play a crucial role in the onset of allergic reactions, and mast cell activation is a typical activator and is closely related to specific immunoglobulin E (IgE) antibodies. IgE binds to the Fcε receptor I (Fc epsilon receptor I, FcεRI) expressed on the surface of mast cells. When FcεRI crosses the antigen on mast cells, an intracellular signaling cascade is induced and Lyn, Syk, protein kinase C (PKC) and mitogen activated protein kinase (PKC) MAPK) and the downstream (downstream) is activated, such media, thereby increasing the cell in Ca ^{2 +} concentration (Ca ion). In addition, the aggregation of IgE-FcεRI induces exocytosis of preformed granules containing histamine, chemokine, cytokine and serine protease, and leukotrienes and prostaglandins Lt; / RTI > These inflammatory mediators cause a variety of pathophysiological symptoms in allergic reactions, including airway smooth muscle contraction, secretion of mucus and recruitment of other inflammatory cells. Thus, regulation of inflammatory mediator production is important for controlling allergic reactions.

Rat basophilic leukemia (RBL) -2H3 cells are rodent mucosal mast cells that secrete inflammatory mediators. The RBL-2H3 cell line does not completely represent the primary mast cell and basophil, but the RBL-2H3 cell line shares the characteristics of the two cells and allows simple subculture. The main characteristic of mast cells is the secretion of histamine. Histamine is involved in allergic and inflammatory responses and plays an important role in mediating interactions among other cells. Histamine is also secreted in RBL-2H3 cells. Thus, RBL-2H3 cells have generally been used for in vitro studies of allergic reactions. Phosphoryl myristate acetate (PMA) and calcium ionophore A23187, an activator of PKC, increased intracellular calcium ion concentration in RBL-2H3 cells and induced activation of the signaling pathway do.

Under various reports of such allergy-related mechanisms, much research is underway to develop more effective antiallergic substances.

U.S. Pat. No. 4,737,517 issued Apr. 12, 1988.

Arabian Journal of Chemistry (2015), doi: http://dx.doi.org/10.1016/ j.arabjc.2015.06.012 (2015.06.03.) Bulletin of the Korean Chemical Society, Volume 37, Issue 6 (2016) 950-953 (Aug. EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 874-880, 2017 (April 17, 2017).

In studying more effective antiallergic substances, the inventors of the present invention examined the anti-allergic effect of coumarin derivatives in RBL-2H3 cells using a combination of PMA and A23187, -2H3 cells were examined for the effect of coumarin derivatives on the pathway for promoting the activation of the intracellular signaling pathway leading to degranulation, and it was found that 2-oxo-2H-chromen- 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate strongly inhibited the degranulation of RBL-2H3 cells, and 2-oxo-2H-chromen- 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate can be used in the treatment of allergic inflammatory diseases.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of an allergic inflammatory disease comprising 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate.

According to one aspect of the present invention there is provided a method of treating an allergic inflammatory disease comprising 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate A pharmaceutical composition for preventing or treating is provided.

In one embodiment, the allergic inflammatory disease may be one or more symptoms selected from the group consisting of allergic itching, fever and pain, and may be caused by immediate allergic reactions, including asthma, fever, allergic rhinitis ), And atopic eczema.

In one embodiment, the prevention or treatment of the allergic inflammatory disease is due to inhibition of degranulation of mast cells, which may be inhibition of release of beta -hexosaminidase or release of histamine, The pharmaceutical composition for preventing or treating diseases may be an antihistamine.

In one embodiment, the 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate may have a therapeutic concentration of 3-25 μM, preferably 25 μM.

According to the present invention, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibits degranulation in mast cells stimulated with pobol myristate acetate as a PKC activator and A23187 as a calcium carrier, , And it was confirmed that it did not show cytotoxicity in the efficacy expression concentration interval.

Therefore, the composition comprising the 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate of the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of allergic inflammatory diseases.

