JP2000178196A - Novel hyaluronidase inhibitor and preparation for external use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject novel hyaluronidase inhibitor that has marked hyaluronidase inhibitory activity with high safety by using a sulfated polysaccharide having specific properties. SOLUTION: This hyaluronidase inhibitor has <=0.05 μg/ml of the 50% inhibitory concentration of the hyaluronic acid hydrolysis activity of the hyaluronidase (preferably lyase type) originating from Staphylococcus aureus (preferably obtained from the skin of a patient with atopic dermatitis under the reaction conditions in 40 mM formate buffer solution (at pH of 4.0) at 37 deg.C for 16 hours. In addition, this inhibitor has the molecular weight of 7,500-17,000 according to the gel filtration method and contains sulfated polysaccharide with a sulfur content of 12.5-18% (for example, sulfated chondroitin sulfate, dextran sulfate, hyaluronic acid sulfate, heparin sulfate, sulfated dermatan sulfate or the like).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention belongs to the technical field of pharmaceutical use of sulfated polysaccharide. More specifically, a hyaluronidase inhibitor containing a specific sulfated polysaccharide as an active ingredient,
And a therapeutic agent for atopic dermatitis and an external preparation containing the same as an active ingredient.

[0002]

2. Description of the Related Art The prior art closest to the present invention will be described. JP-A-7-82131 describes that a hyaluronidase activity inhibitor acts on allergy in a suppressive manner, and that chondroitin sulfate is a hyaluronidase activity inhibitor.

[0003] Trends in Glycoscience and Glycote
chnology 8 (44), 419-434 (1996) describes that heparin is a strong inhibitor of hyaluronidase activity.

[0004] Japanese Patent No. 2723473 exemplifies atopic dermatitis as a disease to which sulfated sugar can be applied. As sulfated sugars, heparin, dermatan,
Chondroitin, glucosaminoglycan, mucopolysaccharide and the like are exemplified, and there is a description that polysulfated saccharide or persulfated saccharide is preferred. It is also described that heparan sulfate, dextran sulfate and the like are hyaluronidase inhibitors.

[0005] However, there is no description or suggestion of a sulfated polysaccharide having a remarkable inhibitory ability against a specific hyaluronidase involved in atopic dermatitis.

There is no description or suggestion that a sulfated polysaccharide having a specific sulfur content and molecular weight has remarkable hyaluronidase inhibitory activity and low toxicity.

There is no description or suggestion that such a specific sulfated polysaccharide is used as an active ingredient of a hyaluronidase inhibitor or a therapeutic agent for atopic dermatitis.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a hyaluronidase inhibitor which has a remarkable hyaluronidase inhibitory activity and contains a highly safe sulfated polysaccharide as an active ingredient.
An object of the present invention is to provide a therapeutic agent for atopic dermatitis and an external preparation.

[0009]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that a sulfated polysaccharide having specific properties has a remarkable hyaluronidase inhibitory activity and is safe. It is also found that the nature is high. In addition, they have found that a sulfated polysaccharide having this specific property can be used for inhibiting hyaluronidase and treating atopic dermatitis. The present invention has been completed based on these findings.

That is, the present invention provides a hyaluronidase inhibitor (hereinafter, also referred to as the inhibitor of the present invention) containing a sulfated polysaccharide having the following property (1) as an active ingredient. (1) Under the following reaction conditions, Staphylococcus aureus
The hyaluronic acid degrading activity of the hyaluronidase from
% Inhibitory concentration is 0.05 μg / ml or less. Temperature: 37 ° C Reaction buffer: 40 mM formate buffer (pH 4.0) Reaction time: 16 hours

In the inhibitor of the present invention, preferred are those in which the sulfated polysaccharide further has the following properties (2) and (3) in addition to the above properties. (2) Molecular weight determined by gel filtration method: 7500-17000 (3) Sulfur content: 12.5% -18%

In the inhibitor of the present invention, those in which the sulfated polysaccharide is sulfated chondroitin sulfate, sulfated dextran, sulfated hyaluronic acid, sulfated heparin, or sulfated dermatan sulfate are further preferred.

Further, the present invention provides a therapeutic agent for atopic dermatitis comprising the inhibitor of the present invention as an active ingredient (hereinafter, also referred to as the therapeutic agent of the present invention).

The present invention further provides an external preparation containing the inhibitor of the present invention (hereinafter also referred to as the external preparation of the present invention).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the inhibitor of the present invention, the therapeutic agent of the present invention and the external preparation of the present invention will be described in more detail.

<1> Inhibitor of the Present Invention The inhibitor of the present invention is a hyaluronidase inhibitor containing a sulfated polysaccharide having the following property (1) as an active ingredient. (1) Under the following reaction conditions, Staphylococcus aureus
The hyaluronic acid degrading activity of the hyaluronidase from
% Inhibitory concentration (hereinafter referred to as a 50% inhibitory concentration of hyaluronidase) is 0.05 μg / ml or less. Temperature: 37 ° C Reaction buffer: 40 mM formate buffer (pH 4.0) Reaction time: 16 hours

Hereinafter, the sulfated polysaccharide which is an active ingredient of the inhibitor of the present invention and other ingredients which can be added as required will be described in detail.

