KR20070057377A - Pharmaceutical composition for treatment of wound and dermatitis comprising glucan as active ingredient - Google Patents

Abstract

A pharmaceutical composition comprising glucan is provided to show excellent treating effect on wound and burn by promoting the proliferation of skin cell and increasing the expression of collagen and show excellent inhibition effect on edema, pruritus and vasodilatation, thereby being usefully used for preventing and treating allergic dermatitis and atopic dermatitis. The pharmaceutical composition for treating dermatitis comprises glucan and a pharmaceutically acceptable carrier as effective ingredients. In the composition, the therapeutically effective amount of the glucan is 0.1-0.2 wt.%. The composition is provided for skin cell regeneration.

Description

Pharmaceutical Composition for Treatment of Wound and Dermatitis Comprising Glucan as Active Ingredient}

Figure 1 shows a PCA response-induced animal model, 2, 1, 0.5, 0.2 and 0.1% concentration of glucan (GLUCAN), 1, 0.5 and 0.1% concentration of dexamethasone (DEXA) and 1 g / kg concentration of Atofam A photograph of the size of Evans blue spots on the skin to which (ATOP) was applied.

Figure 2 shows the media control (A), DEXA 1% (B), 0.5% (C) and 0.1% (D), GLUCAN 2% (E), 1% (F), 0.5 in PCA response-induced animal model Histopathological picture showing local dermal edema change in skin with% (G), 0.2% (H) and 0.1% (I), ATOP 1 g / kg (J). Scale bars are indicated as 200 μm.

FIG. 3 shows GLUCAN at concentrations of 2, 1, 0.5, 0.2, and 0.1%, DEXA at 1, 0.5, and 0.1% concentrations and 1 g / h in a local vasodilator animal model induced by injection of Compound 48/80 (COM). It is the photograph which observed the size of Evans blue spot in the skin which applied ATOP of kg concentration.

FIG. 4 is a media control (A), DEXA 1% (B), 0.5% (C) and 0.1% (D), GLUCAN 2 in a local vasodilator animal model induced by injection of Compound 48/80 (COM). Tissues showing local dermal edema changes in skin with% (E), 1% (F), 0.5% (G), 0.2% (H) and 0.1% (I), ATOP 1 g / kg (J) Pathology picture. Scale bars are indicated as 200 μm.

5 is a cell proliferation result confirmed by MTT analysis after treating glucan to HaCa T cells at concentrations of 0.02 mg / ml and 0.05 mg / ml.

Figure 6 shows the results of collagen expression confirmed by Western blot after treating glucan in HaCa T cells at 20 μM and 50 μM concentrations.

The present invention relates to a pharmaceutical composition for treating dermatitis, and more particularly, to a pharmaceutical composition for treating allergic or atopic dermatitis containing glucan as an active ingredient.

Atopic dermatitis is very common in children but is a chronic, itchy skin condition that occurs at all ages, known as eczema and atopic eczema, and is the most common form of dermatitis. Atopic dermatitis is generally caused in people with an 'atopic tendency', which causes one or both of atopic dermatitis, asthma and high fever (allergic rhinitis). Often this condition also affects parents, children or siblings. Family history of asthma, eczema or high fever is particularly useful for diagnosing atopic dermatitis in infancy. In general, atopic dermatitis is expressed in 15-20% in children, but is limited to 1-2% in adults (Kiczuk et al., Ann Univ Mariae Curie Sklodowska [Med], 2004, 59: 503-7). Foroughi et al., Curr Opin Pediatr, 2005, 17 (5): 658-63; Nakahara and Furue, Nippon Rinsho, 2005, 63: 80-5; Williams HC, N Engl J Med, 2005, 352: 2314- 24).

Various methods are used to treat atopic dermatitis, but topical treatments (lotions, creams and ointments) are used due to the ease of application of the skin. Topical treatments are applied first to inflammation and wounds caused by complications of atopy on the surface of the skin. These treatments are known to slow the progression of atopic dermatitis (Skinner R, J Cutan Med Surg, 2004, 8: 22-31). .

Passive cutaneous anahylaxis (PCA) is known to be a very useful method for evaluating the effectiveness of therapeutic agents on type I allergic dermatitis (Matsuda et al., Biol Pharm Bull, 2002, 25: 809-12). Vasodilatory skin reactions are induced when antibodies are injected intravenously into the skin and then antigens are injected intravenously with dyes. Thus, the extent of vasodilation can be assessed by the amount and concentration of dye leaked by rapidly induced capillary dilation and increased vascular permeability (Yoshida et al., Int Arch Allergy Appl Immunol, 1979, 59: 373-82). PCA is a relatively sensitive reaction that can detect very small amounts of antibodies and is a way to study the mechanism of immediate hypersensitivity. In general, the effectiveness of the drug is assessed based on the diameter of the leaked dye, the amount of pigment leaked and histopathological changes of the skin (Teixeira et al., Br J Pharmacol, 1996, 118: 317-24; Kubo et al., Biol Pharm Bull, 1997, 20: 511-6; Matsuda et al., Biol Pharm Bull, 2002, 25: 809-12).