Figure 1 shows the structure of a coumarin derivative. [ C1 : 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate. C2 : 5,7-Dimethyl-2H-chromen-2-one. C3 : 7-chloro-2H-chromen-2-one. C4: 2H- benzo [h] chromen-2-one (2H-benzo [h] chromen -2-one). C5: 3H- benzo [f] chromene-3-one (3H-benzo [f] chromen -3-one). C6: 2--benzyl-3-phenyl -4H- furo [3,2- c] chromene-4-one (2-benzyl-3-phenyl -4H-furo [3,2- c] chromen-4-one ). C7: 3-Benzyl-2- (4-methoxyphenyl) -4H- furo [3,2- c] chromene-4-one (2-benzyl-3- (4 -methoxyphenyl) -4H-furo [3 , 2- c ] chromen-4-one).]
2 is a graph showing the effect of coumarin derivatives on β-hexosaminidase release and cell survival in RBL-2H3 cells. [ A : Amount of β-hexosaminidase released in RBL-2H3 cells treated with 25 μM coumarin derivative for 1 hour and stimulated with PMA (50 nM) and A23187 (1 μM). B : Cell viability of RBL-2H3 cells treated with coumarin derivatives for 24 hours. The results are expressed as mean ± standard deviation (SD) of three experiments. ** P < 0.05. C1: 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate. C2: 5,7-Dimethyl-2H-chromen-2-one. C3: 7-Chloro-2H-chromen-2-one. C4: 2H-benzo [ h ] chromen-2-one. C5: 3H-benzo [ f ] chromen-3-one. C6: 2-Benzyl-3-phenyl-4H-furo [3,2- c ] chromen-4-one. C7: 2-Benzyl-3- (4-methoxyphenyl) -4H-furo [3,2- c ] chromen-
3 is a graph showing the effect of C1, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on cell survival, beta -hexosaminidase release and histamine release in RBL-2H3 cells. [A: 3, 6.25, 12.5 , or 25 μM for 1 hour C1 treatment and PMA (50 nM) and A23187 (1 μM) to stimulate the RBL-2H3 β- hexyl release of sosami The kinase in cells. The cell viability of RBL-2H3 cells treated with C1 was measured and then stimulated. B : Histamine release in RBL-2H3 cells treated with C1 and loratadine of the same concentration gradient for 1 hour and stimulated with PMA and A23187. The cell viability of RBL-2H3 cells treated with the sample was measured and then stimulated. Lauratadine was used as a positive control. Results were expressed as mean ± SD of three experiments. ** P < 0.05 compared to stimulated RBL-2H3 cells.
4 is a graph showing the effect of C1 on LPS-induced NO production and cytokine expression in MOLT-4 cells in RAW264.7 cells. [ AC : LPS-induced NO production and cell viability in RAW264.7 cells treated with indicated concentrations of C1. A : Cells were treated with Cl for 24 h and activated with 1 μg / ml LPS for 24 h to determine the pro-inflammatory effect. B : Cells were pre-treated with C1 for 1 hour and activated with 1 μg / ml of LPS for 24 hours to determine the anti-inflammatory effect. C : Cell viability of RAW264.7 cells treated with C1 for 24 hours. DF : cytokine expression level and cell viability in MOLT-4 cells treated with indicated concentrations of C1. D : mRNA expression level of IFN-y. E : mRNA expression level of IL-4. F : Cell viability of MOLT-4 cells treated with C1 for 12 hours. Data are expressed as percent of activity as compared to control. Results were expressed as mean ± SD of three experiments. ** P < 0.05 compared to stimulated RBL-2H3 cells.
Figure 5 shows the effect of C1 on phosphorylation of MAPK in RBL-2H3 cells. [ A : Immunoblot analysis of MAPK phosphorylation. B : Immunoblot analysis of MAPKK phosphorylation.
Figure 6 is a schematic of the MAPK pathway inhibited by C1 in RBL-2H3 cells.

The present invention relates to a pharmaceutical composition for the prevention or treatment of an allergic inflammatory disease comprising 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate Lt; / RTI &gt;

In one embodiment of the present invention, the allergic inflammatory disease may be at least one symptom selected from the group consisting of allergic itching, fever and pain, and the symptom is due to immediate allergic reaction, And allergic rhinitis (allergic rhinitis), and atopic eczema. However, the present invention is not limited thereto.