1. Sulfated polysaccharide A sulfated polysaccharide that can be used as an active ingredient of the inhibitor of the present invention is not particularly limited as long as it has the following property (1). (1) Under the following reaction conditions, Staph
The concentration that inhibits the hyaluronic acid-decomposing activity of the hyaluronidase from ylococcus aureus by 50% (hereinafter referred to as the hyaluronidase 50% inhibitory concentration) is 0.05 μg / ml or less. Temperature: 37 ° C Reaction buffer: 40 mM formate buffer (pH 4.0) Reaction time: 16 hours

The hyaluronidase derived from Staphylococcus aureus is not particularly limited as long as it is a hyaluronidase derived from the bacterium.
A lyase-type hyaluronidase from s is preferred. Preferably, Staphylococcus aureus is obtained from the skin of a patient with atopic dermatitis. Such hyaluronidases are known, for example, from the British Journal of Der
matology 90, p525-530 (1974)
The cells can be cultured, but are not limited thereto. Hyaluronidase will be described in detail in <2> below.

The method for determining the 50% inhibitory concentration of hyaluronidase is not particularly limited. For example, a constant concentration of the above hyaluronidase, various concentrations of sulfated polysaccharide, and a constant concentration of hyaluronic acid are brought into contact with each other, and the mixture is brought to 37 ° C. Incubate for 16 hours in 40 mM formate buffer (pH 4.0),
Thereafter, the degree of degradation of hyaluronic acid is measured, and a method of obtaining a 50% inhibitory concentration of hyaluronidase from the result is mentioned.

Contact and incubation of the three members of hyaluronidase, sulfated polysaccharide, and hyaluronic acid is described in Anal.
iochem., 225, 333-340 (1995) is preferred because the reaction operation and detection of the degree of degradation of hyaluronic acid are extremely simple.

For example, a polyacrylamide gel or a polyacrylamide gel containing sodium dodecyl sulfate in which a certain concentration of hyaluronic acid having a molecular weight of about 1,000,000 is uniformly mixed is used, and a certain amount of the above hyaluronidase is electrophoresed using this. . After electrophoresis, the gel is incubated at 37 ° C. for 16 hours in 40 mM formate buffer (pH 4.0) containing various concentrations of sulfated polysaccharide. After the incubation, the gel is incubated in a solution of a reagent for staining a sugar chain (such as Alcian Blue), and the portion where hyaluronic acid is degraded by hyaluronidase appears as a white band. The degree of staining of the white band is measured with a densitometer or the like, and a calibration curve is created for the relationship between the concentration of sulfated polysaccharide and the degree of staining of the band. Using the calibration curve, the concentration of the sulfated polysaccharide at half the level of the staining of the band in the absence of the sulfated polysaccharide may be determined, and this may be determined as the 50% inhibitory concentration of hyaluronidase.

The sulfated polysaccharide which can be used as an active ingredient of the inhibitor of the present invention preferably further has the following properties (2) and (3). (2) Molecular weight determined by gel filtration method: 7500-17000 (3) Sulfur content: 12.5% -18%

The molecular weight can be determined by a gel filtration method according to a method known per se. For example, a standard substance having a known molecular weight is applied to a gel filtration column to prepare a calibration curve for the relationship between elution time and molecular weight, and then a sulfated polysaccharide to be subjected to molecular weight measurement is subjected to gel filtration under the same conditions. The elution time is measured by applying to a column, and the molecular weight of the sulfated polysaccharide can be determined using the measured value and the calibration curve.

The sulfur content can also be measured by a method known per se. For example, it can be measured by a known analysis method for a sulfate group of a sulfated polysaccharide.

As such a method, for example, a turbidimetric method (B
iochem. J., 84, 106 (1962)), benzidic acid method (Acta Chem.
Scand., 16, 1521 (1962)), rhodizonic acid method (Anal.Biochem.,
41,471 (1971)), a method using high-performance liquid chromatography ("Proc. 10th Int. Symp. Glycoconjugates", ed. By N.
Sharon, H. Lis, D. Duskin, I. Kahane, p. 322, Jerusale
m (1989)) et al. (New Chemistry Laboratory Course 3, Carbohydrate II-proteoglycans and glycosaminoglycans-, P37-38 and P81-
82, (1991), Tokyo Kagaku Dojin), but the nephelometry is preferred because it is simple.

As a result of the measurement by the above method, the molecular weight was
Sulfated polysaccharides having a sulfur content of 7500 to 17000 and a sulfur content of 12.5% to 18% can be used as an active ingredient of the inhibitor of the present invention.

