JP2005255678A - Allergic disease treating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an allergic disease treating agent applicable even to patients sensitive to external irritations, slight in irritancy and good in feeling in use. <P>SOLUTION: This allergic disease treating agent comprises, as active ingredient, a galactosaminoglycan containing 20-100 mol% of 2-acetamido-2-deoxy-3-O-(4-deoxy-α-L-threo-hexy-pyranosyluronic acid)-4,6-bis-O-sulfo-D-galactose units or a pharmacologically acceptable salt of the galactosaminoglycan. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an agent for treating allergic diseases comprising galactosaminoglycan having a specific property or a salt thereof as an active ingredient, preferably a disease caused by type I allergic reaction or type IV allergic reaction, particularly allergic dermatitis (atopic) Dermatitis, allergic contact dermatitis, etc.).

  In recent years, allergic diseases have increased and become a major problem in the medical field. This type of disease includes atopic dermatitis, hives, allergic contact dermatitis, hay fever, allergic rhinitis, bronchitis, bronchial asthma, gastrointestinal abnormalities associated with dietary allergies, allergic conjunctivitis, allergic A wide range of diseases such as keratitis, metal allergy, multiple sclerosis are included. For these diseases, various drugs including steroids, non-steroidal anti-inflammatory drugs, antihistamines, and immunosuppressants are used for treatment, but the side effects of these drugs are problematic.

  Among these allergic diseases, atopic dermatitis has been rapidly increasing in number in recent years, causing chronic skin lesions sometimes accompanied by bleeding from the affected area, erythema, wounds and itching of the affected area. Although many unclear points remain regarding the pathophysiology, it is considered that skin barrier dysfunction is involved in the onset in addition to immunological functions mainly consisting of allergic inflammation. Various treatments have been explored focusing on these causes. For example, since there is an immune abnormality, administration of a topical steroid or an immunosuppressant mainly for the purpose of calming skin inflammation has been performed. However, corticosteroids have an inhibitory effect on adrenocortical function, cause infectivity, or develop peptic ulcer, hypertension, osteoporosis, and especially rosacea-like dermatitis when used on the face for a long time as an external preparation. There are a variety of very serious side effects, such as onset. Immunosuppressants also have very serious side effects such as bone marrow suppression, gastrointestinal disorders, liver disorders, interstitial pneumonia, kidney disorders, and hemorrhagic cystitis.

In addition, many histamine antagonists and so-called antiallergic agents, which are chemotransmitter release inhibitors from mast cells, have been used to suppress itching. However, these drugs have side effects such as fatigue and drowsiness, and some drugs are contraindicated for use in pregnant women because of their ability to transfer to breast milk.
In atopic dermatitis, the stratum corneum is destroyed and the moisture in the skin is significantly lower than that of healthy people. For this reason, the chance of contact with foreign allergens through the skin is increased, and the stabilization of the disease state is inhibited.

  In addition, as an external preparation, an ointment containing a moisturizing component, mainly petrolatum, has been widely prescribed so far to keep moisture, but it is not easy to apply to a broken stratum corneum. The feeling of application is not so good due to its strong sexual feeling. Furthermore, in order to maintain the moisturizing effect, it is necessary to apply frequently, and it cannot be said that the quality of life (QOL) of patients in the dry season such as winter is high.

  On the other hand, pharmaceuticals or cosmetics containing chondroitin sulfate and the like, in particular, compositions for external or topical administration or use thereof include water-in-oil emulsion compositions containing sulfated polysaccharides (sulfur oxides of mucopolysaccharides) or salts thereof. (Patent Document 1), an oil-in-water emulsion lotion (Patent Document 2) containing a sulfated polysaccharide (mucopolysulfate, etc.) and / or a salt thereof and an emulsion stabilizer, and a polysulfated mucopolysaccharide ( Preventive or therapeutic agents for allergic diseases containing polysulfated chondroitin sulfate (such as polysulfated chondroitin sulfate) (Patent Document 3), selectin-dependent cell adhesion comprising polysulfated mucopolysaccharide (polysulfated chondroitin sulfate, etc.) as an active ingredient Due to allergic diseases, autoimmune diseases, adhesion and metastasis using inhibitors (Patent Document 4), glycosaminoglycans and at least one transdermal absorption agent in blood. Prevention or treatment for a disease that occurs (Patent Document 5) is known, and chondroitin sulfate E binds to P- and L-selectin to suppress leukocyte infiltration (Non-Patent Document 1). However, galactosaminoglycans having specific properties as contained in the allergic disease treatment agent of the present invention are effective for the prevention or treatment of allergic diseases, particularly atopic dermatitis. It was not known.

JP 7-118306 A JP 11-180821 JP-A-11-335288 JP2001-335490 WO02 / 09728 Miyasaka et al., J. Biol. Chem. 277 (15) 12921-12930, 2002

  The present invention is a safe and practical allergic disease treatment agent having an antiallergic action, and particularly applicable to patients who are sensitive to external stimuli. It aims at providing a disease treating agent.

  The present inventors have developed NC / Nga mice (Matsuda H. et al, Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC / Nga mice., International Immunology. Vol. 9), an animal model of atopic dermatitis. , No. 3, pp.101-106), the pharmacological action of galactosaminoglycan or pharmacologically acceptable salt thereof was tested. It was found that a salt that is acceptable to the skin has an effect of recovering skin damage such as bleeding, erythema, and wound in dermatitis of the same animal model, and is less irritating and excellent in safety.

  Therefore, the present invention provides a therapeutic agent for allergic diseases (hereinafter also referred to as the therapeutic agent of the present invention) containing galactosaminoglycan having specific properties or a pharmacologically acceptable salt thereof as an active ingredient. The “allergic disease treatment agent” in the present invention means a drug that can be used for the prevention and / or treatment of allergic diseases.

  Galactosaminoglycan which is an active ingredient of the therapeutic agent for allergic diseases of the present invention is 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hexyl-pyranosyluronic acid) -4, A galactosaminoglycan having a 6-bis-O-sulfo-D-galactose unit content of 20 mol% to 100 mol% or a pharmaceutically acceptable salt thereof.

