JP4961772B2 - Compound selected from sulfated cellulose and its salt, and therapeutic agent for dermatitis - Google Patents

Description

  The present invention relates to a compound selected from sulfated cellulose excellent in hydrolysis resistance and a salt thereof, and a dermatitis therapeutic agent and a cosmetic using the same. Specifically, a compound selected from sulfated cellulose and a salt thereof that can be used as an active ingredient of an external preparation for skin, which is specifically intended to prevent, alleviate, ameliorate, or cure atopic skin symptoms, and the same The present invention relates to a dermatitis therapeutic agent and a cosmetic material using the above.

  It is known that heparin, which is a typical biological polymer electrolyte, has various physiological activities such as anticoagulant activity, antiviral activity, and anti-inflammatory activity (see, for example, Non-Patent Document 1). . As an attempt to obtain heparin analogs exhibiting various physiological activities, many natural polysaccharides have been sulfated so far (see, for example, Non-Patent Document 2). Among many sulfated polysaccharides, in particular, chondroitin sulfate, which is a kind of polysulfide mucopolysaccharide, is known as a typical heparin-like substance (see, for example, Patent Document 1). Chondroitin sulfate and its sulfate group-introduced derivatives have hyaluronidase inhibitory activity and skin moisturizing action, so dry skin diseases such as xerosis, sebum deficiency, progressive palmar keratoderma and atopic properties Application to dermatitis has been studied (for example, see Patent Documents 2 and 3). In addition, sulfated polysaccharides have been reported to show the inhibitory activity of Staphylococcus aureus-derived hyaluronidase (see, for example, Patent Document 4), and the possibility of sulfated polysaccharides as therapeutic agents for atopic dermatitis is described. . In addition, studies on inhibition of hyaluronidase activity and anti-inflammatory effect of sulfated sucrose have been carried out (see, for example, Patent Document 5).

  Anti-inflammatory action and tissue regeneration action by hyaluronidase activity inhibition are considered to be due to the following mechanism. Degradation of hyaluronic acid and glycosaminoglycans by hyaluronidase disrupts the cell surface or support matrix material, exposing the cells, and by various agents such as pathogens, inflammatory mediators, inflammatory agents, and preservatives Get damaged. Hyaluronidase activity inhibitors, such as sulfated polysaccharides, promote the regeneration of the cell surface and the protective connective tissue matrix by inhibiting the activity of the enzyme, resulting in anti-inflammatory and tissue regeneration effects. Conceivable.

  Thus, for the purpose of improving or curing atopy-like skin symptoms, sulfates such as hyaluronic acid, chondroitin sulfate, dextran, etc. have been intensively studied using the inhibition of hyaluronidase activity as an index. There has been no detailed study on the inhibitory activity of hyaluronidase. Similarly, there has been no detailed study on the confirmation of the efficacy of sulfated cellulose and its salts against atopic dermatitis. As one of the causes, it is considered that natural cellulose has various structures and molecular weights, and the properties of the obtained sulfated cellulose and its salt are not constant and are likely to change over time due to hydrolysis or the like.

In addition, steroidal anti-inflammatory drugs are used for the treatment of atopic dermatitis in the medical field, but these steroidal drugs have strong side effects and are contraindicated for administration of steroids to particularly mild patients. This is the current situation. On the other hand, non-steroidal anti-inflammatory agents are generally less potent. Therefore, development of a therapeutic agent for atopic dermatitis having a sufficient effect at a low concentration without causing side effects is desired. Moreover, in order to maintain the quality of the product on the formulation even for cosmetics for sensitive skin and atopy-like skin, a base material that is highly safe and exhibits high efficacy at a low concentration is required. Furthermore, from the viewpoint of safety, for natural materials, since the occurrence of BSE (mad cow disease), a person skilled in the art dealing with medical care or cosmetics tends to seek materials originating from other than animal species. In particular, there is a demand for the development of plant-derived substances that have performances higher than those of heparin-like substances described in the Japanese Pharmacopoeia Pharmaceutical Standards and originate from sources other than animal species.
Japanese Examined Patent Publication No. 62-4362 JP-A-8-277224 JP 11-335288 A JP 2000-178196 A Japanese Patent No. 2723473 Int. J. Biochem. Cell. Biol. 29, 709-714, 1997 Trend in Glycoscience and Glycotechnology Vol.15, No.81, pp. 29-46

  An object of the present invention is to provide sulfated cellulose excellent in hydrolysis resistance, which can be used as an active ingredient of a topical skin preparation produced with the intention of preventing, alleviating, ameliorating or curing atopic skin symptoms, for example. And a compound selected from the salts thereof, a dermatitis therapeutic agent and a cosmetic using the same, and the like.

  The inventors of the present invention have intensively studied to solve the above problems. As a result, a compound selected from sulfated cellulose obtained from crystalline cellulose and a salt thereof, and a compound having a solubility in pure water at 20 ° C. of 3 g / L or more is an existing chondroitin sulfate (heparin-like substance), etc. As compared with the above, it was found that it exhibits a high hyaluronidase inhibitory activity and a marked improvement effect on atopic dermatitis, and the present invention was completed based on this finding.