In the present specification, the term " immediate allergic reaction "means Type I, Type II, Type III or Type IV allergic reactions. Type I reaction is a type I reaction in which IgE binds to the surface of mast cells or basophilic cells and binds to specific IgE antibodies, leading to granule escape (degranulation) in mast cells or basophilic cells, leading to histamine, serotonin , And bradykinin are released to cause vascular permeability hyperactivity, mucus secretion promotion, and the like. Symptoms include anaphylaxis, bronchial asthma, allergic rhinitis, and urticaria.

In one embodiment, the prevention or treatment of the allergic inflammatory disease is due to inhibition of degranulation of mast cells, which may be inhibition of release of beta -hexosaminidase or release of histamine, The pharmaceutical composition for preventing or treating a disease may be, but is not limited to, an antihistamine.

2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate was found to inhibit degranulation and inhibit histamine release in a concentration-dependent manner at a therapeutic concentration of 3-25 μM, preferably at 25 μM Respectively. It was confirmed that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate was not cytotoxic in the above concentration range.

In connection with the mechanism that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate exhibits the above-mentioned efficacy, phorbol myristate acetate (PMA) A23187, a calcium ionophore that increases ionic concentration, inhibited the activation of mitogen activated protein kinase (MAPK) pathway. Specifically, phosphorylation of ERK1 / 2 . &Lt; / RTI &gt;

Thus, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate can be used for the prophylactic or therapeutic use of allergic inflammatory diseases.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier and may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, Can be formulated in the form of sterile injectable solutions. The pharmaceutically acceptable carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. It also includes diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Solid form preparations for oral use include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like, and may include a lubricant such as magnesium stearate, talc, and the like. Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may contain diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives and the like. Examples of the non-aqueous solution include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. Non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, ethyl And injectable esters such as oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like. In addition, the pharmaceutical composition of the present invention may contain 0.001 to 50% by weight of the compound, based on the total weight of the composition.

The dosage of the compound contained in the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. For example, the compound may be administered at a dose of 0.0001 to 1000 mg / kg, preferably 0.01 to 1000 mg / kg per day, and the administration may be administered once a day or divided into several times.

The pharmaceutical compositions of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes, for example, oral, intraperitoneal, rectal or intravenous, intramuscular, subcutaneous, intrauterine, intracerebroventricular &Lt; / RTI &gt;

The compound of the present invention is not limited in its production method and can be obtained, for example, by synthesis or separation from natural products.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Examples>

1. Materials and Methods

1.1. material

Coumarin derivatives were synthesized as described in the prior art [AL Hansen, et al., Org. Lett. 7 (2005) 5585-5587 .; H. Choi, et al., 2016. doi: 10.1016 / J.Title 2001.06.06.039; W. Huang, et al., Tetrahedron. 63 (2007) 11636-11643.]. Melting the coumarin derivative in dimethyl sulfoxide (DMSO) and PIPES buffer (25 mM PIPES, 119 mM NaCl , 5 mM KCl, 1 mM CaCl 2, 0.4 mM MgCl 2, 5.6 mM glucose, 0.1% bovine serum albumin, pH 7.2) &Lt; / RTI &gt; The final concentration of DMSO was 0.125% (v / v) and the same concentration of DMSO was used as a control. PMA (phorbol myristate acetate), A23187 (calcium ionophore) and o-phthalaldehyde (OPT) were purchased from Sigma Aldrich (St. Louis, Mo., USA).

Total p38 and phosphorylated p38, extracellular signal-regulated kinase (ERK), c-Jun N terminal kinase (JNK), mitogen activated protein kinase kinase 3 (MKK3) , MAP kinase kinase 1/2 (MEK1 / 2), or MKK4 were purchased from Cell Signaling Technology (Beverly, MA, USA). Finally, anti-beta-actin antibodies were purchased from Abcam (Cambridge, UK).