The galactosaminoglycan that is an active ingredient of the therapeutic agent for allergic diseases of the present invention preferably has the following characteristics.
(A) The number of sulfate groups in the constituent disaccharide is 0.5 to 2.0.
(B) The digestibility with chondroitinase ABC is 60% to 100%.
(C) 2-acetamido-2-deoxy-3-O- (4) represented by ΔDi-diSE in the constituent unsaturated disaccharide composition calculated by digestion with chondroitinase ABC and analysis by high performance liquid chromatography -Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose in 20 mol% to 100 mol% and ΔDi-diSE, ΔDi 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galacto And 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo- represented by ΔDi-6S The sum total with D-galactose is 80 mol% to 100 mol%.

  The galactosaminoglycan that is an active ingredient of the therapeutic agent for allergic diseases of the present invention more preferably has one or more of the following characteristics.

In a constitutive unsaturated disaccharide composition calculated by digesting galactosaminoglycan with chondroitinase ABC and analyzing with high performance liquid chromatography, 2-acetamido-2-deoxy-3-O represented by ΔDi-diSE -(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose is 50 mol% to 70 mol%.
The digestibility of galactosaminoglycan with chondroitinase ABC is 80% to 100%.
The average molecular weight of galactosaminoglycan is 1000 to 100,000 daltons.

The galactosaminoglycan is a galactosaminoglycan obtained from a mollusk or has the same physicochemical properties as the galactosaminoglycan.
Galactosaminoglycan is a galactosaminoglycan obtained from squid cartilage.

  Galactosaminoglycan is a galactosaminoglycan obtained by reducing the molecular weight of galactosaminoglycan obtained from squid cartilage.

The therapeutic agent for allergic disease of the present invention is preferably used for the prevention and / or prevention of diseases caused by type I allergic reaction or type IV allergic reaction, more preferably allergic dermatitis, particularly atopic dermatitis or allergic contact dermatitis. Or it can be used for treatment.
The allergic disease treatment agent of the present invention is preferably in the form of a topical administration agent, and particularly preferably in the form of an external preparation.

  According to the agent for treating allergic diseases of the present invention, diseases caused by type I or type IV allergic reactions, particularly allergic dermatitis (atopic dermatitis, allergic contact dermatitis, urticaria, eczema), hay fever Treatment agents for diseases caused by allergic reactions such as allergic rhinitis, bronchitis, bronchial asthma, gastrointestinal abnormalities associated with dietary allergies, allergic conjunctivitis, allergic keratitis, metal allergy, multiple sclerosis .

  In particular, it has an effect of recovering skin damage such as bleeding, erythema and wounds caused by atopic dermatitis. In addition, it is possible to provide an external preparation with excellent safety that can be applied to a patient sensitive to stimulation, and does not exhibit significant side effects. Moreover, the combined use with other atopic dermatitis therapeutic agents, for example, a corticosteroid hormone, an immunosuppressive agent, etc., can reduce the usage-amount of the medicine which shows these serious side effects.

  In general, “galactosaminoglycan” refers to N-acetyl-D, which is a constitutive sugar, having a repeating skeleton of N-acetyl-D-galactosamine and D-glucuronic acid or L-iduronic acid as a basic skeleton. -A generic term for glycosaminoglycans having a sulfate group at the 4-position or 6-position or 4-position and 6-position of galactosamine and / or 2-position of L-iduronic acid or D-glucuronic acid, meaning dermatan sulfate and chondroitin sulfate (Annu. Rev. Biochem., 60, 443-457, 1991).

  Degradation of galactosaminoglycan with an enzyme (lyase) produces an unsaturated disaccharide represented by the following general formula I. By analyzing the composition of these unsaturated disaccharides, it is possible to analyze the number of sulfate groups and the binding position in the constituent disaccharide units of the galactosaminominoglycan.

More specifically, galactosaminoglycan is enzymatically treated with a lyase that acts on galactosaminoglycan to produce unsaturated disaccharide, and the galactosaminoglycan is formed to reflect the type and amount of the constituent disaccharide. Unsaturated disaccharides can be identified and quantified by high performance liquid chromatography (HPLC).
The lyase used in the unsaturated disaccharide analysis is not particularly limited as long as it can be finally decomposed into the unsaturated disaccharide, and can be appropriately selected by those skilled in the art depending on the galactosaminoglycan to be analyzed. Chondroitinase, heparinase, heparitinase, hyaluronidase and the like can be used.

  The above HPLC analysis method is known to those skilled in the art, and the elution position of unsaturated disaccharide obtained by enzymatic treatment of galactosaminoglycan is compared with the elution position of standard unsaturated disaccharide. Can identify and quantify unsaturated disaccharides. The elution position on HPLC is usually monitored by absorption in the ultraviolet region (for example, wavelength 232 nm), and the content of each disaccharide unit in galactosaminoglycan is the standard value of known concentration of the integrated value (area) of the elution pattern. It can obtain | require by comparing with the integrated value (area) of the elution pattern of unsaturated disaccharide.

  The following ΔDi-4S and ΔDi-6S having the adjacent elution positions are distinguished, and the exact distinction between ΔDi-diSB and ΔDi-diSE is performed by digestion with a specific sulfatase (for example, chondro-6-sulfatase). Can do.

  That is, for example, as a result of treating unsaturated disaccharides with chondro-6-sulfatase, the amount by which the HPLC elution position is shifted to the position of ΔDi-4S is identified as ΔDi-diSE. In addition, use a column (eg, Zorbax SAX column; manufactured by Rockland Technologies, etc.) that can separate unsaturated disaccharides that are close to each other in the elution position, and distinguish these unsaturated disaccharides strictly. Is possible. A structure in which the 1-position of the D-glucuronic acid residue and the 3-position of 4,6-disulfide oxide of the N-acetyl-D-galactosamine residue are glycosidically bonded, and the 1-position of the L-iduronic acid residue A structure in which the N-acetyl-D-galactosamine residue at the 3-position of 4,6-disulfoxide is glycosidically bound is detected as ΔDi-diSE in the disaccharide analysis.