The present invention is constituted as follows.
1) A compound selected from sulfated cellulose obtained from crystalline cellulose and a salt thereof, and having a solubility in pure water at 20 ° C. of 3 g / L or more.
2) The compound according to 1) above, wherein the crystalline cellulose has an average degree of polymerization of 150 to 300.
3) The compound according to 1) or 2), wherein the sulfur content is 6.5 to 19.0% by weight.
4) The compound according to any one of 1) to 3), wherein the weight average molecular weight is 1,000 to 200,000.
5) The compound according to any one of 1) to 4) above, which is a sulfated cellulose calcium salt or a sulfated cellulose sodium salt.
6) A compound selected from sulfated cellulose obtained from crystalline cellulose and a salt thereof, having a sulfur content of 6.5 to 19.0% by weight and a weight average molecular weight of 50,000 to 80,000. A compound having a solubility in pure water at 20 ° C. of 3 g / L or more.
7) The compound according to 6) above, which is a sulfated cellulose calcium salt or a sulfated cellulose sodium salt.
8) In a 0.1 mol / L acetate buffer solution (pH 4.0) at a temperature of 37 ° C., the concentration that inhibits the hyaluronic acid decomposing activity of bovine testis-derived hyaluronidase by 50% is 0.018 mg / mL or less 1) The compound according to any one of items 7 to 7).
9) The compound according to any one of 1) to 7) above, which exhibits a dermatitis inhibitory action against a dermatitis model using NC mice.
10) A dermatitis therapeutic agent containing at least one compound according to any one of items 1) to 9).
11) The dermatitis therapeutic agent according to 10) above, which is for treating atopic dermatitis.
12) A cosmetic comprising at least one compound according to any one of items 1) to 9).

  The compound selected from the sulfated cellulose and the salt thereof of the present invention is excellent in hydrolysis resistance. This therapeutic agent for dermatitis containing a sulfated cellulose compound as an active ingredient is stable in quality and hardly deteriorates over time, and is particularly effective in preventing atopic skin symptoms or reducing, improving or healing the symptoms. is there.

A compound selected from the sulfated cellulose of the present invention and a salt thereof (hereinafter referred to as a sulfated cellulose compound) is a compound obtained by electrolyzing crystalline cellulose to give water solubility or a salt thereof. Since cellulose used as a raw material for the sulfated cellulose compound is crystalline cellulose, the obtained sulfated cellulose compound is excellent in hydrolysis resistance, and the dermatitis therapeutic agent obtained from this sulfated cellulose compound is highly effective. Difficult to decrease over time.
Moreover, the solubility with respect to the pure water of 20 degreeC of the sulfated cellulose compound of this invention is 3 g / L or more, Preferably it is 15 g / L or more and less than 20 g / L. If the solubility is 3 g / L or more, the sulfated cellulose compound of the present invention is electrolyzed and imparts water solubility, and is particularly effective for preventing or alleviating, ameliorating, or curing the symptoms of atopic skin. It is.

The crystalline cellulose used in the sulfated cellulose compound of the present invention may be of any kind as long as it is derived from plants. However, when the average degree of polymerization is 150 to 300, particularly about 240, the crystalline cellulose is highly crystalline and chemically stable. Is easily obtained. The sulfated cellulose compound obtained by using the crystalline cellulose is also chemically stable and excellent in hydrolysis resistance. As a result, the effectiveness of the dermatitis therapeutic agent containing the sulfated cellulose compound as an active ingredient decreases with time. Hard to do.
The crystalline cellulose used in the sulfated cellulose compound of the present invention is preferably a product listed in the 13th revised Japanese Pharmacopoeia, such as cellulose microcrystals (Wako Pure Chemical Industries, Ltd., Osaka City) or crystalline cellulose (trade name). Commercially available products such as Theolas, Asahi Kasei Corporation, Tokyo) can be used. The crystallinity of the crystalline cellulose used in the present invention (measured by X-ray diffractometry [Avicel Times, No. 30, 1973]) is preferably 63% or more.
The weight average molecular weight of the sulfated cellulose compound of the present invention is not particularly limited, but is preferably 1,000 to 200,000, more preferably 50,000 to 80,000. Since the sulfated cellulose compound having a weight average molecular weight in the above range is obtained from the above crystalline cellulose having high crystallinity, it is chemically stable and suitable as an active ingredient of a dermatitis therapeutic agent.

  Although the method for sulfating crystalline cellulose is not particularly limited, for example, it can be performed as follows. First, crystalline cellulose is swollen with a solvent such as pyridine, dimethyl sulfoxide, dimethylformamide, and chlorsulfonic acid, piperidine-N-sulfuric acid, sulfuric anhydride-dimethylformamide complex, sulfur trioxide-pyridine complex, sulfur trioxide as a sulfating agent. -A trimethylamine complex, a sulfuric acid-trimethylamine complex, etc. are dripped. The amount of the sulfating agent used can be arbitrarily selected according to the sulfation rate (or sulfur content) of the desired sulfated cellulose and the reaction conditions, but 1.2 to 3 equivalents are used with respect to the cellulose hydroxyl group. Is appropriate. The reaction varies depending on the solvent and the sulfating agent, but is carried out in an inert gas at 0 to 100 ° C., preferably 20 to 85 ° C., for 0.5 to 24 hours, preferably 0.5 to 10 hours. After the reaction, methanol, ethanol, isopropyl alcohol, acetone or the like is added to precipitate the polymer. Alternatively, the reaction solution may be dropped into methanol, ethanol, isopropyl alcohol, acetone or the like to precipitate the polymer. Alternatively, the reaction may be stopped by adding distilled water and then neutralized with an alkali such as sodium hydroxide. This is filtered or centrifuged, dissolved in distilled water, ethanol, isopropyl alcohol, acetone, etc. are added again to precipitate the polymer, or dropped into ethanol, isopropyl alcohol, acetone, etc., to precipitate the polymer, The sulfated cellulose can be obtained by drying. Alternatively, excess inorganic salt may be removed using an ion exchange resin column, and ethanol, isopropyl alcohol, acetone, or the like may be added to precipitate the polymer. Alternatively, sulfated cellulose can be obtained by dissolving in distilled water, dialyzing, and concentrating to dryness.