1.2. Cell culture

RBL-2H3 cells and RAW264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin and 1% streptomycin (Dulbecco's modified Eagle's medium, Hyclone Logan, UT, USA). MOLT-4 cells were cultured in RPMI-1640 medium (Hyclone Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. The cells were cultured in a humidified atmosphere containing 5% CO ₂ at 37 ° C.

1.3. Degranulation  evaluation

In order to confirm the effect of the compound on the degranulation, β-hexosaminidase release level was measured as follows. The enzyme is contained in the granules of mast cells and has been used as a marker of granules. The RBL-2H3 cells (2 × 10 ⁵ cells / mL) in 24-well plates and cultured for 24 hours. Next, the medium was removed and the cells were incubated with various concentrations of compound diluted in PIPES buffer for 1 hour at 37 ° C. After incubation, the cells were washed twice with PIPES buffer and stimulated with PMA (50 nM) and A23187 (1 μM) for 30 min at 37 ° C. Subsequently, 20 mu l of the supernatant was reacted with 80 mu l of the substrate buffer (2 mM 4-p-nitrophenyl-N-acetyl- beta -D-glucosaminide in 0.05 M citrate buffer, pH 4.5) at 37 DEG C for 30 minutes. By the addition of stop buffer _{(0.1 M NaHCO 3, pH 10} ) for 200 ㎕ the reaction was stopped. Absorbance was measured at 405 nm using an Epoch microplate spectrophotometer (Biotek, Santa Barbara, Calif., USA).

Analysis of histamine release was performed as follows. The RBL-2H3 cells (2 × 10 ⁵ cells / mL) in 24-well plates and cultured for 24 hours. Compounds and stimulants were treated in the same manner as described above for [beta] -hexosaminidase release. Released histamine was measured by spectrofluorometric procedure. Briefly, histamine reacts with OPT to form fluorophore. In this test, 1 mL of the supernatant, 0.2 mL of 1 N NaOH, and 0.1 mL of 1% OPT were mixed and incubated at room temperature for 5 minutes. The reaction was stopped by adding 0.1 mL of 3 N HCl. Fluorescence intensity was measured at excitation and emission wavelengths of 360 nm and 450 nm, respectively, using a Synergy H1 hybrid multimode microplate reader (Biotek, Santa Barbara, Calif., USA).

1.4. Cell survival analysis

Survival rates of RBL-2H3 cells, RAW264.7 cells and MOLT-4 cells were determined by MTT assay. Each cell was cultured in a 24-well plate at 37 占 폚 for 24 hours. Then, the cells were treated with the compound diluted to various concentrations in the medium. RBL-2H3 cells and RAW264.7 cells were incubated at 37 DEG C for 24 hours, and MOLT-4 cells were incubated at 37 DEG C for 12 hours. After treatment with the compound, the supernatant was discarded and the cells were incubated with MTT solution (5 mg / ml). The supernatant was discarded again and the water-insoluble formazan compound formed in the cells was dissolved in DMSO. The solution was then transferred to a 96-well plate and absorbance was measured at 550 nm using an Epoch microplate spectrophotometer (Biotek, Santa Barbara, Calif., USA).

1.5. Nitrite analysis

The nitrite concentration was measured using the Griess method. RAW 264.7 cells (5 x 10 ⁴ cells / mL) were cultured in a 24-well plate for 24 hours. Cells were treated with Cl and activated with 1 μg / ml of LPS for 24 h to determine the inflammatory effect. The cells were pretreated with C1 and N-iminoethyl-L-lysine (L-NIL) for 1 hour and then treated with 1 μg / ml of LPS for 24 hours Lt; / RTI &gt; L-NIL, a selective inhibitor of inducible nitric oxide synthesis (iNOS), was used as a positive control. Then, 100 μl of the supernatant was reacted with 50 μl of a grease reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in distilled water) at room temperature for 10 minutes. The absorbance of the mixture was analyzed at 550 nm using an Epoch microplate spectrophotometer (Biotek, Santa Barbara, Calif., USA).