  The isomers based on the number of sulfate groups and bonding positions in such unsaturated disaccharides are summarized in Table 1 below together with abbreviations indicating them.

式中、Ｒ¹、Ｒ²、Ｒ³は水素原子またはＳＯ³⁻を示し、Ａｃはアセチル基を示す。

In the formula, R ¹ , R ² and R ³ represent a hydrogen atom or SO ^3- , and Ac represents an acetyl group.

The chemical names of the isomers are as follows.
ΔDi-0S: 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -D-galactose ΔDi-4S: 2- Acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose ΔDi-6S: 2- Acetamide-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-galactose ΔDi-diSD: 2-acetamido- 2-deoxy-3-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-galactose ΔDi-diSE: 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L Threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose ΔDi-diSB: 2-acetamido-2-deoxy-3-O- (4-deoxy-2- O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-glycose ΔDi-triS: 2-acetamido-2-deoxy-3-O- (4- Deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D-glycose

  In the present invention, the “constitutive unsaturated disaccharide composition” of galactosaminoglycan refers to the produced unsaturated disaccharide obtained for each unsaturated disaccharide produced by decomposing galactosaminoglycan with lyase as described above. It means the ratio to the total sugar and is expressed in mol%.

  The galactosaminoglycan having a specific property contained as an active ingredient in the agent for treating allergic diseases of the present invention is 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hexy- Pyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose unit is a galactosaminoglycan (hereinafter referred to as “the galactosaminoglycan of the present invention”) having a content of 20 mol% to 100 mol%. More preferably, in the constituent unsaturated disaccharide composition, ΔDi-diSE is 20 mol% to 100 mol%, particularly preferably about 50 mol% to about 70 mol%, and ΔDi-4S, ΔDi-6S and ΔDi galactosaminoglycans having a total of -diSE of about 80 mol% to 100 mol%, particularly preferably about 80 mol% to about 95 mol%, specifically the 4-position of N-acetyl-D-galactosamine and 6th position also has sulfate group , No sulfate group at the 2-position of D- glucuronic acid, galactosaminoglycan having a basic skeleton a repeating structure of a disaccharide shown below.

  The galactosaminoglycan of the present invention is not particularly limited as long as it has the above-mentioned properties. Usually, mollusks such as squid cartilage are used as raw materials, and crushing, boiling, extraction, enzymatic degradation, organic solvent precipitation, salt It can be produced by separating and purifying by appropriately combining methods commonly used for separation and purification of galactosaminoglycan or glycosaminoglycan, such as precipitation, salt solution, various types of chromatography and the like. Preferably, for example, squid cartilage is hydrolyzed with protease to degrade protein, the hydrolyzed solution is filtered, and the filtrate is obtained by precipitating a galactosaminoglycan fraction using alcohol such as ethanol in the presence of sodium chloride. be able to. If necessary, the protein and peptide contents in the extract are reduced by multiple protease treatments, aqueous sodium hydroxide treatment, alcohol precipitation, filtration, and desalting.

Thus, the average molecular weight of the galactosaminoglycan extracted and purified from mollusks is about 10,000 to 100,000 daltons. Moreover, as long as this invention galactosaminoglycan is obtained, the kind of said squid is not specifically limited, For example, mica, squid, squid, squid, etc. are mentioned.
The method for separating the galactosaminoglycan of the present invention is not limited to the above-mentioned method, and known methods described in JP-B-60-9042, JP-B-61-21241, JP-A-9-202731, etc. It can also be obtained by extraction and purification according to the method.

  In addition, the galactosaminoglycan of the present invention has been reduced in molecular weight obtained by combining the extraction or purification from mollusks and the method for reducing the molecular weight according to the conventional method for reducing the molecular weight of polysaccharides as described above. It may be a galactosaminoglycan. Such low molecular weight methods include known chemical hydrolysis methods using acids or alkalis, galactosaminoglycan degrading enzymes derived from microorganisms, mammals, etc. (chondroitinase ABC, chondroitinase AC, hyaluronidase, etc. ) Is used. Enzymatic degradation is preferred, and in particular, a method using lyase derived from microorganisms such as chondroitinase ABC, chondroitinase AC or animal-derived testicular hyaluronidase (hydrolyzing enzyme) is preferred. When lyase is used, the non-reducing end becomes an unsaturated sugar. The molecular weight of galactosaminoglycan thus reduced in molecular weight is usually about 1,000 to 30,000 daltons.

The salt of the galactosaminoglycan of the present invention is not particularly limited as long as it is a pharmacologically acceptable salt, but alkali metal salts and alkaline earth metal salts such as sodium salt and potassium salt , Lithium salts, calcium salts and the like are preferable, and sodium salts are particularly preferable (hereinafter, unless otherwise specified, the term “galactosaminoglycan” is used in the meaning including pharmacologically acceptable salts thereof).
The weight average molecular weight of the galactosaminoglycan of the present invention is not particularly limited, but is usually about 1,000 to about 100,000 daltons (Da), preferably about 3,000 to about 90,000 Da, more preferably about 5,000 to 70,000 Da, and more preferably It is about 5,000 to about 26,000 Da.

  Further, the digestibility when chondroitinase ABC is allowed to act on the galactosaminoglycan of the present invention is usually about 60% by weight to about 100% by weight, preferably about 80% by weight to 100% by weight. Here, the digestibility when chondroitinase ABC is applied is a value obtained by analyzing the chondroitinase ABC digestion pattern, and the galacto before digestion of the unsaturated disaccharide produced by chondroitinase ABC digestion. Percentage (% by weight) relative to saminoglycan.