Sulfated cellulose can also be used as a pharmaceutically and physiologically acceptable salt form. For example, a salt form obtained by a salt formation reaction using an alkaline earth metal salt such as calcium or an inorganic salt such as an alkali metal salt such as sodium or potassium, or an amine such as cyclohexylamine can be used. In particular, calcium salts and sodium salts are more preferable because of simplicity in the production process. Among these, calcium salts are expected to be effective against various dermatitis symptoms.
The sulfated cellulose calcium salt can be obtained, for example, by adding a calcium chloride aqueous solution to a sulfated cellulose aqueous solution and neutralizing it. In this case, the concentration of the sulfated cellulose aqueous solution is not particularly limited, but is preferably about 5 to 20% by weight. The concentration of the aqueous calcium chloride solution is not particularly limited, but a saturated aqueous solution is preferred. In order to precipitate and precipitate the sulfated cellulose calcium salt, an alcoholic or other amphiphilic solvent may be used, but methanol, ethanol, and isopropyl alcohol are preferable.

  The sulfur content of the sulfated cellulose compound is preferably 6.5 to 19.0% by weight, and more preferably 12 to 15% by weight. If the sulfur content is in the above range, it exhibits remarkable hyaluronidase inhibitory activity.

The sulfated cellulose compound of the present invention can exhibit hyaluronidase inhibitory activity. The inhibitory activity of hyaluronidase can be determined by the Reissig method (J. Biol. Chem., 217, 959, 1955) obtained by improving the Morgana-Elson method. For example, the method for determining the hyaluronidase 50% inhibitory concentration can be performed by the method described in Japanese Patent No. 3066484.
In addition, the measurement of the inhibitory activity of hyaluronidase due to the difference in the degree of sulfation is carried out by adding a certain concentration of hyaluronidase to a solution composed of hyaluronic acid as a substrate and a sulfated cellulose compound of various sulfation degrees, followed by reaction for a certain period of time It can be determined by analyzing the degradation rate of hyaluronic acid.
The sulfated cellulose compound of the present invention has a concentration of 0.018 mg / l in a 0.1 mol / L acetate buffer solution (pH 4.0) at a temperature of 37 ° C., which inhibits the hyaluronic acid degradation activity of hyaluronidase derived from bovine testicles by 50%. It is preferably at most mL, more preferably at most 0.015 mg / mL. If the concentration is 0.018 mg / mL or less, it is generally recognized that there is a significant hyaluronidase inhibitory activity, and a high effect as a dermatitis therapeutic agent can be expected.

  The medicinal effect of the sulfated cellulose compound of the present invention against atopic dermatitis can be confirmed by an animal test using NC mice, for example, as in the case of Hirasawa et al. (Hirasawa et al. Pharmacometrics, 59, 123- 134, 2000). NC mice are model animals that spontaneously develop symptoms very similar to human atopic dermatitis, using mites and Staphylococcus aureus as antigens when bred in a conventional environment, and are widely used for the development of drugs for the treatment of atopic dermatitis (CRJ Letters, 11, 1-8, 1998). A certain amount of the sulfated cellulose compound of the present invention is administered at regular time intervals to the affected part of NC mice that have developed symptoms, and the skin condition is observed. The state of dermatitis can be scored by a dermatitis score determined based on an evaluation standard or the like in clinical symptoms of human atopic dermatitis. In that case, the efficacy comparison with the sulfated cellulose compound of the present invention can be made by using existing chondroitin sulfate or the like as a control group. In the present invention, the expression of a dermatitis inhibitory effect on a dermatitis model using NC mice means that the progression of dermatitis symptoms is suppressed by histopathological findings.

  The therapeutic agent for dermatitis of the present invention contains at least one sulfated cellulose compound as an active ingredient. The content of the sulfated cellulose compound in the therapeutic agent for dermatitis varies depending on the form and application site of the therapeutic agent for dermatitis, the method and frequency of application, and can vary widely depending on the severity of symptoms. The content of the sulfated cellulose compound with respect to the total amount of the dermatitis therapeutic agent is preferably 0.001 to 5% by weight, more preferably 0.005 to 1.0% by weight.

  The dermatitis therapeutic agent containing the sulfated cellulose compound of the present invention as an active ingredient may contain other ingredients as long as the pharmacological action is not substantially impaired. Examples of those contained include preservatives, stabilizers, humectants, fragrances, surfactants and the like commonly used in medicines and cosmetics.

