JP2001151680A - Antipruritic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antipruritic which is capable of effectively preventing and treating an itch on chronic disease, e.g. psoriasis vulgaris or the like, accompanied with atopic dermatitis, urticaria, eczema, pruritus cutaneus, prurigo or pruritus and exhibits no significant adverse drug reactions. SOLUTION: This antipruritic is obtained by including galactosamino glycan or its pharmacologically permissible salt with a specific uronic acid composition, chondroitinase digestibility, sulfate group number and disaccharide composition as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antipruritic agent which can be used for the prevention and treatment of itching of the skin and mucous membranes, in particular, itching induced by allergy. More specifically, atopic dermatitis, urticaria, eczema, pruritus cutaneous, pruritus or pruritus in pruritus or psoriasis vulgaris with pruritus etc. can be prevented and treated, and undesired effects caused by scratching behavior on itch, For example, the present invention relates to an antipruritic agent capable of preventing bacterial infection and the like due to destruction of the skin barrier function.

[0002]

2. Description of the Related Art Atopic dermatitis, urticaria, eczema, pruritus, prurigo or psoriasis vulgaris accompanied by pruritus have chronic itchy skin lesions. Although much remains to be known about its pathology, skin barrier dysfunction is thought to be involved in its onset, in addition to immunological functions mainly involving allergic inflammation. Various treatments have been sought. For example, due to immunological abnormalities, topical steroids or immunosuppressants are mainly administered for the purpose of calming skin inflammation. However, corticosteroids have a suppressive effect on adrenocortical function, cause susceptibility to infection, or develop peptic ulcers, hypertension, osteoporosis, and rosacea-like dermatitis especially when used on the face as a topical agent for a long time. There are a variety of very serious side effects such as onset. In addition, immunosuppressants also have extremely serious side effects such as myelosuppression, gastrointestinal disorders, liver disorders, interstitial pneumonia, renal disorders, and development of hemorrhagic cystitis.

[0003] In addition, it is attempted to avoid contact with allergens as a two-dimensional treatment, but avoidance of contact with allergens often hinders daily life.

In atopic dermatitis, urticaria, eczema, pruritus cutis, prurigo or psoriasis vulgaris accompanied by pruritus,
It causes itching and curettage and exacerbates the rash. Controlling pruritus in the sense of eliminating the pruritus-curette-rash exacerbation cycle is a major point in treating these diseases. Histamine antagonists, mainly for the purpose of preventing itching,
So-called antiallergic drugs such as inhibitors of chemical mediator release from mast cells are used, and specifically, zazydene, azeptin, Celtect, Intal, Rizaben (all trade names) are used. However, these drugs have side effects such as fatigue and drowsiness, and some of them have the ability to transfer into breast milk, so their use in pregnant women is contraindicated.

[0005]

Therefore, the present invention is intended to alleviate itching of the skin and mucous membranes, and in particular, to chronic atopic dermatitis, urticaria, eczema, pruritus, prurigo or psoriasis vulgaris accompanied by pruritus. It is an object of the present invention to provide an antipruritic agent that can effectively prevent and treat pruritus of a disease that has passed, and that does not exhibit significant side effects.

[0006]

Means for Solving the Problems The present inventors have developed NC / Nga mice (Matsuda H. et al.) Which are animal models of atopic dermatitis.
et al, Development of atopic dermatitis-like skin
lesion with IgE hyperproduction in NC / Nga mice., I
nternational Immunology. Vol. 9, No. 3, pp.101-10
6) was tested for the antipruritic effect of galactosaminoglycan or a pharmacologically acceptable salt thereof, and as a result, a specific galactosaminoglycan or a pharmacologically acceptable salt thereof was found to have a low dose. Even when administered, it effectively inhibits the curettage caused by itch and delays the progress of the disease, that is, it has a significant antipruritic effect, is excellent in safety, and can be used for a long time. Was.

Accordingly, the present invention provides an antipruritic comprising galactosaminoglycan or a pharmaceutically acceptable salt thereof as an active ingredient.