The sulfated polysaccharide which can be used as an active ingredient of the inhibitor of the present invention is not particularly limited as long as it has the above-mentioned property (1) (preferably further has the above-mentioned properties (2) and (3)). However, for example, sulfated chondroitin sulfate, sulfated dextran, sulfated hyaluronic acid, sulfated heparin, sulfated dermatan sulfate, sulfated keratan sulfate, sulfated starch, sulfated glycogen, sulfated cellulose, sulfate having the above properties Pullulan, sulfated inulin, sulfated chitin, sulfated pectin, sulfated cyclodextrin, and the like.

The sulfated polysaccharide that can be used as an active ingredient of the inhibitor of the present invention includes sulfated chondroitin sulfate, sulfated dextran, sulfated hyaluronic acid, sulfated heparin, and sulfated dermatan sulfate having the above properties. Among them, sulfated chondroitin sulfate, sulfated dextran, sulfated hyaluronic acid or sulfated heparin having the above properties is more preferred.

Among these sulfated polysaccharides, the following sulfated polysaccharides are particularly preferred as the active ingredient of the inhibitor of the present invention. (a) having a molecular weight of 2 × 10 ^{3 to 4} × 10 ⁴ and a sulfur content of 8.5
~ 15.0%, preferably 10.0-13.0% sulfated chondroitin sulfate. (b) a molecular weight of 3 × 10 ^{3 to} 1.2 × 10 ⁴ and a sulfur content of 1
Sulfated dextran sulfate which is 0.0-20.0%, preferably 15.0-18.0%. (c) having a molecular weight of 1 × 10 ^{4 to} 2 × 10 ⁶ and a sulfur content of 3.5
硫酸 15.8%, preferably 6.0-14.0% sulfated hyaluronic acid. (d) a molecular weight of 5 × 10 ^{3 to} 2 × 10 ⁴ and a sulfur content of 12.
Heparin sulphate which has been reduced after periodate oxidation by 5 to 15.0%, preferably 13.0 to 14.0%. (e) having a molecular weight of 2 × 10 ³ to 2 × 10 ⁴ and a sulfur content of 8.5
~ 19.0%, preferably 10.0-18.0%, sulfated dermatan sulfate.

As described above, the preferred molecular weight and sulfur content differ depending on the type of sulfated polysaccharide.
Although those having a molecular weight specified in (2) and a sulfur content specified in (3) are included, among the above-mentioned sulfated polysaccharides, those having the properties of (2) and (3) above are included. It is to be understood that this is highly preferred.

"Sulfated chondroitin sulfate" (sulfated oxide of chondroitin sulfate) is a product obtained by artificially introducing a sulfate group into chondroitin sulfate, and is higher than ordinary chondroitin sulfate derived from natural products. It has a sulfate group content (sulfur content). Similarly, for example, “sulfated dextran” (sulfated oxide of dextran), “sulfated hyaluronic acid” (sulfated oxide of hyaluronic acid) and “sulfated heparin” (sulfated oxide of heparin), “sulfated dermatan sulfate” (dermatan sulfate) Sulfuric acid sulfate) is a product obtained by artificially introducing a sulfate group into dextran, hyaluronic acid, heparin and dermatan sulfate, respectively, and has a higher sulfate group than dextran, hyaluronic acid, heparin and dermatan sulfate, respectively. Content (sulfur content).

The method for artificially introducing a sulfate group is not particularly limited, and examples thereof include a method for chemically introducing a sulfate group and a method for introducing a sulfate group using a suitable sulfotransferase (sulfotransferase). It is preferable to use a method of introducing the target.

The method for chemically introducing a sulfate group is not particularly limited as long as the sulfate group is introduced into the polysaccharide.
For example, Japanese Patent Publication No. 6-99485 and Carbohydr.
s., 158, 183 (1986). In this method, a polysaccharide salt is sulfated by reacting it with a sulfating reagent in a polar organic solvent. The polar organic solvent that can be used in this reaction may be any one that does not affect the reaction, for example, N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMAC), pyridine And trimethylamine. The sulfating reagent is, for example, sulfur trioxide-
A pyridine complex (SO ₃ -py), a sulfur trioxide-N, N-dimethylformamide complex, a sulfur trioxide-trimethyl (or triethyl) amine complex, or the like can be used. Those skilled in the art can appropriately set the amount of the reagent to be used, the reaction conditions, and the like according to the target sulfur content (degree of sulfation) and the like.

When the molecular weight of the sulfated polysaccharide thus obtained is larger than the target molecular weight, the polysaccharide to be used as a raw material or the sulfated polysaccharide obtained in advance is subjected to high-temperature and high-pressure treatment in an autoclave or the like. The molecular weight can be reduced by a treatment, a shear treatment at a high rotation speed, a treatment with an alkali, an acid or an enzyme, or the like, and adjusted to a target molecular weight range.

The sulfated polysaccharide thus obtained can be isolated from the reaction mixture by an ordinary method for separating sugar chains, and can be purified if necessary. The specific method for chemically introducing a sulfate group will be described in detail in Examples below.