1.6. Quantitative real-time Reverse transcription  Enzyme chain reaction qRT - PCR )

Total cellular RNA was isolated from MOLT-4 cells using X-ZOL RNA reagent (PhileKorea, Seoul, Korea) and the RNA concentration was measured with an Epoch microplate spectrophotometer (Biotek, Santa Barbara, CA, USA). In order to analyze the gene expression levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) mRNA, a PCR kit (ReverTra Ace qPCR RT Master Mix, TOYOBO, Osaka, Japan) ng was reverse transcribed with cDNA. Relative gene expression was measured with THUNDERBIRD SYBR qPCR Mix (TOYOBO) and the house-keeping gene HPRT (hypoxanthine guanine phosphoribosyltransferase) was used for standardization. (Antisense 5'-TCCTTCTCAGTTGTGTTCTTGG-3 ') and HPRT (antisense 5'-GGGTCACCTGACACATTCAA-3'), IL-4 (sense, 5'- CACCTTACAGGAGATCATCAAAACT-3 'and antisense 5'-CCAACCTAAGCAAGATCCCA- Sense, 5'-CCTGGCGTCGTGATTAGTG-3 'and antisense 5'-TGAGGAATAAACACCCTTTCCA-3') were used to analyze gene expression levels. qRT-PCR was performed using a StepOne Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).

1.7. Western Blotting

RBL-2H3 cells were harvested by centrifugation and resuspended in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing protease and phosphatase inhibitor (Quartett Immunodiagnostika und Biotechnologie Vertriebs GmbH, Berlin, Germany) (Lysis). A bicinchoninic acid assay was performed to quantify the total protein from each pot. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then performed on the 10% acrylamide gel with the same amount of protein (30 μg). The protein isolated from the acrylamide gel was transferred to a polyvinylidene difluoride membrane (Amersham Hybond-P; GE Healthcare, Buckinghamshire, UK). Immediately after transfer, the membrane was immersed in 1% skim milk in Tris buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) for 1 hour. The membrane was then incubated with the primary antibody at 4 DEG C for 16 hours. After 3 washes in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. Next, the membranes were washed again in TBS-T and visualized for protein bands using WesternBright Peroxide chemiluminescence detection reagent (Advansta, Menlo Park, CA, USA). Finally, the membranes were incubated at 60 ° C for 10 minutes in a stripping solution (10% SDS, 0.5 M Tris-Cl, 0.8% β-mercaptoethanol) and reused.

1.8. Statistical analysis

All data for the tests of the present invention were expressed as mean ± SD (SD) of the experiment three or more times. Statistical differences between groups were assessed by one-way ANOVA followed by Bonferroni's test using SPSS (v.21, for Windows). ** P <0.05 was considered significant.

2. Results

2.1. RBL - 2H3  Cell On degranulation  Oxo-2H- Kromen -4-yl 4- Methylbenzene sulfo  Nate's inhibitory effect

In the present invention, β-hexosaminidase assay was performed to measure the effect of coumarin derivatives on the degranulation in RBL-2H3 cells (FIG. 1). RBL-2H3 cells were treated with 7 coumarin derivatives at a concentration of 25 [mu] M, respectively, and stimulated with PMA and A23187 (Fig. 2A). 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate reduced the release of [beta] -hexosaminidase by about 50% (Figure 2A), but other derivatives were observed not to affect the cells . Survival of RBL-2H3 cells was measured by MTT assay (Fig. 2B). Survival of RBL-2H3 cells was observed to be unaffected by coumarin derivatives at a concentration of 25 μM. Thus, from these results, it was found that coumarin derivatives do not show cytotoxicity to the cell line, and that only 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibits degranulation without cytotoxicity (Fig. 2B). Thus, the dose-dependent effect of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on the degranulation and cytotoxicity in RBL-2H3 cells was investigated (FIG. 3A). Treatment of different concentrations of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate significantly inhibited degranulation in a concentration-dependent manner compared to the untreated control (FIG. 3A). It should be noted that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate showed no cytotoxic effect in the concentration interval used in this test (FIG. 3A).

In addition, histamine release was measured by comparing 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate with loratadine, a selective H1 receptor antagonist. As a result, it was confirmed that both 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate and loratadine inhibited histamine release dose-dependently without cytotoxicity (FIG. 3B).