[0008] Galactosaminoglycan, which is an active ingredient of the antipruritic agent of the present invention, has the following properties. (A) The molar ratio of iduronic acid to glucuronic acid in the constituent sugars is from about 40:60 to about 100: 0. (B) The digestibility by chondroitinase B is about 40% to about 100% by weight. (C) The number of sulfate groups per constituent disaccharide is from about 0.9 to about 1.3.
It is. (D) In the constituent disaccharide composition calculated by digestion with chondroitinase ABC and analysis by high performance liquid chromatography, 2-acetamido-2-deoxy-3-O- (4-deoxy) represented by ΔDi-0S -Α-L-threo-hex-4-enopyranosyluronic acid) -D-galacto-
2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D represented by ΔDi-4S
-Galactose and 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L- represented by ΔDi-6S
Threo-hex-4-enopyranosyluronic acid) -6
The total amount of O-sulfo-D-galactose is about 71 mol%
It is about 94 mol%.

Japanese Patent Application Laid-Open No. 9-286731 discloses a polysulfate ester of chondroitin sulfate (usually an organic sulfate group having 26
(Including about 37% by weight) as an active ingredient, but since the polysulfate of chondroitin sulfate is used as the active ingredient, the anticoagulant effect may be a problem.

It has also been known that chondroitin polysulfate is used in combination with zinc oxide as an external preparation for skin to prevent recurrence of atopic dermatitis (JP-A-11-604).
94) However, galactosaminoglycan, which is an active ingredient of the antipruritic agent of the present invention, is different from chondroitin polysulfate in the point that it contains iduronic acid and in the content of sulfate group. It shows an excellent effect that it is sufficiently effective when used alone at a dose.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Generally, "galactosaminoglycan" has a basic skeleton consisting of a repeating structure of a disaccharide composed of N-acetyl-D-galactosamine and D-glucuronic acid or L-iduronic acid. N-acetyl-
Glycosaminoglycan having a sulfate group at the 4-position or 6-position of D-galactosamine, and / or the 2-position of L-iduronic acid or D-glucuronic acid, which means dermatan sulfate and chondroitin sulfate (Annu. Rev. . Bi
ochem., 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 the unsaturated disaccharide, it is possible to analyze the number and the bonding position of the sulfate groups in the constituent disaccharide unit of galactosaminoglycan. The isomers of such unsaturated disaccharides according to the number of sulfate groups and the bonding positions are summarized in Table 1 below.

[0013]

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

[0014]

[Table 1]

The chemical names of the above 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-galacto-
ΔDi-6S: 2-acetamido-2-deoxy-3-O-
(4-deoxy-.alpha.-L-threo - hex-4-eno pyranosyl uronic acid) -6-O-sulfo -D- galactose ΔDi-diS _D: 2- acetamido-2-deoxy -3-O
-(4-Deoxy-2-O-sulfo-α-L-threo-
Hex-4-eno pyranosyl uronic acid) -6-O-sulfo -D- galactose ΔDi-diS _E: 2- acetamido-2-deoxy -3-O
-(4-Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D
-Galactose ΔDi-diS _B : 2-acetamido-2-deoxy-3-O
-(4-Deoxy-2-O-sulfo-α-L-threo-
Hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose 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-galactose

The above galactosaminoglycan is usually used for intestinal mucosa, skin, lung, kidney, liver, pancreas, aorta, mammals such as cows and pigs or birds such as chicken, duck, turkey and duck.
Separation of glycosaminoglycans using spleen, brain, thymus, cartilage, umbilical cord, crown or other tissues or organs as raw materials, crushing, extraction, enzymatic degradation, organic solvent precipitation, salting out, salt solution, various types of chromatography, etc. It can be produced by separating and purifying by appropriately combining methods usually used for purification.
Preferably, for example, it is obtained by hydrolyzing a cockscomb with a protease to decompose a protein, filtering the hydrolyzed solution, and precipitating a galactosaminoglycan fraction using an alcohol such as ethanol in the presence of sodium chloride. Can be. If necessary, use heparin and heparan sulfate (Hep /
When the HS content is to be reduced, the recovered precipitate is treated with nitrous acid (Shively and Conrad, Biochemis
try, 15, 3932-3942 (1976)) or ion-exchange chromatography (WO95 / 09188), or a combination of these two treatments to remove impurities such as Hep / HS. By filtering and desalting, galactosaminoglycan can be obtained.