In addition, the number of sulfate groups per constituent disaccharide of the galactosaminoglycan of the present invention is usually in the range of about 0.5 to 2.0, preferably in the range of about 0.9 to 1.8. In addition, the average number of sulfate groups is represented by the average for the entire constituent disaccharide.
The total of ΔDi-4S, ΔDi-6S and ΔDi-diSE of the galactosaminoglycan of the present invention is usually about 80 mol% to about 100 mol%, preferably about 80 mol% to about 95 mol%.

Further, in the composition ratio of the constituent unsaturated disaccharide of the galactosaminoglycan of the present invention, ΔDi-diSE is usually about 20 mol% to about 100 mol%, preferably about 50 mol% to about 70 mol%, more preferably About 55 mol% to about 65 mol%. ΔDi-6S is usually about 1 mol% to about 25 mol%, preferably about 5 mol% to about 20 mol%, and ΔDi-4S is usually about 5 mol% to about 30 mol%, preferably about 10 mol%. ~ About 25 mol%.
The target of application of the therapeutic agent for allergic disease of the present invention is not particularly limited as long as it is an allergic disease, but it can be used particularly for the prevention and / or treatment of diseases caused by type I or type IV allergic reactions.

  As used herein, type I allergic reaction has the meaning understood by this term in the art and typically induces the production of IgE specific for the antigen and is activated by this induced IgE. Various mediators such as histamine, eosinophil migration factor (ECF-A), leukotriene, platelet activating factor (PAF), thromboxane are produced from released mast cells and neutrophils, and are allergic by this mediator. It means a biological reaction that induces inflammatory inflammation (Nikkei Bio Latest Glossary of Terms, 5th edition, pages 42-44, September 17, 2002, published by Nikkei BP).

  Examples of diseases caused by type I allergic reactions include allergic dermatitis (atopic dermatitis, hives, eczema, etc.), hay fever, allergic rhinitis, bronchitis, bronchial asthma, digestive tract associated with dietary allergy Examples include abnormalities, allergic conjunctivitis, and allergic keratitis, and allergic dermatitis, particularly atopic dermatitis, is preferable as a disease to be used for the therapeutic agent for allergic diseases of the present invention.

  On the other hand, type IV allergy is also called delayed type allergy and is a reaction that does not depend on antibodies. The reaction between the antigen and lymphocytes (T cells) releases the chemical mediator lymphokine. As a result, phagocytic cells and leukocytes aggregate and vascular permeability increases, resulting in an inflammatory reaction. (The Nikkei Bio Latest Dictionary, 5th Edition, pages 42-44, September 17, 2002, published by Nikkei BP)

  Examples of diseases caused by type IV allergic reactions include allergic dermatitis (atopic dermatitis, allergic contact dermatitis, urticaria, etc.), metal allergy, multiple sclerosis, etc. Dermatitis and allergic contact dermatitis are preferred as diseases for which the therapeutic agent for allergic diseases of the present invention is used.

  The administration method of the treatment agent of the present invention is not particularly limited as long as the effect of the treatment agent of the present invention on allergic diseases is exhibited. For example, injection (intravenous, subcutaneous, intradermal etc.), nasal, oral, transdermal And oral and parenteral routes of administration such as inhalation. The administration method is appropriately selected depending on the disease or site to be applied, such as direct administration to a specific site by injection or administration by infusion.

  According to such administration route and administration method, the galactosaminoglycan of the present invention or a pharmaceutically acceptable salt thereof can be appropriately formulated and used as the treatment agent of the present invention. Dosage forms include injections (solutions, suspensions, emulsions, solid preparations for use), parenterals such as suppositories, tablets, capsules, liquids, granules, powders, liposizing agents Oral preparations such as inhalation powder, topical administration, especially ointments, plasters, patches, liquids, lotions, creams, gels, aerosols, sprays (sprays), pasta, gels, topical use External preparations such as powders and eye drops may be mentioned.

  A known method can be used for formulating the therapeutic agent for allergic diseases of the present invention. In addition, other pharmaceutical active ingredients (steroidal agents, antihistamines, steroids, antihistamines, etc.) are used as long as they do not adversely affect the galactosaminoglycan of the present invention or a pharmaceutically acceptable salt thereof and do not affect the effects of the present invention. Immunosuppressants, antibacterial agents, antibiotics, anti-inflammatory agents, etc.) and pharmaceutically acceptable ordinary stabilizers, emulsifiers, solubilizers, thickeners, surfactants, osmotic pressure regulators, pH regulators Etc. can be blended as appropriate.

  For example, when the allergic disease treatment agent of the present invention is used as an external preparation for the treatment of allergic dermatitis, particularly atopic dermatitis, the galactosaminoglycan of the present invention is usually mixed and dissolved in an appropriate base, An external preparation is formed by dispersing and the like, and the external preparation is transdermally applied to damaged skin or mucous membrane by coating, sticking, spraying or the like. The dosage form of the external preparation is not particularly limited, but as described above, ointment, plaster, patch, liquid, lotion, cream, gel, aerosol, spray (spray), pasta, gel Agents, powders for external use, and the like.

  As a base for external preparations, bases usually used for external preparations can be used. However, if a non-stimulant indication designation component such as paraben or lanolin is used, the galactosaminoglycan of the present invention is used. Since it is not itself irritating, the therapeutic agent for allergic diseases of the present invention can be applied to patients sensitive to stimulation, which is preferable.

  Commonly used bases include polyethylene glycol, carboxyvinyl polymer, beeswax, white petrolatum, plastibase, higher fatty acid or higher alcohol, hydrophilic ointment, burnishing cream, hydrophilic petrolatum, euselin, neoserin, water-absorbing ointment, hydrophilic plastibase, liquid paraffin Isopar, silicon oil, fatty acid ester, vegetable oil, squalane, polyhydric alcohol fatty acid ester, polybasic ester, alkyl glyceryl ether, soybean lecithin, stearic acid and the like.

  In the case of an external preparation, the galactosaminoglycan of the present invention is based on a concentration of about 0.1 to 3.0% by weight, preferably about 0.3 to 1.5% by weight, based on the weight of the allergic disease treatment agent of the present invention. Is blended into.