Topical steroid application has been commonly chosen to treat various skin diseases, but its use has been limited because of the potential for severe local and systemic side effects such as skin atrophy and hypothalamic-pituitary-adrenal (HPA) axis inhibition. (Gupta and Chow, J Cutan Med Surg, 2004, 8: 244-7). Among the glucocorticoids, dexamethasone is one of the widely used topical steroids for atopic dermatitis (Ganir et al., Acta Paediatr Jpn, 1996, 38: 702-4; Matsumoto et al., Skin Res Technol, 2005, 11: 209 -17), it has been frequently used as a control drug in the development of new anti-allergic drugs (Shichinohe et al., J Vet Med Sci, 1996, 58: 419-23; Ohtsuka et al., J Pharmacol Sci, 2003, 91 : 263-6).

Ahtopam ^TM (ATOPALM ^TM) is a cream for the treatment or prevention of various skin diseases including atopic dermatitis, because it contains a variety of active plant extracts such as purslane (Portulaca oleracea) extract, its anti-allergenic activity is relatively well known (Rashed et al., J Ethnopharmacol, 2003, 88: 131-6; Malek et al., J Ethnopharmacol, 2004, Jul 03 (1): 57-62).

However, these therapeutic agents not only have a low therapeutic effect, but also cause problems of resistance, and thus, there is an urgent need for the development of new atopic dermatitis therapeutics.

Accordingly, the present inventors have conducted a study to overcome the problem of the low therapeutic effect of the conventional dermatitis treatment, glucan is effective in skin cell regeneration, high treatment than all the conventional treatment for allergic dermatitis and atopic dermatitis The present invention was completed by confirming the effect.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating wounds and dermatitis comprising glucan as an active ingredient.

Other objects and advantages of the present invention will become apparent from the following detailed description and claims.

In order to achieve the above object, the present invention provides a therapeutic pharmaceutical composition comprising a glucan and a pharmaceutically acceptable carrier thereof as an active ingredient.

The pharmaceutical composition for treating wounds and dermatitis of the present invention includes glucan as a therapeutically active ingredient, and allergic dermatitis or atopic dermatitis may be applied as dermatitis.

As a type of glucan, beta-glucan is a fiber-type complex sugar (polysaccharide) isolated from numerous medicinal mushrooms such as the cell walls and maitake of yeast, oats and barley used in baking. Two primary uses of beta-glucan are to improve the immune system and lower blood cholesterol levels. Numerous experimental studies in vitro and in animals have confirmed that beta-glucan activates leukocyte cells. In fact, hundreds of research papers on beta-glucan have been published since 1960. These studies indicate that beta-1,3-glucan is particularly effective in activating macrophages and white blood cells known as neutrophils. These cells provide one of the immune systems that primarily defends against external invasion. Beta-glucan activated macrophages or neutrophils leukocytes recognize and kill tumor cells, remove cellular debris resulting from oxidative damage, accelerate the rate of recovery of damaged tissues and further activate other parts of the immune system. Beta-glucan is an important factor in the cholesterol-lowering action of oat cereal. Binding to cholesterol by beta-glucan, along with other water-soluble fibers, thereby removing these molecules in the stool is very useful for reducing blood cholesterol. Numerous double-blind methods using beta-glucan derived from oats or yeast show a significant decrease at least 4 weeks after use, with a 10% reduction for approximately total cholesterol and 8% reduction for LDL and HDL cholesterol It is known to result in an increase of 0-16%.

The composition of the present invention, by including the glucan as an active ingredient, exhibits an effect of inhibiting vasodilation and edema resulting therefrom, an allergic pruritus inhibitory effect, and an effect of inhibiting edema and pruritus of contact dermatitis, and allergic dermatitis and It is effective in the prevention and treatment of various dermatitis including atopic dermatitis.

In addition, the composition of the present invention by containing glucan as an active ingredient, promotes the proliferation of skin cells, increases the expression of collagen, a skin cell constituent, effectively regenerates skin cells. Therefore, the composition of the present invention is effective in the treatment of wounds, wounds and burns, which are conditions related to skin cell regeneration.

The therapeutically effective dose of glucan included in the composition of the present invention is 0.1 to 0.2% by weight, preferably 0.5 to 1% by weight. According to the present invention, glucan shows an effective therapeutic effect against allergic dermatitis and atopic dermatitis even at a minimum concentration of 0.1%.

Glucan of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the glucan of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used Is prepared using. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one pharmaceutically acceptable carrier in one or more glucan compounds. The carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone , Cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, or the like.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

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

However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.

1. Test substance and test method

(1) Experimental animals and breeding

Twenty male Wistar rats (6 weeks old, SLC, JAPAN) were used after 10 days acclimation. The test animals were placed in polycarbonate cages of five animals at a kennel maintained at a temperature of 20-25 ° C. and a humidity of 30-35%. The light: dark cycle was maintained at 12 hours: 12 hours, free diet (Samyang, Korea) and watered. All animals were PCA induced. In this experiment, animals were divided back into two subgroups to detect vasodilatation changes and histological changes. In each subgroup, 10 animals were used. All experimental animals used in the present invention are the Guide for the Care and Use of Laboratory Animals by Institute of Laboratory Animal Resources, Commission on Life Science, National Research Council, USA on 1996, Washington, DC).