The method for separating galactosaminoglycan is not limited to the above method, but is disclosed in Japanese Patent Publication No. 60-90.
42, and can be obtained by extraction and purification according to known methods described in JP-B-61-21241.

Dermatan sulfate, which is an example of galactosaminoglycan, may have different average molecular weight, sulfate content, bond position of sulfate group, and D-glucuronic acid content depending on the species of animal, tissue or organ. Are known. Table 2 below shows examples of the physical properties and disaccharide composition of dermatan sulfate obtained from typical raw materials (see Reference Example 1).

[0020]

[Table 2] Dermatan sulfate in the above table is an example of the galactosaminoglycan of the present invention. In addition, the above-mentioned cattle intestine, pig intestine, pig skin,
Dermatan sulfate derived from cockscomb was obtained by the method described in WO95 / 09188.

The galactosaminoglycan as an active ingredient of the antipruritic agent of the present invention is not particularly limited as long as it has the above-mentioned properties (A) to (D), but is preferably a bird tissue or organ. And more preferably those extracted and separated from cockscomb.

The salt of galactosaminoglycan used in the antipruritic agent of the present invention is not particularly limited as long as it is a pharmacologically acceptable salt, and examples thereof include alkali metal salts and alkaline earth salts. Metal salts, for example, sodium salts, potassium salts, 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 sense including its pharmacologically acceptable salts).

The galactosaminoglycan used in the present invention includes not only the above-mentioned substances extracted and purified from raw materials but also those obtained by chemically modifying or decomposing these substances. For example, a molecular weight of 1600 Da to 10,000 obtained by decomposing dermatan sulfate derived from mammals
Dermatan sulfate having a relatively low molecular weight of about Da is also known (Dol, F. et al., J. Lab. Clin. Med., 1990, vo
l. 115, 43-51, Bianchini, P. et al., Thrombosis an
d Heamostasis, 1991, vol. 65, 1315 or Barban
ti, M. et al., Thrombosis and Heamostasis, 1993, v
ol. 69, 147-151), and such a low molecular weight dermatan sulfate is also included in the galactosaminoglycan of the present invention as long as it has the above-mentioned properties (A) to (D).

In the present invention, the term "constituent disaccharide composition" refers to a composition comprising galactosaminoglycan and chondroitinase ABC.
(Chondroitin ABC lyase) breaks down into unsaturated disaccharides,
A known method (Yoshida K. et al., Supra) which fractionates enzymatic degradation products by high performance liquid chromatography using a polyamine silica carrier column and analyzes the content of each unsaturated disaccharide isomer.
Anal. Biochem. 1989, vol. 177, No. 2, 327-332) refers to the unsaturated disaccharide composition (mol%) obtained.

The above galactosaminoglycan has a weight average molecular weight of about 1,600 Da to about 100,000 Da, preferably about 23 kDa to about
45 kDa.

The molar ratio of iduronic acid to glucuronic acid in the sugar constituting the galactosaminoglycan is about 4%.
The range is from 0:60 to about 100: 0, preferably from about 65:35 to
It is about 90:10.

When chondroitinase B is allowed to act on the galactosaminoglycan, the digestibility is about 40% to about 100% by weight, preferably about 65% to 95% by weight.
It is. Here, the digestibility when chondroitinase B is acted is a value obtained by analyzing the chondroitinase B digestion pattern, and means the percentage (% by weight) of the digestion by chondroitinase B digestion. .

Further, the number of sulfate groups per constituent disaccharide of the galactosaminoglycan is in the range of about 0.9 to 1.3, preferably in the range of about 0.9 to 1.1.

Further, the total of ΔDi-0S, ΔDi-4S and ΔDi-6S of the galactosaminoglycan is about 71 mol% or less.
About 94 mol%, preferably about 90 mol% to about 94 mol%.