  The dermatitis treatment agent of the present invention is intended to prevent atopic skin symptoms, or to alleviate, improve, or cure the symptoms, but it is not only atopy-like but also itching from sensitive skin and dry skin. The intention of prevention, prevention of exacerbation, symptom relief, and treatment for inflammation and allergy-like symptoms is also included.

The therapeutic agent for dermatitis of the present invention is used in the form of lotion, emulsion, ointment and the like. Specific examples include ointments, plasters, aerosols, solutions, suspensions, patches, lotions and the like. Moreover, a liquid agent etc. can also be used by impregnating gauze, absorbent cotton, a wound dressing, an adhesive plaster, etc.
In addition, the dermatitis therapeutic agent of this invention is further used with forms of cosmetics, such as a lotion, a milky lotion, and a cream.

The present invention will be described in more detail, but the present invention is not limited to these examples. Moreover, unless otherwise indicated,% is weight%. The evaluation test methods used in the examples and comparative examples are described below.
1) Solubility Solubility was determined by measuring the weight dissolved in 10 mL of water at a temperature of 20 ° C.
2) Sulfur content The sulfur content was measured by oxygen flask combustion-ion chromatography.
3) Weight average molecular weight of sulfated cellulose compound The weight average molecular weight of the sulfated cellulose compound was measured by gel filtration. That is, pullulan (Pullulan Shodex Standard P-82) is used as the molecular weight standard, the substance to be measured is Shodex OHpak SB-804HQ as the column, 0.2 mol / L sodium chloride solution is used as the moving bed, and the flow rate is 0.5 mL / min. The eluate was analyzed with a differential refractometer, the elution time was measured, and the weight average molecular weight was determined.
4) Average degree of polymerization of crystalline cellulose The average degree of polymerization of crystalline cellulose was measured by the method of confirmation test (3) described in D-586 to 589, published in the 13th revised Japanese Pharmacopoeia Manual (1996, published by Yodogawa Shoten). did.

(Synthesis of sulfated cellulose calcium salt)
Synthesis example 1
In a 3 L (liter) separable flask equipped with a thermometer, a dropping funnel, a stirrer, and a nitrogen gas inlet tube, 180.0 g of crystalline cellulose (Theolus PH-101 (trade name), average polymerization degree 242) and dimethylformamide 160 mL was added and stirred overnight. Next, 1220.75 g of 18% -sulfuric anhydride-dimethylformamide complex was added over 35 minutes under ice-cooling, and the reaction vessel was immersed in a constant temperature bath at 20 ° C., and the temperature was raised over 20 minutes. Reacted.
After completion of the reaction, the reaction vessel is ice-cooled to 5 ° C., the reaction solution is gradually poured into 1220 mL of water over 75 minutes while cooling with ice, and then calcium hydroxide is added until the pH of the reaction solution reaches 7. It was gradually introduced. At this time, calcium hydroxide required for neutralization was 70.82 g. The resulting precipitate was filtered off, the filtrate was poured into 20 L of isopropyl alcohol to precipitate a polymer, filtered, and the filtrate was dried under reduced pressure at 40 ° C. for 8 hours to obtain a sulfated cellulose calcium salt. The yield of the obtained sulfated cellulose calcium salt was 130 g, the solubility in pure water at 20 ° C. was 15 g / L, the sulfur content was 14.0% by weight, and the weight average molecular weight was 77,000.

Synthesis example 2
Into a 3.0 L separable flask equipped with a thermometer, a dropping funnel, a stirrer, and a nitrogen gas inlet tube, 15.0 g of crystalline cellulose (Theolus PH-101) and 75 mL of dimethylformamide are added and stirred at room temperature for 15 hours. did. Next, under ice cooling, 337.5 g of an anhydrous 18.8 wt% sulfuric acid-dimethylformamide solution was added, and then the reaction vessel was immersed in a constant temperature bath at 24 ° C., and the temperature was raised over 20 minutes. Reacted for hours.
The reaction vessel was ice-cooled to 5 ° C., and 375 mL of isopropyl alcohol was added while ice-cooling to obtain a precipitate. After stirring for 30 minutes, the precipitate was filtered off, dissolved in 375 mL of ion-exchanged water cooled to 10 ° C. or lower, and stirred for 15 minutes. Next, 104.0 g of a saturated aqueous calcium chloride solution and 750 mL of isopropyl alcohol were added to cause precipitation. This was filtered off and the filtrate was dried under reduced pressure at 40 ° C. for 8 hours to obtain a sulfated cellulose calcium salt. The yield of the obtained sulfated cellulose calcium salt was 40.3 g, the solubility in pure water at 20 ° C. was 15 g / L, and the sulfur content was 15.4% by weight. The weight average molecular weight was 76,000.
Thereafter, the sulfated cellulose calcium salt of Synthesis Examples 3 to 7 was obtained by reacting in the same manner as in Synthesis Example 2 except that the amount of anhydrous sulfuric acid-dimethylformamide solution used was changed as shown in Table 1. Table 1 shows the solubility, sulfur content, and weight average molecular weight of the sulfated cellulose calcium salt obtained in Synthesis Examples 2 to 7 in pure water at 20 ° C.