(2) Administration of test sample

Glucan and DEXA (Sigma, USA) and ATOP (Neopharm, Korea) were used for this experiment. Glucan was stored in a 4 ° C. refrigerator to block light and prevent deterioration.

Dilute 2.5% glucan (β-glucan, Glucan Corp. Ltd., Korea) to distilled water to concentrations of 2, 1, 0.5, 0.2 and 0.1%, and DEXA to 0.5% sodium carboxymethyl cellulose (CMC · Na; Sigma, USA), to concentrations of 1, 0.5 and 0.1%. ATOP was used in commercially available formulations. All test substances were applied topically twice a day for 2 days to the sensitization site on the shaved dorsal side and applied once more before 1 hour of antigen exposure.

To determine the anti-allergic effect of glucan and the treatment of atopic dermatitis, 1) passive dermal anaphylaxis (PCA) induced model, 2) compound 48/80 (COM) induced local vasodilation model, 3) COM induced pruritus model, 4) platelet Active factor (PAF) induced pruritus model, 5) protease (PA) induced pruritus model, 6) mosquito salivary gland extract (SGE) induced allergic pruritus model, 7) DNFB induced contact dermatitis model and 8) picryl chloride ( PC) The experiment was divided into induced delayed contact dermatitis model, and the dose and dose schedule administered are shown in Table 1 below.

TABLE 1

(3) allergy  Inducing and evaluating response or contact dermatitis

(3-1) Induced Passive Skin Anaphylaxis (PCA) Response

The effect of GLUCAN on atopic dermatitis was evaluated using a Passive Cutaneous Anaphylaxix (PCA) model induced by mouse OVA (egg white protein) antiserum.

Anti-OVA serum from rats containing IgE was prepared using Stoland's and Share's method (Stotland and Share, Can J Physiol Pharmacol, 1974, 52: 1119-25). 0.5 ml saline suspension containing OVA (1.0 mg; Sigma, USA) and aluminum hydroxide gel (10 mg; Aldrich, USA) was injected into the dermis followed by an inert Bordetella pertussis bacterium. 1.0 ml of the suspension (2 × 10 ¹⁰ cells / ml) was sensitized by injecting the rat intraperitoneally. After 7 days, 0.5 ml saline suspension was injected intramuscularly, followed by 1.0 ml of inert bacterial suspension injected intraperitoneally to sensitize again. After 14 days of the first sensitization, the mice were anesthetized with pentobarbital, bleeding from the carotid artery, and the anti-OVA serum of the mice was obtained according to the usual method. Titers of anti-OVA IgE antibodies were measured by homogeneous PCA in mice at 48 hours. Titers were measured as diameters of about 5 mm and expressed as mutual ratios to the final dilution resulting in a positive response. The titer of anti-OVA serum containing IgE was 1:32. The serum was stored at -80 ° C until use.

<Sensitization and exposure>

Serum diluted 8-fold with physiological saline was sensitized by intradermal injection into the shaved dorsal skin of rats at a dose of 0.05 ml / site.

48 hours after sensitization, the mice were challenged by injecting 0.5 ml of a saline solution containing OVA (2.0 mg) and Evans blue (5.0 mg; Sigma, USA) through the tail vein.

(3-2) Induction of Local Vasodilation Using Compound 48/80

The effect of glucans on atopic dermatitis was assessed using a local vasodilation model induced with the compound 48/80 (COM), a histamine free promoter. SD rats were used as experimental animals.

The test substance was applied to the dorsal hair removal site twice a day for 3 days, and on the day of COM administration, 1 hour before exposure (total 7 times). GLUCAN at 2, 1, 0.5, 0.2 and 0.1% concentrations were applied at a dose of 1 g / kg, and 50 μl of 10 μg / ml COM (Sigma, USA) was injected intradermally into the epidermal skin to remove the histamine. Local vasodilation was induced. Thirty minutes after the administration of Evans Blue intravenously 30 minutes after COM administration, all experimental animals were sacrificed to observe histopathological changes in the size of the leaked Evans Blue spots, the amount of Evans Blue spots and the triggered sites. Experimental results were compared with DEXA (1, 0.5 and 0.1% in 0.2% CMC · Na) and ATOP application group.

(3-3) Measurement of Evans Blue Spot Diameter and Measurement of Leaked Evans Blue Amount

Thirty minutes after the end of the test, the animals were sacrificed and the dorsal skin removed. The extent of extravasation of Evans Blue due to vasodilation and the diameter of Evans Blue spots were measured using a digital caliper at the mm level.

Circular dorsal skin (10 mm in diameter) was used to measure the amount of Evans blue. The amount of Evans blue leaked (mg / site) was measured using the method of Katayama et al. (Katayama et al., Microbiol Immunol, 1978, 22: 89-101). 1.0 N KOH (1.0 mL) was circularly fused to round skin at 37 ° C. for 48 hours, and then acetone (6.5 mL) was added to the reaction mixture. After vigorous mixing and centrifugation (900 μg, 10 min), the supernatant was measured at 620 nm using a spectrophotometer to observe the amount of dye leaked at mg / site level.