Further, the composition ratio of each of the above constituent disaccharides is not particularly limited, but about ΔDi-0S is about 0 mol% to 15 mol%.
Mol%, preferably about 2 mol% to about 15 mol%. In particular, galactosaminoglycans derived from bird tissues or organs have a relatively high ratio of ΔDi-0S, usually about 2 mol% to about 15 mol%, preferably about 4 mol% to about 10 mol%. About 4 mol% to about 40 mol%, preferably about 4 mol% to about 30 mol% for ΔDi-6S, and about 4 mol% to about 30 mol% for ΔDi-4S.
45 mol% to about 100 mol%, preferably about 60 mol% to about 9 mol%
0 mol%, from about 0 mol% for [Delta] Di-diS _D ~ about 5 mole%,
Preferably, from about 0 mol% to about 2 mol%, from about 1 mole% to about 15 mol% for [Delta] Di-diS _B, preferably, from about 0 mol% to
About 2 mol%, [Delta] Di-diS _E is about 0 mol% to about 3 mol%, preferably, it can be exemplified about 0 mole% to about 2 mole percent ratio.

The route of administration of the antipruritic agent of the present invention containing galactosaminoglycan as an active ingredient as described above is not particularly limited, but parenteral administration is preferable, and it is usually suitable for the skin and mucous membranes exhibiting itching sensation. A galactosaminoglycan mixed, dissolved, dispersed, or the like, is transdermally applied to the preparation by application, sticking, spraying, or the like. It may be administered to mucous membranes such as eyes, nose, and oral cavity.
In addition, it can be injected subcutaneously, intradermally, or intramuscularly after dissolving in an appropriate solvent such as water. The dosage form of the antipruritic agent of the present invention is not particularly limited, and examples thereof include ointments, plasters, patches, liquids, lotions, creams, gels, aerosols, sprays (sprays), nasal drops and injections. Can be

The antipruritic agent of the present invention may contain, in addition to galactosaminoglycan as an active ingredient, a base necessary for forming the above-mentioned dosage form.

Further, the antipruritic agent of the present invention may be used, if necessary, with auxiliaries such as ordinary pharmaceutically acceptable stabilizers, emulsifiers, solubilizers, thickeners, surfactants, osmotic pressure regulators and pH regulators. Can be appropriately contained.

Furthermore, the antipruritic agent of the present invention includes steroids, antihistamines, immunosuppressants, antibacterials, antibiotics,
It can be used in combination with drugs such as anti-inflammatory agents.

The amount and dosage of galactosaminoglycan, which is an active ingredient of the antipruritic agent of the present invention, depend on the method of administration of the preparation, the dosage form, the purpose of use, the specific symptoms of the patient, the weight of the patient, and the like. The amount of galactosaminoglycan to be administered to a patient is, for example, about 1 mg / kg to 2 mg / kg for an adult per day.
00 mg / kg, preferably about 2.5 mg / kg to 150 mg / kg.

The antipruritic agent of the present invention may be administered once a day, 2 to 4 times a day, or more often. The administration can be carried out every day or an appropriate number of days as necessary.

The subject to which the antipruritic agent of the present invention is applied is not particularly limited as long as it is a chronic course of disease accompanied by itch. Atopic dermatitis, hives, eczema, pruritus, prurigo or pruritus or pruritus Pruritus associated with metabolic diseases such as cirrhosis, uremia, chronic renal failure, pruritus associated with endocrine diseases such as diabetes, hyperthyroidism, malignant lymphoma (Hodgkin's disease, mycosis fungoides, etc.) ) It is widely used for pruritus associated with malignant tumors, pruritus associated with parasitism caused by roundworm, duodenum, etc., psychogenic pruritus due to neurosis, senile pruritus, pruritus associated with infections such as vaginal candidiasis and trichomoniasis. Can be applied.

According to the definition of the Japanese Dermatological Association, atopic dermatitis is "a disease in which repetition of hatred and remission is caused mainly by pruritic eczema, and many patients have atopic predisposition." It is a disease in which pruritus is at the top of the diagnostic criteria, and many patients suffer from pruritus that makes it difficult to actually go to bed. By administering the antipruritic agent of the present invention,
By preventing the itch of atopic dermatitis, the curettage can be suppressed, and as a result, bacterial infection and inflammation hatred resulting from the curettage can be prevented, treated or reduced. Also,
Prevention of itching in atopic dermatitis leads to improved quality of life for patients. In addition, bacterial infections resulting from curettage,
Prevention of inflammation hatching is effective in reducing the amount of other therapeutic drugs for atopic dermatitis, for example, drugs showing serious side effects such as corticosteroids and immunosuppressants.