(Synthesis of sulfated cellulose sodium salt)
Synthesis Example 8
A 500 mL separable flask equipped with a thermometer, a dropping funnel, a stirrer, and a nitrogen gas inlet tube was charged with 15.0 g of crystalline cellulose (Ceolus PH-101) and 75 mL of dimethylformamide. Next, 193.8% sulfuric anhydride-dimethylformamide complex 163.8 g was added over 22 minutes under ice-cooling, and then the reaction vessel was immersed in a constant temperature bath at 20 ° C., and the temperature was increased over 20 minutes, followed by 6 hours at 20 ° C. Reacted.
The reaction vessel was ice-cooled to 5 ° C., and 500 mL of isopropyl alcohol was added while cooling with ice to obtain a precipitate. After stirring for 30 minutes, the precipitate was filtered off, dissolved in 200 mL of ion-exchanged water cooled to 10 ° C. or lower, and stirred for 15 minutes. Next, 20% aqueous sodium hydroxide solution was gradually added until the pH of the reaction solution reached 7. Subsequently, 500 mL of isopropyl alcohol was added to precipitate a polymer, which was filtered off. This was dried under reduced pressure at 40 ° C. for 8 hours to obtain a sulfated cellulose sodium salt. The yield of the obtained sulfated cellulose sodium salt was 26.8 g, the solubility in pure water at 20 ° C. was 180 g / L, and the sulfur content was 13.6 wt%.

Synthesis Example 9
A 3.0 L separable flask equipped with a thermometer, a dropping funnel, a stirrer, and a nitrogen gas inlet tube is charged with 75.0 g of crystalline cellulose (Theolus PH-101) and 375 mL of dimethylformamide, and stirred at room temperature for 15 hours. did. Next, 845.9 g of sulfuric acid-dimethylformamide solution having a concentration of 17.7% by weight was added under ice cooling, and the reaction vessel was immersed in a constant temperature bath at 20 ° C., and the temperature was increased over 20 minutes. Reacted for hours.
After completion of the reaction, the reaction solution was poured into 3000 mL of water, and then a 20% aqueous sodium hydroxide solution was gradually added until the pH of the reaction solution reached 7. The generated precipitate was filtered off, and 140.0 g of sodium chloride was added to the filtrate and stirred for 30 minutes. Subsequently, the polymer was precipitated by throwing it into 3 L of methyl alcohol, filtered, and dried under reduced pressure at 40 ° C. for 8 hours to obtain a sulfated cellulose sodium salt. The yield of the obtained sulfated cellulose calcium salt was 128.5 g, the solubility in pure water at 20 ° C. was 180 g / L, and the sulfur content was 13.9% by weight.

Example 1
The sulfated cellulose calcium salt obtained in Synthesis Example 1 was diluted with water for injection in Japanese Pharmacopoeia to prepare a dermatitis therapeutic agent having a sulfated cellulose calcium salt concentration of 0.5% by weight.

Example 2
The sulfated cellulose calcium salt obtained in Synthesis Example 1 was diluted with Japanese Pharmacopoeia water for injection to prepare a therapeutic agent for dermatitis having a sulfated cellulose calcium salt concentration of 0.1% by weight.

Comparative Example 1
As Comparative Example 1, Hirudoid Soft (manufactured by Maruho Co., Ltd., ointment containing 3 mg of sulfated chondroitin sulfate in 1 g) was used.

[Inhibitory effect test 1 on the progression of dermatitis in atopic dermatitis model NC mice]
An NC / Nga male mouse (Japan SLC Co., Ltd.) was carefully shaved from the back of the head to the back of the neck and the back, and 100 μL of the sample prepared in Examples 1 and 2 was prepared in Comparative Example 1. A sample of 100 mg was applied once a day for 10 days with a plastic spatula on the outside of the shaved area and ear. The test was conducted with 6 animals in each group.
For all cases, once a day, the state of symptoms on the outer surface of the test substance application site was observed after the application was completed. Symptoms were scored for the ear and back of the neck based on the following classification expression, and an overall score was calculated by combining the scores of the ear and back of the neck.
0: Asymptomatic 1: Epidermis detachment (scales, desquamation), Redness 2: Edema 3: Crust (erosion / ulcer) is less than 1/4 of test substance application site 4: Crust (erosion / ulcer) is examined Less than 1/2 of the substance application site 5: Scab (erosion / ulcer) more than 1/2 of the test substance application site