  Moreover, when using the therapeutic agent for allergic diseases of the present invention as an external preparation, sodium hyaluronate may be further added. The weight average molecular weight of sodium hyaluronate used is not particularly limited, but preferably 1000 to 6 million daltons, more preferably 300,000 to 3 million daltons, and more preferably 600,000 to 800,000 daltons. preferable. The amount of sodium hyaluronate is preferably about 0.001 to 1.0% by weight, more preferably about 0.01 to 0.1% by weight, based on the weight of the allergic disease treatment agent of the present invention.

  When the allergic disease treatment agent of the present invention is used as an eye drop, in addition to the galactosaminoglycan of the present invention, an isotonic agent (for example, sodium chloride, potassium chloride, etc.) and a buffer (for example, boric acid, phosphorus, etc.) Acid-sodium hydrogen, sodium dihydrogen phosphate, etc.), preservatives (eg, benzalkonium chloride, phenoxyethanol, chlorobutanol, etc.), antibiotics (eg, oxytetracycline) or anti-inflammatory agents (eg, diclofenac, glycyrrhizin, etc.) You may add the chemical | medical agent and additive normally used for eyedrops.

The compounding amount and dosage of galactosaminoglycan, which is the active ingredient of the present invention, are individually determined according to the administration method, dosage form, purpose of use, specific symptoms of the patient, patient weight, etc. Although not particularly limited, the amount of galactosaminoglycan administered to a patient is about 100 μg / kg to 20 mg / kg per administration, for example, for an adult when administered locally as an external preparation, preferably An example is about 250 μg / kg to 15 mg / kg.
In addition, the administration frequency of the present invention may be once a day, or may be divided into 2 to 4 times a day or more, and such administration may be performed daily or as appropriate. It can be administered for a necessary period after a long period of time.

  Examples of animals to which the present invention can be applied include mammals including humans (primates such as humans, pets such as dogs and cats, domestic animals such as cows, pigs, and horses), and birds such as chickens. It can be used to prevent, treat or alleviate the above symptoms of animals.

[Example]
Examples of the present invention will be specifically described below. However, these do not limit the technical scope of the present invention. In the examples, unless otherwise specified, “molecular weight” and “average molecular weight” mean “weight average molecular weight”.

[Reference Example 1] Preparation of Galactosaminoglycan Fraction 1 750 g of cartilage obtained from Japanese squid was shredded, boiled for 10 minutes, and then cooled to 50 ° C. After adjusting to pH 11 using NaOH, extraction was performed overnight under conditions of pH 10 and 50 ° C. using 150 g of Esperase (Novo Novartis).
3.75 g of sodium carbonate was added to the obtained extract and stirred for 1 hour under conditions of pH 10 and 50 ° C., followed by filtration, and the filtrate was concentrated to 750 ml.

16 g of NaOH was added to the obtained concentrated liquid, reacted at 35 ° C. for 2 hours, and then centrifuged. The obtained supernatant was fractionated three times with 1105 ml of ethanol, 600 ml of ethanol + 3% sodium acetate (pH 4.8) and 612 ml of ethanol + 3% sodium acetate (pH 4.8), and then 1500 ml of ethanol was added to obtain crude galactosamino. 18.8 g of glycan was obtained.
10 g of the obtained crude galactosaminoglycan was dissolved in 0.1 M Tris-HCl (pH 8.0), actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) was added, and the protein was further degraded overnight at 37 ° C.

  After decomposition, NaOH was added to bring the total reaction solution to 0.5N and reacted at 40 ° C. for 1 hour. Next, ethanol was added in an amount equivalent to the reaction solution, and the resulting precipitate was dissolved in water. After dissolution, the pH was adjusted to 3 and sodium acetate was added to form a 5% aqueous solution. After adjusting the pH to 4.8 ± 0.2, an equal amount of ethanol was added.

  The resulting precipitate was recovered, dissolved in water, adjusted to pH 4.8 ± 0.2, and a quarter amount of ethanol was added. Next, 2 g of activated carbon was added and the mixture was treated at 50 ° C. for 1 hour and filtered using a filter aid. Sodium acetate was added to the filtrate so that it might become 2%, and it was made pH 5.0-5.5, and filtered.

  An equal amount of ethanol was added to the obtained filtrate to make it white turbid, and the white turbid solution was added to the same amount of ethanol as previously added to cause precipitation. The resulting precipitate was collected and washed with alcohol, and then the precipitate was separated. The precipitate was dried using a vacuum dryer to obtain 6.8 g of purified galactosaminoglycan (average molecular weight: about 90,000 Da).

  1.5 g of the resulting purified galactosaminoglycan was collected and dissolved in phosphate buffered saline (PBS) (pH 5.3). To this solution, 2 mg of sheep-derived testicular hyaluronidase (manufactured by SIGMA) was added and reacted at 37 ° C. A part of the reaction solution was taken over time, and the degree of low molecular weight was examined by GPC-HPLC. When the molecular weight reached about 25 kDa, the reaction solution was boiled to stop the reaction. Activated carbon was added to the obtained reaction solution and reacted at 50 ° C. for 1 hour. The reaction solution was filtered, and sodium acetate was added to the filtrate so as to be 3%. Four times the amount of ethanol was added to this solution to obtain a precipitate. The precipitate was washed with ethanol and dried to obtain 1.3 g of a purified fraction (galactosaminoglycan fraction 1).