Animals to which the antipruritic agent of the present invention is applied include:
Mammals including humans (primates such as humans, companion animals such as dogs and cats, livestock such as cattle, pigs and horses), birds such as chickens, and the prevention of the above-mentioned symptoms of these animals. Can be used for treatment or alleviation.

[0040]

EXAMPLES The present invention will be described below with reference to Examples and Examples, but the present invention is not limited thereto.

Reference Example 1 Preparation of galactosaminoglycan sodium salt (ccgg) 4000 l of water was added to 1,500 kg of cockscomb, minced, and then boiled.
Protease after cooling (Pronase, manufactured by Kaken Pharmaceutical Co., Ltd.)
And hydrolyzed overnight. After adding 32 L of a benzalkonium chloride solution to the hydrolyzed solution, the solution was filtered through diatomaceous earth, and the filtration supernatant was discarded to obtain 180 kg of diatomaceous earth.

The diatomaceous earth was mixed with 350 L of water and 42 L of sodium chloride.
After adding kg, filter and add 250 L of ethanol to the filtrate,
The obtained precipitate was dried to obtain 1.3 kg of powder.

The above powder was dissolved in 1.3 L of water to prepare a 10% solution, and the mixture was treated with nitrite according to the method of Shively and Conrad (supra) to remove heparin and heparan sulfate.

That is, 0.1% of a solution in which the above powder is dissolved
After mixing with an aqueous nitrous acid solution and allowing to stand at room temperature for 10 minutes, the precipitate was removed by filtration. The pH of the filtrate was adjusted to 10.5, sodium chloride was added to a final concentration of 1%, and ethanol was added to the final concentration of 48% with stirring over 30 to 40 minutes. Activated carbon was added to the obtained precipitate, followed by suction filtration. The filtrate was passed through an ion exchange resin Diaion SA-12A (manufactured by Mitsubishi Chemical Corporation) to be desalted. Ethanol was added to the filtrate, and sodium galactosaminoglycan was added. Salt refined product (cc
gg) 105 g were obtained.

Further, 18 lots of purified galactosaminoglycan sodium salt products were prepared in the same manner as described above.

Reference Example 2 Analysis of Galactosaminoglycan

(A) Measurement of Weight Average Molecular Weight The weight average molecular weight was determined by the method of Arai et al. (Biochim. Biophys. Ac
ta, 1117, 60-70, 1992). In other words, elution time in gel filtration (GPC-HPLC) using high performance liquid chromatography using chondroitin sulfate (weight average molecular weight 39100, 18000, 8050) and sodium hyaluronate (weight average molecular weight 104000) with known molecular weights as standard products Determined by The column is TSK gel G4000 PW _XL ,
G3000 PW _XL and G2500 PW _XL (each φ7.8 × 300 mm, manufactured by Tosoh Corporation) were used. Solvent 0.2 mol / L
A sodium chloride solution was used, the flow rate was 0.6 ml / min, and the detector was a differential refractive index detector (RI-8100, manufactured by Tosoh Corporation)
Was used.

(B) Analysis of chondroitinase B digestion pattern Chondroitinase B (0.03 U) was added to 10 μL of buffer A (0.001 mol / L calcium acetate, 0.02 mol / L Tris-HCl, pH 7.5) in 100 μL of ccgg 1% solution. (Seikagaku Corporation) was added and digested at 37 ° C. for 2 hours. Heat for 1 minute in a boiling water bath to stop the reaction, and add 10 μg equivalent to 100 μg of digest.
L was analyzed at 40 ° C. using GPC-HPLC. Column is TSK gel
G4000 PW _XL , G3000 PW _XL and G2500 PW _XL (φ7.8 × 30 each
0 mm, manufactured by Tosoh Corporation). The solvent used was a 0.2 mol / L sodium chloride solution, and the flow rate was 0.6 mL /
The detector used was a differential refractive index detector (RI-8100, manufactured by Tosoh Corporation) and an ultraviolet-visible detector (UV-8020, A230 nm, manufactured by Tosoh Corporation).