On the next day after the final application of the test substance, the blood was lethal under ether anesthesia, and the skin of the head, neck and back was removed and fixed with 10% neutral buffered formalin solution. This skin tissue was embedded in paraffin according to a conventional method, then sliced, and hematoxylin / eosin stained and toluidine blue stained specimens were prepared, (1) inflammatory cell infiltration, (2) dermal fibrosis, (3) flattening Epithelial hyperplasia, (4) erosion, and (5) crust were diagnosed. The diagnostic criteria for each histopathological finding are shown below.
(1) Inflammatory cell infiltration Cell infiltration mixed with lymphocytes, granulocytes, macrophages and the like was defined as inflammatory cell infiltration. Grade classification is mild (+) without tissue destruction, moderate without tissue destruction, moderate (++), without tissue destruction, but beyond the dermis Was defined as altitude (++++). In addition, when cell infiltration was observed only around the erosion, it was not an abnormal finding.
(2) Fibrosis of the dermis When the fibrosis is observed only in the surface layer of the dermis or when there is no change in the thickness of the dermis (+), when the dermis is thickened due to fibrosis of the entire dermis Was moderate (++), and the entire dermis on the specimen was thickened as high (++++).
(3) Squamous epithelial hyperplasia When the thickness of the squamous epithelium (epidermis) is clearly thickened due to cell enlargement or increase in the number of cells, it is mild (+), and moderately accompanied by papillary stroma ( (++), and the case where thickening was observed throughout the specimen was defined as altitude (++++).
(4) Erosion Mild (+) when epidermal defect is observed within 2 places within 200 magnifications and within 1 field or within 1 field. A case where a size exceeding 2 fields of view was observed in one or more places was regarded as moderate (++), and a case where the size was observed in the whole specimen was regarded as altitude (++++).
(5) Scabs Skin covering less than 1/3 of the skin was defined as mild (+) (except for erosion).

Table 2 shows the observation results of the symptoms on the outer surface of the test substance application site. Table 3 shows the diagnostic results of (1) inflammatory cell infiltration, (2) dermal fibrosis, (3) squamous hyperplasia, (4) erosion, and (5) crust.
The inbred NC / Nga mice used in this study spontaneously developed dermatitis with strong sensation on the face, ears, neck, back, etc. at 7-8 weeks of age. Deterioration is seen. This NC / Nga mouse dermatitis with a strong sense of sensation begins with the appearance of scratching behavior, is accompanied by bleeding, and when it progresses, it leads to skin erosion and ulceration, and the skin becomes dry and thick. These are considered very similar to human atopic dermatitis. In the test, animals in which crust formation (score 4) was observed on the neck or back were sequentially used, and the groups were divided so that the symptoms and body weight were uniform.
As shown in Table 2, in this test, by the end of the administration period, the dermatitis treatment agent prepared in Example 1 was prepared in Example 2 in all 6 cases (of which 1 case disappeared). In the dermatitis treatment agent, 5 cases out of 6 cases (disappearance of symptoms in 2 cases) and in the dermatitis treatment agent of Comparative Example 1 in 4 out of 6 cases, the reduction of symptoms was observed. Considering that these results show that the average score tends to increase in the non-treated group (application day 0: 4.0, application day 11: 4.5), it suggests an inhibitory effect on symptom progression. it was thought. Furthermore, as shown in Table 2, it was shown that the therapeutic agent for dermatitis prepared in Example 1 and Example 2 was superior in the effect of suppressing symptom progression as compared with the therapeutic agent for dermatitis of Comparative Example.
In addition, as shown in Table 3, the dermatitis therapeutic agents prepared in Example 1 and Example 2 showed a decrease in the number of solids in which histopathological findings were observed, as compared with the comparative dermatitis therapeutic agent. It was. In particular, (1) In inflammatory cell infiltration, the dermatitis therapeutic agent prepared in Example 2 has a significant decrease in the number of solids in which inflammatory cell infiltration is observed (p) compared to the comparative dermatitis therapeutic agent. <0.01, one-sided nonparametric Tukey's test) was observed.

Examples 3-6, Comparative Examples 2, 3
[Inhibitory effect test 2 on the development of dermatitis in atopic dermatitis model NC mice]
Using the sulfated cellulose calcium salt obtained in Synthesis Examples 2 to 5, the inhibitory effect on the progression of dermatitis in atopic dermatitis model mice NC mice was evaluated. As Comparative Example 2, Hirudoid Soft (manufactured by Maruho Co., Ltd.) and 1 g of sulfated chondroitin sulfate was used, and as Comparative Example 3, water for injection (Otsuka Pharmaceutical Factory) was used.

(Adjustment of 2,4,6-trinitrochlorobenzene for induction)
100 mg of 2,4,6-trinitrochlorobenzene (Tokyo Chemical Industry Co., Ltd.) was weighed, 0.4 mL of acetone (Wako Pure Chemical Industries, Ltd.) was added and dissolved, and then 1.6 mL of ethanol ((Co., Ltd.) was added. ) Wako Chemical) was added to prepare a sensitive 2,4,6-trinitrochlorobenzene. Olive oil (Wako Pure Chemical Industries, Ltd.) was added to this solution so that the final concentration was 0.8% (W / V) to obtain a 2,4,6-trinitrochlorobenzene solution for induction. This operation was performed immediately before administration using a brown glass container under a yellow light.

(Induction of atopic dermatitis by 2,4,6-trinitrochlorobenzene in NC mice)
The abdomen of male NC / Nga Tnd Crj mice (Nippon Charles River Co., Ltd.) that reached 8 weeks of age was shaved with a clipper under ether anesthesia, and the cut abdomen and footpad were sensitive 2 , 4,6-Trinitrochlorobenzene solution 0.15 mL was applied using a plastic rod and sensitized. Induction was performed on the fourth day after sensitization. That is, the back of a sensitized NC mouse was shaved, and 0.15 mL of a 2,4,6-trinitrochlorobenzene solution for induction was applied to the shaved back and left and right ears using a plastic stick. . The NC mouse used in this example is a model mouse in which the total of dermatitis scores according to clinical symptoms described later is 25 when n = 8 by the first induction by this method.