(3-4) Induction of pruritus and evaluation of pruritus frequency using compound 48/80

The effect of GLUCAN on atopic dermatitis was evaluated using a pruritus mouse model induced with the histamine release promoter, Compound 48/80 (COM). The test substance was applied to the nape region once a day for 4 days, and on the COM administration day 1 hour before administration (total 5 times). 1 g / kg GLUCAN at 2, 1, 0.5, 0.2 and 0.1% concentrations were applied, and 100 ml / kg of COM (Sigma, USA) at 3 mg / kg concentration was injected subcutaneously into the dorsal nasal area to allergic to the histamine glass. It caused sexual pruritus. The frequency of pruritus was observed for 20 minutes from 30 minutes after COM administration. Experimental results were compared with DEXA (1, 0.5 and 0.1% in 0.2% CMC · Na) and ATOP application group.

Thirty minutes after the COM injection, the number of pruritus appearing in the whole body and the COM injection site was observed for 20 minutes.

(3-5) Pruritus induction and evaluation of pruritus frequency using platelet activating factor (PAF)

The effect of GLUCAN on atopic dermatitis was evaluated using a pruritus mouse model induced by Platelet Activating Factor (PAF). Dd Y mice were used as experimental animals. The test substance was applied to the nape of the neck once a day for 4 days, and on the PAF administration day 1 hour before administration (total 5 times). 1 g / kg of GLUCAN at 2, 1, 0.5, 0.2, and 0.1% concentrations were applied, and 10 ml / kg of PAF (Funakoshi Co., Ltd, JAPAN) at 1 μg / kg concentration was injected subcutaneously into the dorsal nasal region. It caused sexual pruritus. The number of times of pruritus was observed for 20 minutes from 30 minutes after PAF administration. Experimental results were compared with DEXA (1, 0.5 and 0.1% in 0.2% CMC · Na) and ATOP application group.

(3-6) Induction of pruritus and evaluation of pruritus frequency using protease (PA)

The effect of GLUCAN on atopic dermatitis was evaluated using a pruritus mouse model induced by the mast cell activator protease (PA). Dd Y mice were used as experimental animals. Test substance was applied to the nape of the neck once a day for 4 days, and 1 hour before administration on the day of PA administration (total 5 applications). 1 g / kg GLUCAN at 2, 1, 0.5, 0.2 and 0.1% concentrations were applied and 10 ml / kg of PA (Worthington Biochemical Corp., USA) at a concentration of 1 mg / kg was injected subcutaneously into the dorsal nasal region. It caused pruritus. The number of times of pruritus was observed for 20 minutes from 30 minutes after PA administration. Experimental results were compared with DEXA (1, 0.5 and 0.1% in 0.2% CMC · Na) and ATOP application group.

(3-7) Allergic pruritus induction and evaluation of pruritus frequency using mosquito salivary gland extract

The effect of GLUCAN on atopic dermatitis was assessed using an allergic pruritus mouse model induced by Mosquito Salivary Gland Extracts (SGE). ICR mice were used as experimental animals. The test substance was applied to the SGE administration site twice a day for 4 days, and the final SGE administration was applied 1 hour before administration (total 9 applications). 1 g / kg of GLUCAN at 2, 1, 0.5, 0.2 and 0.1% concentrations were applied and sensitized by intradermal injection twice a week for 4 weeks in the back dorsal area of 10 μg / 50 μl of SGE. On one day, the same amount of SGE was injected intradermal into the nose to cause pruritis. Immediately after the last SGE administration, the number of pruritus during 1 hour was observed. Experimental results were compared with DEXA (1, 0.5 and 0.1% in 0.2% CMC · Na) and ATOP application group.

(3-8) DNFB Induction of Contact Dermatitis and the Frequency of Ear Edema and Pruritus

The effect of GLUCAN on atopic dermatitis was sensitized with DNP-OVA (Dinitrophenyl-derivatized ovalbumin), and then evaluated using a Type I contact dermatitis mouse model induced by DNFB (2,4-dinitofluorobenzene) application. .

Dd Y mice were used as experimental animals, and only animals showing edema 10% or more through preliminary reactions were used for the experiment (n = 10). The experimental animals were divided into two groups: edema measurement and pruritus measurement. 0.1% DNFB (in EtOH) in the ears and soles, respectively, after 7 days of sensitization with 0.2 mL of physiological saline containing DNP-OVA (10 μg) and aluminum hydroxide gel (1 mg). Μl was applied to cause contact dermatitis. The test substance was applied to the antigen-exposed site once a day from the sensitized day for 6 days, and on the antigen-exposed day 1 hour before the exposure (total 7 times). GLUCAN at 2, 1, 0.5, 0.2 and 0.1% concentrations were applied 1 g / kg, and the thickness of the ears was measured immediately before antigen application, 1 hour after application (IPR) and 24 hours (LPR) to evaluate the effect on edema. The frequency of pruritus was evaluated for 1 hour immediately after the antigen was applied to the sole of the foot. Experimental results were compared with DEXA (1, 0.5 and 0.1% in 0.2% CMC · Na) and ATOP application group.