(C) Constituent disaccharide composition analysis The position of the sulfate group is analyzed by a known method,
3, Carbohydrate II, p49-62 (1991, Tokyo Chemical Doujinsha), "Refer to" Structural Analysis Combining 2.8 Glycosaminoglycan Degrading Enzyme and HPLC "" as described below. .

That is, ccgg was converted to chondroitinase ABC
(Seikagaku Kogyo Co., Ltd.) completely digested into disaccharides, and generated disaccharides containing unsaturated bonds (unsaturated disaccharides) by GPC-HP
Analyzed by LC.

Further, ΔDi-diS which cannot be separated under these conditions
To separate the _B and [Delta] Di-diS _E, digest was further digested with chondro-6-sulfatase (Seikagaku Co.), to shift the [Delta] Di-diS _E to [Delta] Di-4S, similarly GPC -HP
Analyzed by LC. The two results were compared and analyzed to calculate the constituent disaccharide composition. Details are described below.

(I) Digestion with chondroitinase ABC 0.5 U in 50 μL of 1% ccgg solution and 10 μL of buffer B (0.01 mol / L sodium acetate, 0.05 mol / L Tris-HCl, pH 7.5)
Add a solution of chondroitinase ABC and add 37 ° C
For 2 hours. The reaction was stopped by heating in a boiling water bath for 1 minute, and impurities were removed by centrifugation. To this degradation product, a solution prepared by dissolving 0.5 U of chondroitinase ABC in 10 μL of buffer B was added and digested at 37 ° C. for 6 hours. The reaction was stopped by heating in a boiling water bath for 1 minute, and insolubles were removed by centrifugation. (Ii) Digestion with chondro-6-sulfatase

100 μl of digested product by chondroitinase ABC
Buffer C (0.02 mol / LTris-AcOH, pH 7.0)
A solution prepared by dissolving 0.25 U of chondro-6-sulfatase (manufactured by Seikagaku Corporation) in 50 µL was added, digested at 37 ° C for 24 hours, and centrifuged to remove insolubles. (Iii) Analysis by HPLC

The above digest, that is, chondroitinase ABC
The digested juice, or the digested juice further digested with chondro-6-sulfatase, was analyzed by HPLC. The column is a YMC-Pack PA-120-S5 ion exchange column (φ
2.6 × 250 mm, manufactured by YMC Corporation) was used.

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

(D) Measurement of Intrinsic Viscosity The measurement of the intrinsic viscosity was carried out in accordance with the thirteenth revised Japanese Pharmacopoeia. As a measuring device, an automatic viscosity measuring device (VMC-052, manufactured by Rigosha Co., Ltd.) was used. The solvent used was a 0.2 mol / L sodium chloride solution, and the same solution was used for measuring the flow time of an Ubbelohde type viscosity tube. The viscosity was measured at 30 ± 0.1 ° C, and one hundredth second of the flow time was rounded off.
Measurements within seconds were used for intrinsic viscosity calculations.

The limiting viscosity is the reduced viscosity (ηred = (ts / t ₀ −1) /
C) [ts: flow time of solution, t ₀ : flow time of solvent, C: sample concentration (% by weight)] on the vertical axis, and the concentration obtained by plotting the concentration on the horizontal axis is extrapolated to concentration 0. It was determined from the section when it was done.

(E) Analysis of the molar ratio of iduronic acid to glucuronic acid Determination of the contents of iduronic acid (IdoA) and glucuronic acid (GlcA) in galactosaminoglycan

This measurement was carried out by the method of Karamanos et al. (Analytica
Biochemistry 172, 410-419, 1988). That is, 5 mg of galactosaminoglycan was dissolved in 500 μg of distilled water, and 50 mg of 1-ethyl-3,3-dimethylaminopropylcarbodiimide (EDC) was added and stirred. 37 ℃
At room temperature for 20 minutes, and then add 40 mM HCl
The pH of the solution was maintained between 4 and 5 by adding 0 μl 6-7 times. In the next hour, 1 ml of a 2 M sodium tetrahydroborate (NaBH ₄ ) solution was added twice to add 50 ml.
The reduction reaction was performed at ℃. After the reaction was completed, the reaction mixture was cooled to room temperature, and glacial acetic acid was added to decompose excess NaBH ₄ . Subsequently, desalting was performed by performing running water dialysis overnight, and the dialysis solution was freeze-dried.