Among the polysaccharides to which a sulfate group is artificially introduced by the above-described method (polysaccharides before the introduction of the sulfate group), those containing no sulfate group have substantially no hyaluronidase inhibitory activity, Is weak but has hyaluronidase inhibitory ability. However, the hyaluronidase inhibitory ability can be increased about 10 to 100 times by artificially introducing a sulfate group by the above method or the like.

The sulfated polysaccharide that can be used as an active ingredient of the inhibitor of the present invention may be a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts of sulfated polysaccharides include, for example, salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt and the like), alkaline earth metal salts and ammonium salts, and diethanolamine salts Among the salts with organic bases such as cyclohexylamine salt and amino acid salt, pharmaceutically acceptable salts can be used.

2. Ingredients that can be added as needed The inhibitor of the present invention may provide the above-mentioned sulfated polysaccharide as it is as the inhibitor of the present invention, but other components may be used as long as the action of the inhibitor of the present invention is not substantially impaired. It may be contained. For example, carriers used in the preparation of ordinary reagents and medicaments may be further added. As such a carrier,
Conventional excipients, binders, lubricants, coloring agents, disintegrants, buffers, tonicity agents, preservatives, stabilizers, etc., can be appropriately selected from components used in ordinary reagents or medicines. .

<2> Therapeutic agent of the present invention and the external preparation of the present invention The therapeutic agent of the present invention is a therapeutic agent for atopic dermatitis containing the inhibitor of the present invention as an active ingredient. The therapeutic agent of the present invention can be used literally for treating atopic dermatitis, but not only for pure therapeutic purposes, but also for the prevention, maintenance (prevention of deterioration),
It can also be used for the purpose of reduction (improvement of symptoms). Therefore, the therapeutic agent of the present invention also includes the concept of a prophylactic agent, a deterioration preventing agent, and a symptom improving agent.

The present inventors found that in patients with atopic dermatitis, 70% of the skin where atopic dermatitis was present
With the probability that Staphylococcus aureus was discovered, it was confirmed that this bacterium produced hyaluronidase. Hyaluronidase was also identified in 46% of the patients' skin from areas without atopic dermatitis. In addition, protease was confirmed from the skin of healthy persons with a probability of 50%, but hyaluronidase was not confirmed with a probability of 100%. Hyaluronidase present in the skin of this atopic dermatitis patient had an activity of degrading chondroitin 4-sulfate, but the activity was 0.5% or less of the hyaluronic acid degrading activity. In addition, this hyaluronidase is chondroitin
6-sulfuric acid, dermatan sulfate, keratan sulfate and heparan sulfate did not decompose.

Staphylococcus aureus isolated from the skin of patients with atopic dermatitis showed hyaluronidase activity as described above, but Staphylococc
us epidermidis, Staphylococcus saprophyticus, Pseu
No hyaluronidase activity was found in culture supernatants of other bacteria such as domonas aeruginosa.

Culture supernatant of bacteria isolated from the skin of patients with atopic dermatitis (hereinafter referred to as 11i in this description)
Hyaluronidase has two optimal pH (pH 4.0 and 7.0)
The degradation product of hyaluronic acid has the same mobility as the unsaturated tetrasaccharide obtained by digestion of hyaluronidase from Streptomyces, and the saturated tetrasaccharide obtained by hyaluronidase from testis Was a different substance. The resulting unsaturated tetrasaccharide further comprises
Digestion with chondroitinase ABC produced saturated and unsaturated disaccharides. Regarding the enzymatic reaction by this chondroitinase ABC, FIG. 1 shows a reducing sugar by the Perk-Johnson method (solid line in FIG. 1), and a reducing end by the Morgan-Elson method.
The result of monitoring N-acetylhexosamine (dotted line in FIG. 1) is shown.

The two absorbance increases are parallel, indicating that the product has N-acetylglucosamine. There was no glucuronic acid at the reducing end. From the above, the degradation product of hyaluronic acid by the culture supernatant 11i is only an unsaturated tetrasaccharide and is a product of lyase-type hyaluronidase (an enzyme that cleaves hyaluronic acid by elimination reaction), and is a product of hydrolytic enzyme-type hyaluronidase. Not a product.

From the above results, it was proved that lyase-type hyaluronidase of Staphylococcus aureus was greatly involved in atopic dermatitis. Based on this finding, the present inventors have searched for inhibitors using a lyase-type hyaluronidase derived from Staphylococcusaureus present in the skin of patients with atopic dermatitis. As a result, the sulfated polysaccharide described in the above <1> was converted to Staphylococc
It was found that lyase type lyase from us aureus has strong inhibitory ability against hyaluronidase.

The therapeutic agent of the present invention is an application of such a potent inhibitory effect of the inhibitor of the present invention on hyaluronidase as a therapeutic agent for atopic dermatitis. As is clear from the results of the examples described below, the sulfated polysaccharide which is an active ingredient of the inhibitor of the present invention is a lyase-type hyaluronidase produced by Staphylococcus aureus present in the skin of patients with atopic dermatitis in a very small amount. Inhibits completely.