(3-9) Evaluation of Delayed Contact Dermatitis and Ear Edema Using Picryl Chloride (PC)

The effect of GLUCAN on atopic dermatitis was evaluated using a Type IV contact dermatitis mouse model induced by picryl chloride (PC).

Dd Y mice were used as experimental animals. 0.1 ml of 7% PC solution (dissolved in EtOH) was applied to the depilated abdominal skin, and after 7 days of sensitization, 0.02ml of 1% PC solution (dissolved in olive oil) was applied to the inner surface of each ear for 24 hours. Post delayed contact dermatitis was induced. The test substance was applied to the ear once a day from the day of sensitization for 6 days, and 16 hours after the exposure on the day of antigen exposure (total 7 times). GLUCAN at 2, 1, 0.5, 0.2 and 0.1% concentrations were applied 1 g / kg, and the thickness of the ears was measured immediately before and 24 hours after antigen application to evaluate the effect on edema. Experimental results were compared with DEXA (1, 0.5 and 0.1% in 0.2% CMC · Na) and ATOP application group.

(4) histopathological examination

At the end of the experiment skin samples were fixed in 10% neutral buffered formalin. After paraffin embedding, sections were sliced to 3-4 μm and stained with Masson Trichrome. Then, the histological structure of each site was observed using an optical microscope.

Histomorphometry: Using an automated image analyzer (DMI 100-D digital CCD system; DMI, Korea), the percentage of edema in the dermis and the long axis length (μm) of the edema are measured and analyzed for PCA response. The extent of the edema site caused by this was evaluated.

(5) statistical analysis

All data were calculated as mean ± S.D (n = 10). Statistical analysis was performed by Mann-Whitney U-Wilcoxon Rank Sum W test using SPSS for Windows (Release 6.1.3., SPSS Inc., USA). The inhibition rate compared to the media control was calculated according to Equation 1 below to determine the effect of the test substance on the difference between the media control and the experimental group.

[Equation 1]

% Change from media control =

[(Data of Test Group-Data of Media Control) / Data of Media Control] × 100

(6) In vitro  Cell proliferation

HaCa T cells were cultured in 96-well plates at 1 × 10 μL / well concentration. Culture conditions were followed the conventional HaCa T cell culture conditions. Β-glucan of the present invention was added to the cultured cells at concentrations of 0.02 mg / ml and 0.05 mg / ml, and then cultured for 1, 2, 3, 4, and 5 days, and cells were obtained to measure absorbance. Three experiments were carried out for each condition, and the measured absorbance values were expressed as mean W SEM.

(7) In vitro  Collagen Expression

HaCa T cells were cultured at 1 × 10 μL / well concentration. Culture conditions were followed the conventional HaCa T cell culture conditions. The cultured cells were incubated for 3 days by adding β-glucan of the present invention at 20 μM and 50 μM concentrations. After culturing, the cells were obtained, the protein was extracted, and the expression level of the collagen protein was confirmed by Western blotting.

2. Experimental Results

(1) Effect on PCA-induced model

(1-1) size change of Evans blue spots

As shown in Table 2 and FIG. 1, the size of Evans blue spots was compared to media control in DEXA 1, 0.5 and 0.1%, GLUCAN 2, 1, 0.5, 0.2 and 0.1%, ATOP 1 g / kg application group, respectively. -46.69%, -38.12%, -33.91%, -53.66%, -43.90%, -37.61%, -34.98%, -20.45% and -33.33%. Thus, a significant (p <0.01) reduction in Evans blue spots caused by chart dose dependent vasodilation was recognized in all test substance application groups.

TABLE 2

N = 10 in the above table; (Mean ± SD); ¹⁾ percent change relative to media control; ^* P <0.01 compared to control by MW test.

(1-2) Change of leaked Evans Blue

As shown in Table 2, the amount of leaked Evans blue was -72.92%, respectively, compared to the media control in the DEXA 1, 0.5 and 0.1%, GLUCAN 2, 1, 0.5, 0.2 and 0.1% and ATOP 1 g / kg application group, -68.61%, -58.44%, -79.71%, -75.15%, -70.27%, -56.57%, -33.99% and -41.74%. Therefore, all test substance application groups recognized a significant (p <0.01) decrease in the amount of Evans Blue leaked by the chart dose dependent vasodilation compared to the media control.

(1-3) Histopathological Test Results

As shown in Table 3 and FIG. 2, significant local dermal edema due to vasodilation was recognized at the site where the OVA antiserum and OVA induced the PCA response in rats. A significant decrease was observed. In addition, the proportion of focal edema in the dermis and the long axis length of the edema were significantly reduced (p <0.01) in all the test substance application groups compared to the media control.

Histopathology: The proportion of focal edema sites in the dermis is -57.55%, -49.35%, -DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP-treated groups, respectively. 35.66%, -67.55%, -54.89%, -40.11%, -34.17%, -32.20% and -34.40%. Long axis length of edema site was -73.58%, -69.95%, -60.27%, -78.65% in DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP groups , -67.28%, -64.17%, -61.75%, -45.01% and -48.83%.