(Administration)
The sulfated cellulose calcium salt of Synthesis Examples 2 to 5 was dissolved in water for injection (Otsuka Pharmaceutical Factory) to prepare a 0.1% (W / V) solution. 0.15 mL of this solution was applied to the induced site of NC mice using a plastic stick. The administration was performed once a day using n = 8 2,4,6-trinitrochlorobenzene-induced NC mice in each group. As Comparative Example 2, Hildoid Soft 100 mg was applied, and as Comparative Example 3, 0.15 mL of water for injection (Otsuka Pharmaceutical Factory) was applied once a day in the same manner. The symptoms on the seventh day after the induction were observed, and the skin symptoms of NC mice were scored according to the following. The results are shown in Table 4.
The dermatitis score is (a) pruritus, (b) redness, bleeding, (c) edema, (d) abrasion / tissue loss, (e) scab formation / drying, asymptomatic (0 points), mild (1 point), moderate (2 points), and altitude (3 points) were scored and the total was used. A larger total score means more severe atopic symptoms in model mice.
From the results of this Example, it is found that the sulfated cellulose calcium salt having a sulfur content of 9.8% or more works effectively for a mouse having a very mild symptom in which the initial atopic symptom is developing. I understood.
In addition, the present Example was implemented according to the precedent of Hirasawa et al. (Hirasawa et al. Pharmacometrics, 59, 123-134, 2000).

Examples 7-12
[Test of inhibition of hyaluronidase activity from bovine testis 1]
Using the sulfated cellulose calcium salts having different sulfur contents obtained in Synthesis Examples 2 to 7, the activity inhibition test of bovine testis-derived hyaluronidase was performed. The reagents used are manufactured by Wako Pure Chemical Industries, Ltd. unless otherwise specified.
The following six types of solutions were prepared.
Solution A: A 0.1 mol / L acetate buffer (pH 4.0) solution (concentration: 2.83 mg / mL) of bovine testis-derived hyaluronidase (manufactured by Sigma).
Solution B: 0.3 mol / L sodium chloride-0.1 mol / L acetate buffer solution (pH 4.0).
Solution C: A 0.1 mol / L acetic acid buffer (pH 4.0) solution (concentration 1.83 mg / mL) of sodium hyaluronate (manufactured by Chisso Corporation).
Solution D: 0.4 mol / L sodium hydroxide aqueous solution.
Solution E: 0.8 mol / L sodium borate aqueous solution.
Solution F: A solution obtained by adding 1.25 mL of 10N hydrochloric acid and 98.75 mL of acetic acid to 1 g of para-dimethylaminobenzaldehyde.

(Preparation of test solution)
Solution A 0.025 mL was mixed with Solution B 0.2 mL and held at 37 ° C. for 20 minutes. To this, sulfated cellulose calcium salts with different sulfur contents obtained in Synthesis Examples 2 to 7 were added 0.001 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.1 mg / mL, 1.0 mg / mL, And 0.1 mL of aqueous solution prepared to the density | concentration of 10.0 mg / mL was added, respectively, and it left still to a thermostat at 37 degreeC for 20 minutes. Furthermore, 0.2 mL of solution C was added, and it was left still in a thermostat at 37 ° C. for 20 minutes. Next, 0.1 mL of solution D and 0.1 mL of solution E were added and boiled for 3 minutes, followed by cooling, 3.0 mL of solution F was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes to prepare a test solution. With respect to this test solution, the absorbance Q _E at 585 nm was measured with pure water as a control using N-acetylhexosamine at the reducing end produced by the degradation of hyaluronidase as an index.

(Preparation of Control Solution 1)
A control solution was prepared in the same manner as in the preparation of the above test solution except that 0.1 mol / L acetate buffer (pH 4.0) was used instead of solution A, and pure water was used instead of the sulfated cellulose calcium salt aqueous solution. 1 was prepared. For this control solution 1, the absorbance Q ₁ at 585 nm was measured in the same manner as in the case of the test solution.

(Preparation of Control Solution 2)
A control solution was prepared in the same manner as in the preparation of the above test solution except that pure water was used instead of the sulfated cellulose calcium salt aqueous solution and 0.1 mol / L acetate buffer (pH 4.0) was used instead of solution C. 2 was prepared. For this control solution 2, the absorbance Q ₂ at 585 nm was measured in the same manner as in the test solution.

Hyaluronidase activity inhibition rate is determined according to the following formula, and the concentration of sulfated cellulose calcium salt and the inhibition rate are plotted to inhibit the hyaluronic acid decomposing activity of sulfated cellulose calcium salt obtained in Synthesis Examples 2 to 50 by 50%. The concentration (50% inhibitory concentration) was determined.
Inhibition rate (%) = {(Q ₂ −Q ₁ ) − (Q _E −Q ₁ )} / (Q ₂ −Q ₁ )
Table 5 shows the 50% inhibition concentration and the inhibition rate at 0.015 mg / mL of sulfated cellulose calcium salt solutions having different sulfur contents.

  All the sulfated cellulose calcium salts obtained in Synthesis Examples 2 to 7 showed high inhibitory activity.