The form of the therapeutic agent of the present invention is not particularly limited as long as it contains at least the sulfated polysaccharide which is an active ingredient of the inhibitor of the present invention. That is, the above-mentioned sulfated polysaccharide may be provided as it is as the inhibitor of the present invention, and does not substantially impair the action of the therapeutic agent of the present invention, is physiologically acceptable, and has such a purity that it can be used as a medicament,
In addition, other pharmaceutically active ingredients and other pharmaceutical carriers may be further added as long as they do not substantially contain pharmaceutically unfavorable ingredients. Such pharmaceutical carriers include components commonly used in medicine such as conventional excipients, binders, lubricants, coloring agents, disintegrants, buffers, isotonic agents, preservatives, and soothing agents. Can be selected as appropriate.

The therapeutic agent of the present invention can be used as an external preparation such as an ointment, a cream, a liquid, a lotion, an emulsion, a gel, a patch, etc., thereby providing the external preparation of the present invention. The external preparation of the present invention can also be used as a cosmetic or an additive for cosmetics, particularly as a skin cosmetic.

In order to provide the therapeutic agent of the present invention or the external preparation of the present invention as such a dosage form, a known pharmaceutical carrier or a cosmetic carrier such as waxes, lanolin, surfactants and cetanol is usually used. It is preferable to formulate by a method.

Further, in order to provide the therapeutic agent of the present invention or the external preparation of the present invention in such a dosage form, oily components, water-soluble components, preservatives, pH adjusters, thickeners, and the like which are commonly used in external preparations for skin are used. An agent, an antioxidant, a surfactant, a coloring agent, a flavoring agent, and the like can be added as necessary.

Examples of the oily component include liquid paraffin, petrolatum, squalane, microcrystalline wax, almond oil, olive oil, hardened oil, jojoba oil,
Coconut oil, castor oil, palm oil, beeswax, lanolin, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, octyldodecanol, behenyl alcohol, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, oleic acid,
Isopropyl myristate, myristyl myristate,
Isopropyl palmitate, isopropyl adipate and the like can be blended.

Examples of the water-soluble component include glycerin, propylene glycol, sorbitol, polyethylene glycol, 1,3-butylene glycol, ethanol,
Isopropanol and the like can be blended.

Examples of preservatives include parabens, benzoic acids, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, alkyltrimethylammonium chloride, alkyldiaminoethylglycine hydrochloride,
Chlorhexidine hydrochloride, chlorobutanol, salicylic acids, domiphen bromide, phenoxyethanol and the like can be blended.

Examples of the pH adjuster include citric acid,
Acids such as tartaric acid and lactic acid, and alkalis such as sodium hydroxide, potassium hydroxide and triethanolamine can be blended.

As the thickener, for example, sodium carboxymethylcellulose, sodium alginate, carboxyvinyl polymer, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polyvinyl alcohol and the like can be blended.

Examples of the antioxidant include tocopherol, erythorbin, propyl gallate, BHT and BH
A, NDGA, etc. can be blended.

Examples of the surfactant include fatty acid glycerin ester, sorbitan fatty acid ester, sucrose fatty acid ester, polyglycerin fatty acid ester, polyhydric alcohol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether, and polyoxyethylene. Ethylene fatty acid ester, polyoxyethylene sorbitan ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene hardened castor oil and the like can be blended.

The amount of the active ingredient (sulfated polysaccharide) in the therapeutic agent of the present invention or the external preparation of the present invention, and the amount and interval of use of the therapeutic agent or the external preparation are determined by the dosage form of the therapeutic agent or the external preparation. It is individual depending on the specific symptom of the patient (degree of disease) and can be increased or decreased as appropriate. Examples can be given. The amount of the therapeutic agent of the present invention or the external preparation of the present invention is 10 per adult per day.
About 0 to 500 mg can be exemplified. In addition, the usage interval is exemplified by a frequency of about 1 to 3 times a day and about once a day to 3 days.

[0059]

The present invention will be specifically described below with reference to examples. However, these should not limit the technical scope of the present invention. First, a method commonly used in this embodiment will be described.

(1) Measurement method of molecular weight The molecular weight of the sulfated polysaccharide was measured as follows. The sulfated polysaccharide to be measured is converted to a gel filtration column TSK gel G6000PW (7.5
mm ID x 30 cm x 2) (manufactured by Tosoh Corporation), and eluted with 0.2 M NaCl at a flow rate of 0.5 ml / min.The eluate was analyzed with a differential refractometer to measure the elution time of the sulfated polysaccharide. .

A sulfated polysaccharide having a known molecular weight (hereinafter also referred to as a standard) was similarly analyzed, and a calibration curve was prepared for the relationship between the elution time and the molecular weight. The molecular weight of the sulfated polysaccharide to be measured was determined using the analysis result of the elution time of the sulfated polysaccharide to be measured and the calibration curve.

When the sulfated polysaccharide to be measured was sulfated chondroitin sulfate, chondroitin sulfate was used as a standard product. Similarly, dextran was used as a standard in the case of sulfated dextran, hyaluronic acid in the case of sulfated hyaluronic acid, and heparin in the case of sulfated heparin.