Next, 200 μl of 2 MTFA (trifluoroacetic acid) was added to 700 μg of the reduced product of galactosaminoglycan, and the tube was sealed and hydrolyzed at 105 ° C. for 6 hours. After completion of the reaction, 3 ml of distilled water was added and freeze-dried. Next, after adding 500 μl of distilled water to the dried product, the mixture was subjected to Dowex 50-X8 (H ⁺ type) equilibrated with distilled water, and the non-adsorbed fraction was added to the mixture.
Collected in ml of distilled water. After lyophilizing this, 500
μl of a pyridine solution (1 g of benzoic anhydride and 0.5 g of 4-dimethylaminopyridine adjusted to 10 ml with pyridine) was added, and the mixture was heated at 37 ° C. for 90 minutes. After stopping the reaction by adding 4.5 ml of distilled water and stirring, SepPak-C
Desalting was performed using an 18 cartridge column.

With respect to the benzoylated product of the neutral sugar derived from uronic acid of galactosaminoglycan produced as described above, HP
Analyzed by LC. That is, using a Shimadzu HPLC system equipped with a Cosmosil-5C18-P column (φ4.6 × 150 mm) equilibrated with 75% acetonitrile, a flow rate of 1 ml / min, 40
The analysis was performed with UV detection at 230 nm under the condition of column open temperature of ° C. Benzoylated 1,6-
Anhydroidose and benzoylated glucose were used.

As a result, benzoylated 1,6-anhydroidose was eluted as a single peak at a retention time of about 5.7 minutes, while benzoylated glucose was converted to α, β at a retention time of about 12 minutes. It was eluted as a peak corresponding to the anomer (see FIG. 1). In addition, the detection wavelength UV 230 nm
Then, assuming that the detection intensity of α, β benzoylated glucose is 5, that of benzoylated 1,6-anhydroidose is 3. Considering the peak area and the detection intensity, the uronic acid composition is IdoA: GlcA (iduronic acid: glucuronic acid) = 7
6.5: 23.5 was calculated.

(F) Measurement of Number of Sulfate Groups The number of sulfate groups per constituent disaccharide was determined for each galactosaminoglyca
Multiply the unsaturated disaccharide composition (mol%) of
It is obtained by the following formula
Was. Number of sulfate groups per constituent disaccharide (pieces) = [model of ΔDi-0S
% × 0 + (mol% of ΔDi-6S + mol% of ΔDi-4S) × 1 +
(ΔDi-diS_DMole% + ΔDi-diS_BMole% + ΔDi-diS _E
Mole%) × 2 + mol% of ΔDi-triS × 3] ÷ 100

(G) Results Table 3 shows the results for ccgg prepared in Reference Example 1, and Table 4
The above (a) to (f) are for 18 lots of galactosaminoglycan sodium salt other than ccgg prepared in Reference Example 1.
The results obtained by the analysis in the same manner as described above are summarized.

[0065]

[Table 3]

[0066]

[Table 4]

Reference Example 3 Analysis of Dermatan Sulfuric Acid Derived from Other Than a Cock As an example of galactosamine derived from a material other than a cockscomb, dermatan sulfate was produced by a method similar to Reference Example 1 using pig skin or bovine kidney as a raw material. The analysis was performed by the method of 2.
The dermatan sulfate derived from pig skin used here is
It is different from the dermatan sulfate derived from pig skin described in 2.

[0068]

[Table 5]

Example 1 Investigation of the Itching Inhibitory Effect of Galactosaminoglycan Using Atopic Dermatitis Model Mice (NC / Nga Mice) (1) Experimental Materials

As the animal, a 6-week-old male NC / Nga mouse was used as an atopic dermatitis model mouse. As the drug, the galactosaminoglycan sodium salt (ccgg) derived from the cockscomb prepared in Reference Example 1 was sterilized and dissolved in physiological saline for use.