[Reference Example 2] Preparation of galactosaminoglycan fractions 2 to 4 4.0 g of the purified galactosaminoglycan (average molecular weight: about 90,000 Da) was collected and dissolved in PBS (pH 5.3). To this solution, 200 mg of sheep-derived testicular hyaluronidase was added and reacted at 37 ° C. overnight. The resulting reaction solution was divided into two parts, A and B, and 80 mg of hyaluronidase was further added to B and reacted at 37 ° C. overnight. A and B were each filtered with a laboratory scale TFF device (Millipore) having a molecular weight of 30,000 cut. After concentrating the filtrates of A and B, the filtrate was applied to a column packed with Sephadex G25 (manufactured by Amersham Biosciences) and eluted with 0.1M NaCl as a solvent. The eluate was fractionated by a fraction collector, and the obtained fraction was analyzed by the carbazole method to confirm the elution position of galactosaminoglycan. Moreover, the galactosaminoglycan molecular weight of each fraction was measured by GPC-HPLC as described later in Reference Example 3 (1). From the fraction obtained from A, fractions having an average molecular weight of about 6 kDa were collected. Further, from the fraction obtained from B, fractions having a molecular weight of 2.5 kDa or less were collected. The fractions obtained from A and B were concentrated, desalted and then freeze-dried to obtain powdered galactosaminoglycan fractions 2 and 3, respectively.
In addition, purified galactosaminoglycan (average molecular weight 69,950 Da) was obtained according to Reference Example 1 (galactosaminoglycan fraction 4). This fraction was subjected to the test without reducing the molecular weight by enzyme digestion.

[Reference Example 3] Analysis of galactosaminoglycan (1) Measurement of weight average molecular weight Weight average molecular weight was determined according to the method of Arai et al. (Biochim. Biophy. Acta, 1117, 60-70, 1992). . That is, chondroitin sulfate (weight average molecular weight 52,200, 31,400, 20,000, 10,200, 6,570, 459) with a known molecular weight and sodium hyaluronate (weight average molecular weight 104,000, 62,000) as standard products and gel filtration using high performance liquid chromatography ( GPC-HPLC) was determined by elution time. A column to which TSK gel G4000PWXL, G3000PWXL, and G2500PWXL (each φ7.8 × 300 mm, manufactured by Tosoh Corporation) was connected was used. The solvent is a 0.2 mol / L sodium chloride solution, the flow rate is 0.6 mL / min, and the detector is a differential refractive index detector (RI-8100, manufactured by Tosoh Corporation) and an ultraviolet-visible detector (UV-8010, Tosoh). (Made by Co., Ltd.).

(2) Determination of digestibility with chondroitinase ABC Add Tris-HCl buffer (pH 8.0) and 0.25 U chondroitinase ABC (Seikagaku Corporation) to 100 μL of galactosaminoglycan 1% solution. Digested overnight at 37 ° C. The reaction was stopped by heating in a boiling water bath for 3 minutes, the digestion equivalent to 50 μg was analyzed by GPC-HPLC, and the digestibility was calculated by comparing with the enzyme undigested galactosaminoglycan.

(3) Constitutive unsaturated disaccharide composition analysis The analysis of the sulfate group position can be carried out by a known method described in “2 .-- Seminar in Experimental Chemistry 3, Carbohydrate II, p49-62 (1991, published by Tokyo Kagaku Dojin). According to “From Structural Analysis Combining HPLC with Glycosaminoglycan Degrading Enzyme and HPLC”, it was carried out as follows.
That is, galactosaminoglycan is completely digested into disaccharides by chondroitinase ABC (manufactured by Seikagaku Corporation), and the resulting disaccharides (unsaturated disaccharides) containing unsaturated bonds are anion-exchanged. Analyzed by HPLC.

  Furthermore, in order to separate ΔDi-diSB and ΔDi-diSE that cannot be separated under these conditions, the digest was further digested with chondro-6-sulfatase (manufactured by Seikagaku Corporation), and ΔDi-diSE was converted to ΔDi- Shifted to 4S and similarly analyzed by anion exchange-HPLC. The two results were compared and analyzed to calculate the constituent unsaturated disaccharide composition. Details are described below.

(I) Digestion with chondro-6-sulfatase In contrast to 100 μg of digestion product with chondroitinase ABC, 0.25 U of chondro-6-sulfatase (raw) in 50 μL of buffer solution C (0.02 mol / L Tris-AcOH, pH 7.0) Chemicals (manufactured by Chemical Industry Co., Ltd.) were added, digested at 37 ° C. for 24 hours, and insolubles were removed by centrifugation.

(Ii) Analysis by HPLC The above digested product, that is, chondroitinase ABC digested solution or digested solution obtained by further digesting it with chondro-6-sulfatase was analyzed using HPLC. The column used was a YMC-Pack PA-120-S5 ion exchange column (φ2.6 × 250 mm, manufactured by YMC Corporation).

  0.8 mol / L sodium hydrogen phosphate was flowed in a linear concentration gradient from 2% to 100% in 60 minutes at a flow rate of 1.5 mL / min. Based on the elution position of the unsaturated chondro-disaccharide kit (Seikagaku Corporation), various unsaturated disaccharides eluted during this period were identified at 232 nm, and the peak area identified as unsaturated disaccharide The unsaturated disaccharide composition was calculated by calculating the sum as 100%.

(4) Measurement of the number of sulfate groups The number of sulfate groups per constituent disaccharide is obtained by multiplying the unsaturated disaccharide composition (mol%) of galactosaminoglycan by the coefficient of the number of sulfate groups, and is obtained by the following formula. It was.
Number of sulfate groups per unit disaccharide (number) = [mol% of ΔDi-0S × 0 + (mol% of ΔDi-6S + mol% of ΔDi-4S) × 1 + (mol% of ΔDi-diSD + ΔDi-diSB Mol% + ΔDi-diSE mol%) x 2 + ΔDi-triS mol% x 3] ÷ 100

(5) Results For the galactosaminoglycan fractions 1 to 4 prepared in Reference Examples 1 and 2, the weight average molecular weight determined by the above analysis method, digestibility by chondroitinase ABC, constituent unsaturated disaccharide composition and constitution The number of sulfate groups per disaccharide is summarized in Table 2 below.