(2) Method for Measuring Sulfur Content The sulfur content was measured by a turbidimetric method (Dodgson-Price turbidimetric method; Biochem.
J., 84, 106 (1962), Shinsei Chemistry Laboratory Course 3, Carbohydrate II -proteoglycan and glycosaminoglycan-, P37-38, (19
91), Tokyo Chemical Dojin). Specifically, it was measured as follows.

(1) Preparation of barium chloride-gelatin reagent:
1 g of gelatin was dissolved in 200 ml of water while warming to 60-70 ° C. After standing at 4 ° C overnight, add 1 g of barium chloride,
Leave at room temperature for at least 4 hours before use. (2) Preparation of gelatin reagent: (1) without adding barium chloride
Was prepared in exactly the same way as (3) Sample hydrolysis: 2 to 4 mg of the sample (sulfated polysaccharide to be measured) has a final concentration of sulfate ion (SO ₄ ^2- ) of sulfated polysaccharide of 40.
Add 1N hydrochloric acid to a concentration of ~ 90μg / 0.2ml and place in a sealed tube 105
Hydrolyzed at ~ 110 ° C for 5 hours. (4) To 0.2 ml of the hydrolyzed solution obtained in the above (3), 3.8 ml of 3% (w
/ v) Trichloroacetic acid (TCA) or 0.1N hydrochloric acid was added and mixed. Subsequently, 1.0 ml of barium chloride-gelatin reagent was added and mixed again, left at room temperature for 15 to 20 minutes, and then measured at 360 nm or 500 nm within 1 hour.

On the other hand, as a control, 0.2 ml of the hydrolyzed solution obtained in the above (3) was subjected to the same operation as above using a gelatin reagent in place of the barium chloride-gelatin reagent, and the obtained value was calculated. It was subtracted from the value resulting from using the barium chloride-gelatin reagent.

The calibration curve used potassium sulfate, 20-200 μg
It was determined for SO ₄ ²⁻ /0.2 ml.

The sulfur content was determined from the sulfate group content thus determined.

(3) Method for measuring 50% inhibitory concentration of hyaluronidase The hyaluronidase activity was determined by Anal. Biochem., 225, 333-340.
(1995). As the hyaluronidase, a culture supernatant of Staphylococcus aureus obtained from the skin of an atopic dermatitis patient was used. This hyaluronidase is a lyase-type hyaluronidase.

Chondroitinase ABC (Seikagaku Corporation)
Hyaluronidase (corresponding to 1 μl of culture supernatant) equivalent to 75 μunit of a product (manufactured by Co., Ltd.) is uniformly mixed with hyaluronic acid having a molecular weight of 1,000,000, and is a 1 mm thick polyacrylamide gel containing 8% sodium dodecyl sulfate ( Hereinafter, referred to as SDS gel) or 7.0% polyacrylamide gel (hereinafter, native g
(Helonic acid concentration in the gel is 170 μg)
/ ml). Electrophoresis was performed in 0.025 M Tris-0.192 M glycine (pH 8.3) at 4 ° C. and 20 V for 30 minutes. After electrophoresis, wash the gel with 2.5% Triton X-100.
Incubation buffer (various concentrations (0, 0.063, 0.1
25, 0.5, 2, 10, 12.5 and 50 μg / ml) in a 40 mM formate buffer (pH 4.0) containing sulfated polysaccharides at 4 ° C. for 15 minutes, and then in fresh incubation buffer at 37 ° C. Incubated for 16 hours.

After incubation, the gel was washed for 20 minutes with a 20% ethanol-10% acetic acid solution, then stained with a 0.5% Alcian blue (20% ethanol-10% acetic acid) solution for 1 hour, and then stained for 20 minutes. Washed with 10% ethanol-10% acetic acid solution. As a result, a portion where hyaluronic acid was degraded by hyaluronidase was observed as a white band.

The degree of staining of the white band was determined using a densitometer (Shima
It was measured at a wavelength of 615 nm using a dzu CS-900 densitometer) and quantified. A calibration curve was prepared for the relationship between the concentration of sulfated polysaccharide and the degree of staining of the band, and this calibration curve was used to determine the half of the degree of band staining at a sulfated polysaccharide concentration of 0 μg / ml. The concentration of the sulfated polysaccharide was determined, and this was defined as a 50% inhibitory concentration of hyaluronidase.