TABLE 3

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to media control; All histopathology is performed using an automated image analyzer; ^* P <0.01 compared to control by MW test.

In conclusion, GLUCAN is considered to be very effective for allergic dermatitis caused by PCA reaction, and the possibility of developing it as a coating agent for allergic dermatitis and atopic dermatitis is recognized to some extent. The application of GLUCAN was observed to show similar vasodilation and resulting edema inhibition effect with the same amount of DEXA application, with a similar effect to ATOP in 0.2% GLUCAN. A very good effect was recognized in GLUCAN 0.1%, so the effective concentration for allergic dermatitis caused by PCA response is considered to be around 0.1%.

(2) Effect on COM-induced local vasodilation model

(2-1) Evans blue spot size change

As shown in Table 4 and Figure 3, the size of Evans blue spots was -46.08, -41.70 in DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP applied groups, respectively, compared to the media control group. , -27.28, -54.00, -45.56, -38.57, -32.22, -21.61 and -32.11%. Therefore, a significant (p <0.01) reduction in Evans blue spots caused by chart dose dependent vasodilation was recognized in all test substance application groups.

TABLE 4

N = 10 in the above table; (Mean ± SD); ¹⁾ percent change relative to media control; ^* P <0.01 compared to media control by MW test.

(2-2) Change in the amount of leaked Evans Blue

As shown in Table 4, the amount of leaked Evans blue was -52.88, -42.77, -34.45 in the DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP applied groups, respectively, compared to the media control group. , -68.98, -55.21, -49.70, -37.87, -23.18 and -36.89%. Thus, a significant (p <0.01) reduction in the amount of Evans Blue leaked by the chart dose dependent vasodilation was recognized in all test subject application groups.

(2-3) Histopathological Test Results

As shown in Table 5 and FIG. 4, local dermal edema due to vasodilation was recognized at the site of COM administration, but such local dermal edema was observed to be significantly reduced compared to the medium control group in all test substance application groups. In addition, in all the test substance application groups, the proportion of the local edema site and the long axis length of the edema site were significantly reduced (p <0.01 or p <0.05) depending on the application compared to the medium control group.

Histopathology: The proportion of focal edema sites in the dermis is -60.17, -46.20, -23.87, DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP group compared to the media control, respectively. Changes of -60.07, -51.37, -39.35, -29.42, -16.01, and -33.72%. Long axis length of edema site was -62.20, -52.02, -35.56, -66.20, -63.90, DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP group Changes of -54.24, -44.17, -27.66 and -44.21%.

TABLE 5

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to media control; All histopathology is performed using an automated image analyzer; ^* P <0.01 and ^** p <0.05 compared to control by MW test.

(2-4) Consideration

Taken together, GLUCAN is considered to be very effective for allergic dermatitis caused by COM intradermal administration, and the possibility of developing it as a coating agent for allergic dermatitis and atopic dermatitis is recognized to some extent. The application of GLUCAN was observed to show similar vasodilation and resulting edema inhibition effect as the same amount of DEXA application, with a similar effect to ATOP in 0.2% GLUCAN. A very good effect is also recognized in GLUCAN 0.1%, so the effective concentration for histamine free allergic dermatitis caused by COM is considered to be around 0.1%.

(3) Effects in COM-Induced Pruritus Model

(3-1) changes in pruritus frequency

As shown in Table 6, the frequency of pruritus showed a change of 1461.02% in the medium control group compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP application The group showed changes of -74.00, -68.22, -59.72, -76.06, -73.34, -60.10, -47.83, -41.15 and -27.69%, respectively, compared to the medium control group. Therefore, the media control group showed a significant increase in the frequency of pruritus (p <0.01) compared to the normal group, but in all test substance application groups, the dose-dependent allergy was significant (p <0.01 or p <0.05) compared to the media control group. Reduction in the frequency of sexual pruritus was recognized, respectively.

TABLE 6

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to normal media control (sham); ^* P <0.01 compared to normal medium control by MW test; And ^# p <0.05, ^## p <0.05 compared to normal media control by MW test.

(3-2) Consideration

Taken together, GLUCAN is considered to be very effective for allergic pruritus caused by subcutaneous administration of COM, and is likely to be developed as an application for external skin diseases such as allergic dermatitis and atopic dermatitis. It is admitted. It was observed that the application of GLUCAN showed an antipruritic effect similar to that of the same amount of DEXA application, and an excellent antipruritic effect was observed compared to ATOP even at the minimum concentration of GLUCAN 0.1% used in this experiment. A very good effect was also recognized in GLUCAN 0.1%, so the effective concentration for histamine free allergic pruritis induced by COM is considered to be less than 0.1%.

(4) effects in PAF-induced pruritus model

(4-1) Frequency change of pruritus

As shown in Table 7, the frequency of pruritus showed a change of 606.93% in the medium control group compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP application The group showed changes of -70.59, -51.68, -37.39, -71.57, -56.02, -51.12, -44.12, -38.80 and -28.15%, respectively, compared to the media control. Therefore, in the media control group, a significant (p <0.01) pruritic frequency was observed compared to the normal group, but in all test substance application groups, a significant (p <0.01 or p <0.05) table dose-dependent relationship was observed. Reductions in the frequency of allergic pruritus were recognized, respectively.