  Therapeutic effect of a topical skin preparation containing galactosaminoglycan on atopic dermatitis

  In order to test the therapeutic effect of a topical skin preparation containing the galactosaminoglycan of the present invention on atopic dermatitis, the galactosaminoglycan fractions 1 to 3 of the present invention obtained in Reference Examples 1 and 2 and chondroitin sulfate derived from shark cartilage Test substances I to IV were prepared using sodium as follows. The shark cartilage-derived sodium chondroitin sulfate contains 60.44 mol% of ΔDi-6S, 20.99 mol% of ΔDi-4S, and 2.01 mol% of ΔDi-SE as a constituent unsaturated disaccharide composition, and does not correspond to the galactosaminoglycan of the present invention. Test substance IV is a comparative example.

<Preparation of external preparation for skin>
2 parts by weight of dimethylpolysiloxane, 4 parts by weight of squalane, 2 parts by weight of isopropyl palmitate, 1.5 parts by weight of isohexadecyl palmitate, 0.5 parts by weight of behenyl alcohol, 0.5 part by weight of glyceryl tri-2-ethylhexanoate, An oil component containing 0.5 part by weight of phenoxyethanol was weighed into a container, a small amount of an emulsifier was added, and the mixture was heated to about 70 ° C. and dissolved by stirring. In a separate container, 3 parts by weight of glycerin, 3 parts by weight of 1,3-butylene glycol, 1 part by weight of a galactosaminoglycan fraction (any of the above galactosaminoglycan fractions 1 to 3) or chondroitin sulfate derived from shark cartilage Aqueous components containing 0.45 parts by weight of sodium, 78.25 parts by weight of purified water, and a small amount of other water-soluble substances such as other known surfactants were weighed into a container and dissolved by stirring at about 70 ° C. When the water-based components are sufficiently dissolved, the oil-based components are gradually added and uniformly emulsified, then cooled to room temperature while cooling with water, and test substances I, II, III, and IV (galactosami), which are oil-in-water skin external preparations, are used. Noglycan fractions 1, 2, and 3 and shark cartilage-derived sodium chondroitin sulfate were prepared).

<Therapeutic effect of topical skin preparation on spontaneous model mice with atopic dermatitis>
Each of the test substances I to IV was applied to a conventional atopic dermatitis model mouse conventional NC / Nga mouse (Japan SLC Co., Ltd.), and the effect on atopic dermatitis was measured. Preliminary breeding was performed from 6-week-old mice, observed twice a week, and the dermatitis state was evaluated according to the evaluation criteria shown in Table 4 below. Animals showing a dermatitis score of 1 or 2 more than once were allocated to each group. The distribution was made sequentially so that the dermatitis score and average body weight of each group were as equal as possible. The number of specimens was 8 in each group.

  The test substance dose (0.10 g) per mouse was weighed in advance and applied to the affected dermatitis site at the site of dermatitis. The number of applications was once a day and up to 21 times (3rd week). After the test substance application was started, the dermatitis state was observed twice a week before the test substance application, and observation was performed until the third week (6 observations). Similarly, the state of dermatitis was evaluated according to the evaluation criteria shown in Table 3 below.

  An untreated group was used as a negative control. The mouse body weight was measured once a week during the preliminary breeding period and the administration period.

  The test results are shown in FIG. In the graph of FIG. 1, the vertical axis represents the average evaluation score of each group, and the horizontal axis represents the number of observations. As is clear from these results, in the galactosaminoglycan fraction 1, fraction 2, and fraction 3 combination skin external preparation administration group, a clear difference in dermatitis evaluation score was obtained compared to the untreated group. . Thereby, it was confirmed that the skin external preparation containing the galactosaminoglycan fraction of the present invention is effective against atopic dermatitis.

Sensory test of skin external preparation containing galactosaminoglycan About the feeling of use as an external preparation, test substance I according to the present invention described in Table 1 and galactosaminoglycan (condoitine sulfate) not corresponding to the allergic disease treatment agent of the present invention Comparison was made with Polymcon S (manufactured by Seikagaku Corporation) and Hirudoid Soft (manufactured by Maruho Co., Ltd.) which are external preparations for skin.

The test was conducted on 30 healthy men and women who did not have a wound at their test site in their 20s and 40s. The sex ratio was not fixed.
For each subject, preference was given to the skin external preparation A (Polymucon S), skin external preparation B (Hildoid Soft, Lot 37612), and skin external preparation C (test substance I) in that order, including their feeling of use and appearance ( Liked and disliked) As the index of the sensory test for the feeling of use, familiarity, feeling of blockage, and calmness were used and evaluated together with palatability. Tables 4 to 7 below show evaluation criteria for familiarity, occlusion, calmness, and palatability.

  The results are shown in FIG. From these results, it was found that the external preparation for skin of the present invention is an external preparation with good familiarity, little occlusion after application, and little stickiness. However, since palatability (likes and dislikes) varies from person to person, no difference was found in palatability. As described above, since the external preparation for skin of the present invention has good familiarity, less occlusion after application, and less stickiness, it is preferable for use during the day and also for systemic application.

  In addition, in the above test, 10% (3) of the subjects showed redness and inflammatory reaction while using hirudoid soft. These were found to be stimulating base ingredients such as parabens and lanolin contained in hirudoid soft. In view of this, it is considered that the topical skin preparation of the present invention with less irritation is more preferable for sensitive atopic dermatitis patients.

Histamine release inhibitory action The histamine release inhibitory action of fractions 1 and 2 prepared in Reference Examples 1 and 2 and polysulfated chondroitin sulfate (MPS: JP-A-11-335288) was measured.
Mast cells collected from the abdominal cavity of a Spargue-Dawley 12-week-old female rat were separated with 60% Percoll (Amersham) and prepared to 1 × 10 ⁵ cells / ml. Fraction 1, fraction 2 or MPS was added thereto (final concentration 10, 30 or 100 μl / ml) and incubated at 37 ° C. for 10 minutes. A buffer solution was added to the control group. Thereafter, compound 48/80 (manufactured by Sigma, final concentration 0.1 μg / ml) was added, followed by incubation at 37 ° C. for 30 minutes. Thereafter, the culture vessel was transferred onto ice to stop the reaction, and then the culture solution was centrifuged, and the supernatant was collected to obtain a sample for measuring free histamine. Distilled water was added to the precipitate (cells), the cells were broken by freezing and thawing, centrifuged, and the supernatant was used as a sample for measuring intracellular histamine. Histamine was measured using a commercially available ELISA kit (Oxford Biomedical Res.). From the measured amount of free histamine and the amount of intracellular histamine, the histamine release rate was determined by the following formula.