[0072]

Example 1 Sulfated Chondroitin Sulfate An aqueous solution of 10 g of sodium chondroitin sulfate (sulfur content: 6.8%, molecular weight: 8,000; hereinafter, referred to as CS) derived from shark cartilage was passed through a cation exchange resin, and trinormal butylamine (tri-butylamine) was added. n-butylamine) salt, and then water was removed to give an N, N-dimethylformamide (DMF) solution.
To this, 12.7 g of sulfur trioxide-pyridine complex (SO ₃ -py) was added and reacted at 80 ° C. for 1 hour. Ethanol was added to the liquid after the reaction, and the resulting aqueous solution of a white powder was converted into a sodium salt with a cation exchange resin. Ethanol was added to obtain 11 g of white powdered sulfated chondroitin sulfate (hereinafter referred to as CPS). The molecular weight, sulfur content, and 50% inhibitory concentration of hyaluronidase of CPS were as follows. Molecular weight (gel filtration method): 10000 Sulfur content: 12.5% Hyaluronidase 50% Inhibitory concentration: 0.05 μg / ml or less

FIG. 2 shows the relationship between the concentration of CPS and CS (raw material for producing CPS) and the hyaluronidase inhibition rate. In FIG. 2, ■ indicates CPS, and ● indicates CS. As is clear from FIG. 2, CPS was shown to have a hyaluronidase inhibitory ability several tens times that of CS.

[0074]

Example 2 Sulfated dextran 10 g of dextran (molecular weight: 45,000) was cooled and cooled.
The reaction was carried out in 0 ml of concentrated sulfuric acid for 2 hours, the reaction solution was poured into ice water and neutralized, and then free sulfuric acid was removed using barium carbonate. A sodium salt was formed with a cation exchange resin, and alcohol was added to obtain 20 g of white powdered sulfated dextran (hereinafter referred to as Dex-S). Dex-S molecular weight, sulfur content,
And the 50% inhibitory concentration of hyaluronidase was as follows. Molecular weight (gel filtration method): 7500 Sulfur content: 17.8% Hyaluronidase 50% Inhibitory concentration: 0.05 μg / ml or less

[0075]

Example 3 Sulfated Hyaluronic Acid An aqueous solution of 10 g of sodium hyaluronate (molecular weight: 10,000) was passed through a cation exchange resin to form a pyridine salt, and water was removed to obtain a DMF solution. To this, 39.4 g of sulfur trioxide-pyridine complex was added and reacted at 80 ° C. for 1 hour.

An aqueous solution of a white powder formed by adding ethanol to the liquid after the reaction was converted into a sodium salt with a cation exchange resin, and ethanol was added thereto to add 14 g of a white powder of sulfated hyaluronic acid (hereinafter referred to as HA- S). The molecular weight, sulfur content, and 50% inhibitory concentration of hyaluronidase of HA-S were as follows. Molecular weight (gel filtration method): 15000 Sulfur content: 13.4% Hyaluronidase 50% Inhibitory concentration: 0.05 μg / ml or less

[0077]

Example 4 Sulfated heparin A solution of heparin in 0.05 M sodium phosphate buffer (pH 7.0) (1 g / 100 ml) was added to a solution of 40 mM sodium periodate in 0.05 M
Add sodium phosphate buffer solution (pH 7.0) and add
The reaction was performed in the dark for 0 hours. 500 mg of glucose was added to the reaction solution, and dialyzed in running water at room temperature for 6 hours. 20g in dialysis solution
Was reduced at room temperature for 6 hours, acetic acid was added to adjust the pH to 4.5, and the reaction was stopped. Ethanol was added to the liquid after the reaction to obtain 0.65 g of a heparin oxidation / reduction compound (hereinafter referred to as O / R-Hep) as a white powder. The sulfur content of O / R-Hep was 10.6%.

500 mg of O / R-Hep was dissolved in DMF,
Add 200 mg of sulfur trioxide-pyridine complex and add
Allowed to react for hours. The aqueous solution of the white powder produced by adding ethanol to the liquid after the reaction was converted into a sodium salt with a cation exchange resin, and ethanol was added thereto to add 460 mg of sulfated heparin of the white powder (hereinafter referred to as O / R-HepS). ). O / R-He
The molecular weight, sulfur content, and 50% inhibitory concentration of hyaluronidase of pS were as follows. Molecular weight (gel filtration method): 17000 Sulfur content: 13.5% Hyaluronidase 50% Inhibitory concentration: 0.05 μg / ml or less

[0079]

Example 5 Acute toxicity test As an example of a sulfated polysaccharide, dextran having various molecular weights was used as a raw material, and sulfated dextran having various molecular weights was produced in the same manner as in Example 2 (sulfur content: 18%).
Was used to perform an acute toxicity test.

The acute toxicity (LD ₅₀ ) was examined in male ddY mice by three different administration methods: oral administration, intravenous injection and subcutaneous administration. Table 1 shows the results.

[0081]

[Table 1]

From Table 1, it was found that in the case of the sulfated polysaccharide having a sulfur content of 18%, the toxicity rapidly increased when the molecular weight reached 10,000 or more.

[0083] Note that in other sulfated polysaccharides not only dextran sulfate, high anticoagulant activity as content sulfate group (sulfur content) is large, the (highly toxic) low value of LD ₅₀ it is known I have. Considering the results of the above examples and these factors, (1) the molecular weight determined by gel filtration is 7500-17000, and (2) the sulfur content is 1
It was considered that the sulfated polysaccharide of 2.5% to 18% had remarkable hyaluronidase inhibitory ability and high safety (low toxicity).