2.2. RAW264 .7 cells and MOLT-4 cells were treated with 2-oxo-2H- Kromen -4-yl 4- Methyl benzene sulphate Effect of Phonate

The effect of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on macrophages and T cells was measured (Figure 4). Representative cell lines RAW264.7 and MOLT-4 cells were used, respectively. First, nitrite analysis was performed to determine the effect of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on inflammatory or anti-inflammatory effects in RAW264.7 cells. RAW 264.7 cells were treated with serially diluted 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate and stimulated with LPS. As a result, NO production was increased in LPS-stimulated RAW264.7 cells, but no effect of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate was observed in RAW264.7 cells (Figures 4A-B). Next, T-cell associated cytokines were examined in MOLT-4 cells after treatment with 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate by qRT-PCR. However, no significant changes in mRNA expression levels of IFN-y and IL-4 were observed, and the results were not statistically significant (Fig. 4D-E). No cytotoxicity was observed in the 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate treated cells (Fig. 4C, F).

2.3. RBL - 2H3  Lt; RTI ID = 0.0 &gt; 2-oxo-2H- Kromen -4-yl 4- Methylbenzenesulfonate MAPK  Effect on activation pathway

It was confirmed that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate together with A23187 inhibited degranulation of PMA-stimulated RBL-2H3 cells. Therefore, in order to elucidate the mechanism of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate for the expression of inflammatory cytokines, 2-oxo-2H- Day 4-methylbenzenesulfonate on the activation of MAPK (Fig. 5). PMA and A23187 treatment induced activation of three types of MAPK, p38, JNK and ERK, in RBL-2H3 cells. In this test, RBL-2H3 cells were treated with various concentrations of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate and stimulated with PMA and A23187. Three major MAPKs (ERK, JNK, p38) were activated by PMA and A23187. However, ERK1 / 2 phosphorylation alone was dose-dependently inhibited in cells treated with 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate (Fig. 5A). On the other hand, phosphorylation of p38 and JNK was not inhibited compared to untreated control. In addition, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate has been shown to have an inhibitory effect upstream of the MAPK pathway. When RBL-2H3 cells were stimulated with PMA and A23187, activation of MKK was observed. However, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate did not inhibit the phosphorylation of MKK dose-dependently (FIG. 5B). These results suggest that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibits phosphorylation of the ERK1 / It can be seen that Nate inhibits mast cell activation at least in part through modulation of the MAPK pathway.

3. Discussion

The prevalence of allergic diseases such as allergic rhinitis, asthma and atopic dermatitis causes serious health problems worldwide. However, the cause of allergic diseases is unclear, and the current treatment strategy is not sufficient. Therefore, it is important to establish an accurate understanding of the pathophysiology of allergic diseases in developing new therapies for allergic diseases.

Mast cells are granulocytes that contain typical mediators such as histamine, heparin, and protease, and are widely distributed in connective tissue. The degranulation of mast cells is induced by various stimuli and the mediator played an important role in the induction of allergic inflammation. Mast cell activation is regulated by aggregation of IgE receptor (FcεRI) on mast cells. In addition, PMA and A23187 induce biochemical and ionic symptoms that lead to increased PKC activation and intracellular calcium influx. Increased intracellular calcium concentration induces degranulation leading to the secretion of inflammatory mediators such as histamine, serotonin, heparin, proteases and cytokines. Overall, various biochemical reactions associated with mast cells produce allergic symptoms.

In this study, the effect of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on mast cell degranulation was examined by measuring the release of β-hexosaminidase. Β-hexosaminidase assay was performed to determine the effect of coumarin derivatives on the degranulation in RBL-2H3 cells. Among the derivatives used, the degranulation inhibitory effect of C1, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate was the highest at 25 μM (FIG. 2A). In addition, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibited degranulation in a concentration-dependent manner in RBL-2H3 cells (Fig. 3A). In particular, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate showed an inhibitory effect on degranulation without inducing cytotoxicity as compared with the control (Fig. 3A). Histamine release assays were also performed to confirm that RBL-2H3 cells were the appropriate pathophysiological model for this study (Fig. 3B). Laurethadine was used as a positive control in this assay. In previous studies, loratadine has proved effective as a selective H1 receptor antagonist to inhibit degranulation of mast cells. As a result, both 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate and loratadine effectively inhibited histamine release in a concentration-dependent manner in RBL-2H3 cells (Fig. 3B). Thus, it has been experimentally demonstrated that RBL-2H3 cells can be used as degranulation models.