  The test results are shown in FIG. The histamine release rate from mast cells is shown on the vertical axis, and the histamine release rate from mast cells corresponding to each sample and concentration shown below is shown by mean value ± SE (n = 4). * Indicates p <0.05, and ** indicates p <0.01, indicating a significant difference from the control group. As can be seen from these results, significant suppression of the histamine release rate was observed in fractions 1 and 2. On the other hand, MPS did not significantly suppress the histamine release rate. This confirmed that the galactosaminoglycan of the present invention suppresses histamine release from mast cells, suggesting that it is effective in the treatment of allergic diseases.

Inhibitory effect on type I allergic reaction by mouse passive skin anaphylactic reaction The effects of fraction 1, fraction 2 and fraction 4 prepared in Reference Examples 1 and 2 on mouse passive skin anaphylactic reaction were measured. As a positive control, ketotifen (manufactured by Sigma), which is a commercially available antiallergic agent, was used.

  20 μl of an anti-dinitrophenyl monoclonal antibody (manufactured by Seikagaku Corporation) solution was intradermally administered to one place where the hair was removed from a BALB / c 8-week-old female mouse. After 24 hours, fraction 1 100 mg / kg, fraction 2 100 mg / kg, fraction 4 100 mg / kg or ketotifen 1 mg / kg was administered intravenously. Physiological saline was administered to the control group. Thirty minutes later, 1 mg / ml dinitrophenylated bovine serum albumin (manufactured by Sigma) and 0.5% Evans blue (manufactured by Wako Pure Chemical Industries, Ltd.) in an equal volume were administered into the tail vein in an amount of 10 ml / kg. After 30 minutes, the back skin blue-stained part was removed, and the pigment in the tissue was quantified.

  The test results are shown in FIG. The amount of dye leaked by passive skin anaphylaxis reaction is shown on the vertical axis, and the amount of dye leaked by passive skin anaphylaxis reaction obtained by each sample described below with each bar is shown as an average value ± SE (n = 13) . * Indicates p <0.05, and ** indicates p <0.01, indicating a significant difference from the control group. As can be seen from these results, significant suppression of pigment leakage was observed in fraction 1, fraction 2 and fraction 4. As a result, it was confirmed that the galactosaminoglycan of the present invention is effective against passive skin anaphylactic reaction which is a type I allergic reaction.

It is a graph which shows the effect with respect to atopic dermatitis of the galactosaminoglycan addition external preparation for skin using NC / Nga mouse. It is a graph which shows the result of the usability | use_condition test of this invention external preparation for skin. It is a graph which shows the result of the histamine release inhibitory action test of this invention glycosaminoglycan. It is a graph which shows the result of the type I allergic reaction inhibitory action test of the glycosaminoglycan of the present invention.

Claims (13)

  The content of 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hexyl-pyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose unit is 20 mol% to A therapeutic agent for allergic diseases comprising 100 mol% of galactosaminoglycan or a pharmacologically acceptable salt thereof as an active ingredient. The agent for treating allergic diseases according to claim 1, wherein the galactosaminoglycan has the following properties.
(A) The number of sulfate groups in the constituent disaccharide is 0.5 to 2.0.
(B) The digestibility with chondroitinase ABC is 60% to 100%.
(C) 2-acetamido-2-deoxy-3-O- (4) represented by ΔDi-diSE in the constituent unsaturated disaccharide composition calculated by digestion with chondroitinase ABC and analysis by high performance liquid chromatography -Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose in 20 mol% to 100 mol% and ΔDi-diSE, ΔDi 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galacto And 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo- represented by ΔDi-6S The sum total with D-galactose is 80 mol% to 100 mol%.   In a constitutive unsaturated disaccharide composition calculated by digesting galactosaminoglycan with chondroitinase ABC and analyzing with high performance liquid chromatography, 2-acetamido-2-deoxy-3-O represented by ΔDi-diSE -(4-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose is 50 mol% to 70 mol% galactosamino It is a glycan, The allergic disease treatment agent of Claim 1 or 2 characterized by the above-mentioned.   The allergic disease according to any one of claims 1 to 3, wherein the galactosaminoglycan is a galactosaminoglycan having a digestibility with chondroitinase ABC of 80% to 100%. Treatment agent.   The galactosaminoglycan is a galactosaminoglycan having an average molecular weight of 1,000 to 100,000 daltons, according to any one of claims 1 to 4.   The galactosaminoglycan is a galactosaminoglycan obtained from a mollusk, or a galactosaminoglycan having the same physicochemical properties as the galactosaminoglycan. The therapeutic agent for allergic diseases according to any one of the above.   The agent for treating allergic diseases according to claim 6, wherein the galactosaminoglycan is a galactosaminoglycan obtained from squid cartilage.   The galactosaminoglycan is a galactosaminoglycan obtained by reducing the molecular weight of a galactosaminoglycan obtained from squid cartilage, The therapeutic agent for allergic diseases according to claim 6 or 7.   The agent for treating allergic disease according to any one of claims 1 to 8, wherein the allergic disease is a disease caused by a type I allergic reaction or a type IV allergic reaction.   The agent for treating allergic disease according to claim 9, wherein the allergic disease is allergic dermatitis.   The allergic disease treatment agent according to claim 10, wherein the allergic disease is atopic dermatitis or allergic contact dermatitis.   The agent for treating allergic diseases according to any one of claims 1 to 11, which is a locally administered agent. The allergic disease treatment agent according to claim 12, which is an external preparation.

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