As is clear from the above Examples, such a sulfated polysaccharide has a remarkable ability to inhibit hyaluronidase, and the 50% hyaluronidase inhibitory concentration is 0.05 μl.
g / ml or less.

As is clear from the above examples, it was revealed that such a sulfated polysaccharide is particularly preferably sulfated chondroitin sulfate, sulfated dextran, sulfated hyaluronic acid or sulfated heparin.

[0086]

Example 6 Formulation Example 100 mg of freeze-dried Dex-S produced in Example 2, mineral oil 4
g, petroleum jelly 8 g, mixed methyl / propyl paraban 60
mg, 1 g of a nonionic surfactant and 30 g of purified water were uniformly mixed, and the mixture was filled in a container to produce a therapeutic agent for atopic dermatitis (an ointment).

[0087]

The sulfated polysaccharide, which is an active ingredient of the inhibitor of the present invention, the therapeutic agent of the present invention and the external preparation of the present invention, has remarkable hyaluronidase inhibitory activity and high safety (low toxicity). , A hyaluronidase inhibitor, a therapeutic agent for atopic dermatitis, a skin cosmetic and the like.

The inhibitor of the present invention has a remarkable hyaluronidase inhibitory activity and is highly safe, and thus is useful as a reagent or as a raw material for medicines and skin cosmetics for diseases caused by hyaluronidase activity.

The therapeutic agent of the present invention is useful as a therapeutic agent for atopic dermatitis like the inhibitor of the present invention, and the external preparation of the present invention is particularly useful as a skin cosmetic.

[0090]

[Brief description of the drawings]

FIG. 1 shows the results of monitoring reducing sugars and N-acetylhexosamine at the reducing end.

FIG. 2 shows the ability of CPS and CS to inhibit hyaluronidase.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 7/48 A61K 7/48 A61P 17/00 31/00 617 43/00 643D // C08B 37/00 C08B 37/00 H 37/02 37/02 37/08 37/08 Z 37/10 37/10 (72) Inventor Katsuyoshi Sakurai 2-527-6 Kurashiki, Higashiyamato-shi, Tokyo F-term (reference) 4C083 AC012 AC482 AC791 AD211 AD212 AD311 AD312 AD331 AD341 BB51 CC03 DD22 EE11 EE13 4C086 AA01 AA02 EA26 EA27 MA01 MA04 MA63 NA14 ZA89 ZB11 ZB13 ZC20 4C090 AA09 BA12 BA64 BA66 BA67 BA68 BD36 BD37 BD41 CA34 DA09 DA23 DA26

Claims (10)

[Claims] 1. A hyaluronidase inhibitor comprising a sulfated polysaccharide having the following property (1) as an active ingredient. (1) Under the following reaction conditions, Staphylococcus aureus
The hyaluronic acid degrading activity of the hyaluronidase from
% Inhibitory concentration is 0.05 μg / ml or less. Temperature: 37 ° C Reaction buffer: 40 mM formate buffer (pH 4.0) Reaction time: 16 hours 2. The sulfated polysaccharide further has the following property (2) and
The hyaluronidase inhibitor according to claim 1, having (3). (2) Molecular weight determined by gel filtration method: 7500-17000 (3) Sulfur content: 12.5% -18% 3. The hyaluronidase inhibitor according to claim 1, wherein the sulfated polysaccharide is sulfated chondroitin sulfate, sulfated dextran, sulfated hyaluronic acid, sulfated heparin, or sulfated dermatan sulfate. 4. The hyaluronidase inhibitor according to claim 1, comprising a sulfated chondroitin sulfate having the following properties as an active ingredient. Molecular weight determined by gel filtration: 2,000 to 40,000 Sulfur content: 10.0% to 13.0% 5. The hyaluronidase inhibitor according to claim 1, comprising a sulfated dextran sulfate having the following properties as an active ingredient. Molecular weight determined by gel filtration method: 3000-12000 Sulfur content: 15.0% -18.0% 6. The hyaluronidase inhibitor according to claim 1, comprising a sulfated hyaluronic acid having the following properties as an active ingredient. Molecular weight determined by gel filtration: 10,000 to 2,000,000 Sulfur content: 6.0% to 14.0% 7. An active ingredient comprising sulfate of heparin, which has been subjected to periodate oxidation and then reduced after the oxidation, having the following properties:
The hyaluronidase inhibitor according to claim 1. Molecular weight determined by gel filtration: 5000-20000 Sulfur content: 13.0% -14.0% 8. The hyaluronidase inhibitor according to claim 1, comprising a sulfated dermatan sulfate having the following properties as an active ingredient. Molecular weight determined by gel filtration method: 2000-20000 Sulfur content: 10.0% -18.0% 9. A therapeutic agent for atopic dermatitis, comprising the hyaluronidase inhibitor according to any one of claims 1 to 8 as an active ingredient. An external preparation containing the hyaluronidase inhibitor according to any one of claims 1 to 8.