Example 13
[Test of inhibition of hyaluronidase activity from bovine testis 2]
(Preparation of test solution)
In the same manner as in Examples 7 to 12, 0.025 mL of solution A was mixed with 0.2 mL of solution B and kept at 37 ° C. for 20 minutes, and then the sulfated cellulose sodium salt obtained in Synthesis Example 8 was added to 0.001 mg / mL. , 0.01 mg / mL, 0.02 mg / mL, 0.1 mg / mL, 1.0 mg / mL, and 10.0 mg / mL of an aqueous solution prepared at a concentration of 10.0 mg / mL, respectively, and added at 37 ° C. for 20 minutes It left still in the thermostat. Furthermore, 0.2 mL of solution C was added, and it was left still in a thermostat at 37 ° C. for 20 minutes. Next, 0.1 mL of solution D and 0.1 mL of solution E were added and boiled for 3 minutes, followed by cooling, 3.0 mL of solution F was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes to prepare a test solution. With respect to this test solution, the absorbance Q _E at 585 nm was measured with pure water as a control using N-acetylhexosamine at the reducing end produced by the degradation of hyaluronidase as an index. Control solution 1 and control solution 2 were prepared in the same manner as in Examples 7-12.
Hyaluronidase activity inhibition rate was determined according to the following formula, the concentration of sulfated cellulose sodium salt and the inhibition rate were plotted, and the concentration of the sulfated cellulose sodium salt obtained in Synthesis Example 8 to inhibit the hyaluronic acid decomposing activity by 50% ( 50% inhibitory concentration).
Inhibition rate (%) = {(Q ₂ −Q ₁ ) − (Q _E −Q ₁ )} / (Q ₂ −Q ₁ )
Table 6 shows the 50% inhibition concentration and the inhibition rate at 0.015 mg / mL of sulfated cellulose sodium salt solutions having different sulfur contents.

  As shown in Table 6, the sulfated cellulose sodium salt obtained in Synthesis Example 8 showed high inhibitory activity.

Comparative Examples 4, 5, 6
Hildoid Soft (Maruho Co., Ltd., 1 g) containing 3 mg of sulfated chondroitin sulfate in place of sulfated cellulose calcium salt or sulfated cellulose sodium salt. Was prepared by calculating the component content of the ointment) and two chondroitin sulfates, which are representative compounds of heparinoids that are said to have high hyaluronidase inhibitory activity, in the same manner as in Examples 7-13. Hyaluronidase activity inhibition test was conducted. The results are shown in Table 7.

That is, only about 1/3 to 1/4 of the sulfated cellulose calcium salt was observed in hirudoid soft, and about 1/50 to 1/100 of inhibitory activity was found in chondroitin sulfate. In addition, only about 1/6 of the inhibitory activity of sulfated cellulose sodium salt was found in hirudoid soft, and about 1/50 to 1/100 of inhibitory activity of sulfated cellulose sodium salt was found in chondroitin sulfate.
As is apparent from Tables 5 to 7, the sulfated cellulose calcium salt of Examples 7 to 12 having a sulfur content of 6.5 to 19.0% by weight has a low value of 50% inhibition concentration of 0.018 mg / mL or less. It shows a significant hyaluronidase activity inhibitory effect compared to the compounds of Comparative Examples 4-6. In addition, the sulfated cellulose sodium salt of Example 13 has a 50% inhibitory concentration of 0.011 mg / mL, and exhibits a remarkable hyaluronidase activity inhibitory effect as compared with the compounds of Comparative Examples 4 to 6.

Since the sulfated cellulose compound of the present invention has hyaluronidase inhibitory activity, it is useful as a dermatitis therapeutic agent or cosmetic. The sulfated cellulose compound of the present invention is particularly useful for the prevention of atopy-like skin symptoms, or the alleviation, improvement or healing of the symptoms.

Claims (10)

A compound obtained is selected from sulfated cellulose and salts thereof from crystalline cellulose state, and are solubility 3 g / L or more in pure water of 20 ° C., and a sulfur content of 13.1 to 15.4 wt% skin external preparation containing der Ru compound as an active ingredient. The skin external preparation according to claim 1, wherein the average degree of polymerization of the crystalline cellulose is 150 to 300. Weight average molecular weight of 1,000 to 200,000, according to claim 1 or 2 skin external preparation as described in the compound. The skin external preparation of any one of Claims 1-3 whose weight average molecular weights of the said compound are 50,000-80,000. The skin external preparation of any one of Claims 1-4 whose said compound is sulfated cellulose calcium salt or sulfated cellulose sodium salt. In a 0.1 mol / L acetate buffer solution (pH 4.0) at a temperature of 37 ° C., the concentration that inhibits the hyaluronic acid degradation activity of bovine testis-derived hyaluronidase by 50% is 0.018 mg / mL or less. The external preparation for skin according to any one of 5 . The skin external preparation according to any one of claims 1 to 5 , which exhibits a dermatitis inhibitory action against a dermatitis model using NC mice. The external preparation for skin according to any one of claims 1 to 7 , which is a therapeutic agent for dermatitis . The external preparation for skin according to claim 8 , which is used for treating atopic dermatitis. The external preparation for skin according to any one of claims 1 to 7 , which is a cosmetic .