TABLE 7

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to normal media control (sham); ²⁾ percent change relative to media control; ^* P <0.01 compared to normal medium control by MW test; And ^# p <0.05, ^## p <0.05 compared to media control by MW test.

(4-2) Consideration

In conclusion, GLUCAN is considered to be very effective for allergic pruritus caused by subcutaneous administration of PAF, and it is recognized that it can be developed as an application for external skin diseases such as allergic dermatitis and atopic dermatitis. do. The application of GLUCAN showed an effect of inhibiting pruritus similar to the application of DEXA of the same amount, except for the highest concentration of 1% group. It became. In the case of GLUCAN 0.1%, a very good effect is recognized, and the effective concentration for allergic pruritus induced by PAF is considered to be 0.1% or less.

(5) Effects in PA-Induced Pruritus Model

(5-1) Frequency change of pruritus

As shown in Table 8, the frequency of pruritus showed a 808.33% change in the media control compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP application The group showed changes of -67.50, -60.81, -57.01, -70.90, -62.39, -58.98, -49.41, -38.79 and -25.69%, respectively, compared to the media control. Therefore, in the media control group, a significant (p <0.01) pruritic frequency was observed compared to the normal group, but in all test substance application groups, a significant (p <0.01 or p <0.05) table dose-dependent relationship was observed. Reduction in the frequency of allergic pruritus was recognized, respectively.

TABLE 8

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to normal media control (sham); ²⁾ percent change relative to media control; ^* P <0.01 compared to normal medium control by MW test; And ^# p <0.05, ^## p <0.05 compared to media control by MW test.

(5-2) Consideration

Taken together, GLUCAN is considered to be very effective for allergic pruritus caused by subcutaneous administration of PA. It is admitted. It was observed that the application of GLUCAN showed an antipruritic effect similar to that of the same amount of DEXA application, and an excellent antipruritic effect was observed compared to ATOP even at the minimum concentration of GLUCAN 0.1% used in this experiment. A very good effect was also found in GLUCAN 0.1%, and the effective concentration for allergic pruritus caused by PA is considered to be less than 0.1%.

(6) Effects of Mosquito Salivary Gland Extract Induced Allergic Pruritus Model

(6-1) Frequency change of pruritus

As shown in Table 9, the frequency of pruritus showed a change of 384.16% in the media control group compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP application The group showed changes of -54.18, -43.56, -32.24, -53.38, -51.34, -42.60, -32.30, -24.89 and -20.23%, respectively, compared to the media control. Therefore, in the media control group, a significant (p <0.01) pruritic frequency was found to be higher than that in the normal group, but in all the test substance application groups, the graphic dose-dependent allergic pruritus was significant (p <0.01) compared to the media control group. The decrease in the frequency of was recognized respectively.

TABLE 9

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to normal media control (sham); ²⁾ percent change relative to media control; ^* P <0.01 compared to normal medium control by MW test; And ^# p <0.05 compared to media control by MW test.

(6-2) Consideration

In conclusion, GLUCAN is considered to be very effective for allergic pruritus caused by intradermal administration of SGE, and it is possible to develop it as an application for external skin diseases such as allergic dermatitis and atopic dermatitis. It is admitted. The application of GLUCAN showed an antipruritic effect similar to that of the same amount of DEXA application, and even better than that of ATOP, GLUCAN 0.1%, the minimum concentration used in this experiment, was recognized. In GLUCAN 0.1%, a very good effect is recognized, and the effective concentration for allergic pruritus caused by insect bites is considered to be 0.1% or less.

(7) Effect in DNFB-induced contact dermatitis model

(7-1) Changes in Ear Edema

As shown in Table 10, the change in ear thickness at IPR (after 1 hour of application) showed a change of 223.19% in the medium control group compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5 , 0.2, 0.1% and ATOP applied group showed -43.44, -36.82, -23.62, -45.86, -38.58, -33.82, -26.64, -20.17 and -17.85%, respectively, compared to the media control group. Changes in ear edema at LPR (24 hours after application) showed a change of 561.27% in the medium control group compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP The application group showed changes of -42.38, -34.41, -22.50, -44.57, -38.43, -28.39, -12.93, -13.50 and -6.54%, respectively, compared to the media control group. Therefore, in the media control group, a significant (p <0.01) increase in ear thickness was observed compared to the normal group, but in all test substance application groups, a significant decrease in graph dose-dependent ear edema was observed in IPR and LPR, respectively, compared to the media control group. It became.

TABLE 10

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to normal media control (sham); ²⁾ percent change relative to media control; ^* P <0.01 compared to normal medium control by MW test; And ^# p <0.01 and ^## p <0.05 compared to media control by MW test.