In addition, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate was treated with other immune cells, RAW264.7 cells and MOLT-4 cells, but no significant effect was observed (FIG. Therefore, it was concluded that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate has a specific effect on RBL-2H3 cells among various immune cells.

The MAPK cascade is an important signaling pathway involved in the immune response. MAPK cascades, including the p38, ERK, and JNK pathways, are an appropriate target for studying inflammatory responses because they play an important role in immune signaling regulation. Addition of PMA and A23187 to mast cells induces phosphorylation of Syn and MAPK as well as increased calcium influx. In addition, the expression of cytokines is activated by stimulation. In order to further investigate the effect of chemical treatment on the signaling cascade, the inhibitory activity of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on the MAPK pathway was evaluated. The results of this study confirmed that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate significantly reduced the phosphorylation of the ERK pathway (FIG. 5).

In the present invention, it was investigated whether the coumarin derivative, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate, contributes to the regulation of PMA and A23187 stimulated responses in RBL-2H3 cells. In conclusion, the present study confirmed that the inhibitory effect of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on RBL-2H3 degranulation occurs through inhibition of ERK phosphorylation ). Therefore, 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate can be used as a therapeutic agent for allergic inflammatory diseases.

4. Summary

Mast cells play a crucial role in the initiation of allergic inflammatory responses by releasing various mediators such as histamine, cytokines and leukotrienes. In addition, signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway contribute to the regulation of mast cell degranulation. Thus, various research strategies have been pursued to develop anti-inflammatory and anti-allergy drugs by modulating these signaling pathways. Development of new drugs that inhibit degranulation of mast cells may be helpful in the treatment of allergies. In this study, we investigated the effect of coumarin derivatives on mast cell degranulation. The influence of coumarin derivatives on degranulation in rats with basophilic leukemia (RBL) -2H3 cells was determined by β-hexosaminidase and histamine assays. The 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate coumarin derivative 1 (C1) inhibited the degranulation in a dose-dependent manner and exhibited the maximum therapeutic effect when used at 25 μM. In addition, these compounds inhibited the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. From these results, it can be seen that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibits the activation of ERK pathway and inhibits mast cell degranulation. Provide a potential treatment strategy.

Claims (10)

2-oxo-2H-chromene-4-yl 4-methylbenzenesulfonate. 2. A pharmaceutical composition for preventing or treating allergic inflammatory diseases comprising 2-oxo-2H-chromene-4-yl 4-methylbenzenesulfonate. The pharmaceutical composition according to claim 1, wherein the allergic inflammatory disease is at least one symptom selected from the group consisting of allergic itching, fever and pain. The pharmaceutical composition according to claim 1, wherein the allergic inflammatory disease is caused by an immediate allergic reaction. The pharmaceutical composition according to claim 1, wherein the allergic inflammatory disease is one selected from the group consisting of asthma caused by immediate allergic reaction, fever fever (allergic rhinitis), and atopic eczema. The pharmaceutical composition according to claim 1, wherein the prevention or treatment of the allergic inflammatory disease is by inhibiting degranulation of mast cells. The pharmaceutical composition according to claim 1, wherein the prevention or treatment of the allergic inflammatory disease is inhibition of the release of beta -hexosaminidase. The pharmaceutical composition according to claim 1, wherein the prevention or treatment of the allergic inflammatory disease is inhibition of release of histamine. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition for preventing or treating the allergic inflammatory disease is an antihistamine. 2. The pharmaceutical composition according to claim 1, wherein the 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate has a therapeutic concentration of 3-25 μM. 2. The pharmaceutical composition according to claim 1, wherein the 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate has a therapeutic concentration of 25 μM.

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