(7-2) Frequency change of pruritus

As shown in Table 11, the incidence of pruritus showed a change of 159.59% in the medium control group compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP application The group showed changes of -73.03, -63.29, -51.81, -73.11, -68.08, -65.33, -53.38, -36.01 and -19.56%, respectively, compared to the media control. Therefore, in the media control group, a significant (p <0.01) pruritic frequency was observed compared to the normal group, but in all the test substance application groups, a significant (p <0.01 or p <0.05) chart dose-dependent compared to the media control group. A decrease in the frequency of allergic pruritus was recognized, respectively.

TABLE 11

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to normal media control (sham); ²⁾ percent change relative to media control; ^* P <0.01 compared to normal medium control by MW test; And ^# p <0.01 and ^## p <0.05 compared to media control by MW test.

(7-3) Consideration

Taken together, GLUCAN is considered to be very effective for allergic dermatitis and pruritis caused by DNFB, and the possibility of developing it as a coating agent for allergic dermatitis and atopic dermatitis is recognized to some extent. The application of GLUCAN showed similar edema and pruritus-inhibiting effects similar to the application of DEXA, and even at 0.1% GLUCAN, the minimum concentration used in this experiment, superior edema and pruritus-inhibitory effects were recognized. In the case of GLUCAN 0.1%, a very good effect is recognized, and the effective concentration for allergic dermatitis caused by DNFB is considered to be around 0.1%.

(8) Effects on PC-induced delayed contact dermatitis

(8-1) Changes in Ear Edema

As shown in Table 12, changes in ear edema showed 573.20% change in the media control group compared to the normal group, while DEXA 1, 0.5, 0.1%, GLUCAN 2, 1, 0.5, 0.2, 0.1% and ATOP application The group showed changes of -73.45, -63.10, -51.24, -72.37, -60.21, -56.25, -47.27, -25.37 and -31.82%, respectively, compared to the media control. Thus, a significant (p <0.01) increase in ear thickness was observed in the media control group compared to the normal group, but in all test substance application groups a significant (p <0.01 or p <0.05) table dose compared to the media control group. Reduction of dependent ear edema was recognized, respectively.

TABLE 12

In the above table, n = 10; (Mean ± SD); ¹⁾ percent change relative to normal media control (sham); ²⁾ percent change relative to media control; ^* P <0.01 compared to normal medium control by MW test; And ^# p <0.01 and ^## p <0.05 compared to media control by MW test.

(8-2) Consideration

Taken together, GLUCAN is expected to be very effective for PC-induced delayed allergic dermatitis (type 4 allergic dermatitis, and it is possible to develop it as an application for external allergic dermatitis and atopic dermatitis). The application of GLUCAN was observed to have similar edema and pruritus inhibitory effect as the same amount of DEXA application, and the minimal concentration used in this experiment, GLUCAN 0.1%, showed a similar effect to ATOP. The effect was recognized to some extent, and the effective concentration for delayed allergic dermatitis induced by PC is considered to be about 0.1%.

(9) Glucan promotes skin cell regeneration

As shown in Figure 5, GLUCAN increased the degree of cell proliferation in a dose-dependent manner compared to the control. In addition, as shown in Figure 6, GLUCAN increased the synthesis of collagen, an epidermal cell component.

Therefore, it can be seen that GLUCAN is a substance that promotes cell regeneration, and it can be effectively used for cell regeneration such as wound healing, wound healing and burn healing.

The pharmaceutical composition comprising the glucan of the present invention as an active ingredient having excellent therapeutic effects against allergic dermatitis and atopic dermatitis and having an excellent effect on promoting skin cell regeneration can be administered parenterally and orally. Formulations were prepared as syrups and tablets.

Preparation Example 1 Preparation of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of glucan, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Glucan 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

Preparation Example 2 Preparation of Syrup

Syrup containing an acid addition salt of glucan of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) is prepared by the following method.

Acid addition salts of glucan, saccharin and sugars were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to this mixture to 100 ml.

The components of the syrup are as follows.

2 g of acid addition salt of glucan

Saccharin: 0.8 g ················

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·····················

Preparation Example 3 Manufacturing Method

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of glucan was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Glucan 250 g

Lactose ···················· 175.9 g

Potato starch ·············· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch · 160 g

Talc · 50 g

Magnesium stearate ·········· 5

As described above, the present invention is a pharmaceutical composition for the prevention and treatment of dermatitis comprising glucan as an active ingredient is excellent in the skin cell regeneration effect and the suppression of allergic edema and pruritus, the treatment of skin damage such as wounds and burns It is very effective in treating allergic and atopic dermatitis.

Claims (5)

A pharmaceutical composition for treating dermatitis comprising glucan and a pharmaceutically acceptable carrier thereof as an active ingredient. The pharmaceutical composition for treating dermatitis according to claim 1, wherein the dermatitis is allergic or atopic dermatitis. The pharmaceutical composition for treating dermatitis according to claim 1, wherein the therapeutically effective dose of glucan is 0.1 to 0.2% by weight. The pharmaceutical composition for treating dermatitis according to claim 1, wherein the composition is provided for skin cell regeneration. The pharmaceutical composition for treating dermatitis according to claim 4, wherein the composition is provided for wound healing, wound treatment and burn treatment.

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