(2) Experimental method Eight NC / Nga mice were divided into two groups of four mice. All mice were placed in cages one by one and reared under mite-positive rearing conditions. Of these, as a non-treated control group, only one group of physiological saline was applied to one group of 4 animals, and the remaining group of 4 animals was subcutaneously subcutaneously three times a week (Monday, Wednesday, Friday) so as to have a ccgg of 60 μg / mouse. It was injected subcutaneously. After injection, they were observed for 10 minutes, and the number of times of curettage for 10 minutes was recorded. Here, the mite-positive breeding conditions refer to conditions that are bred in an isolation facility and that the presence of ticks in animal colonies has been confirmed.

(3) Results FIG. 2 shows the results of measurement of the curettage on the first day of administration and on days 14, 21, and 28 after administration. The error bar in the figure shows the value of the standard error.

As shown in FIG. 2, immediately after the start of the administration, there is no difference in the number of times of curettage between the groups. Raising animals in a tick-positive environment for 14 days or more increases the number of curettage in the untreated control group. This is a characteristic of NC / Nga mice and exhibits allergic symptoms derived from the breeding environment. Specifically, after birth
From around 7 weeks of age, erythema, alopecia, bleeding, and crusting, especially around the neck and auricles, are seen. It is reported in the above-mentioned literature that these symptoms do not appear when reared in a mite-negative environment.

In the 60 μg / mouse administration group, galactosaminoglycan significantly reduced the number of times of scraping on days 14 and 21 (p <0.05). On the 28th day after the start of administration, galactosaminoglycan showed an improving effect. In the galactosaminoglycan administration group, no gross findings (such as subcutaneous hemorrhage at the injection site) were observed during the administration period.

Example 2 (Formulation Example) A 5% aqueous solution of galactosaminoglycan sodium salt (ccgg) obtained in Reference Example 1 was prepared, mixed with hydrophilic petrolatum having the following composition until uniform, and used as an antipruritic ointment. An agent was prepared.

Salami beeswax (manufactured by Cera Rica NODA) 80 g stearyl alcohol or cetanol (manufactured by NOF Corporation) 30 g cholesterol (manufactured by Kanto Chemical Co., Ltd.) 30 g white petrolatum (manufactured by Shima Trading Co., Ltd.) Suitable amount Total amount 1000 g

[0077]

According to the antipruritic agent of the present invention, itching in atopic dermatitis, urticaria, eczema, skin pruritus, prurigo or psoriasis vulgaris accompanied by pruritus can be effectively reduced with a low dose of galactosaminoglycan. It can be prevented and treated, and has no significant side effects. By using the antipruritic agent of the present invention, it is possible to reduce the amount of other therapeutic agents for atopic dermatitis, for example, agents having serious side effects such as corticosteroid hormones and immunosuppressants.

[Brief description of the drawings]

FIG. 1 is a view showing an elution pattern of uronic acid in galactosaminoglycan produced in Reference Example 1 by high performance liquid chromatography.

FIG. 2 is a graph showing the results confirming the antipruritic effect of administering the antipruritic agent of the present invention to mice grown under pruritus-inducing conditions.

Claims (7)

[Claims] 1. An antipruritic agent comprising, as an active ingredient, galactosaminoglycan or a pharmacologically acceptable salt thereof having the following properties; (A) the molar ratio of iduronic acid to glucuronic acid in the constituent sugars is about 40:60. (B) the digestibility by chondroitinase B is from about 40% by weight to about 100: 0.
(D) Chondroitinase A, wherein (C) the number of sulfate groups per constituent disaccharide is from about 0.9 to about 1.3,
In the constituent disaccharide composition calculated by digestion with BC and analysis by high performance liquid chromatography, 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo represented by ΔDi-0S -Hex-4-enopyranosyluronic acid) -D-galactose, 2-di represented by ΔDi-4S
Acetamide-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose and ΔDi-6S
2-acetamido-2-deoxy-3-O- represented by
The total of (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-galactose is from about 71 mol% to about 94 mol%. 2. The properties of galactosaminoglycan are as follows:
(A) the molar ratio of iduronic acid to glucuronic acid in the constituent sugars is about 65:35 to about 90:10, (B) the digestibility by chondroitinase B is about 65% to about 95% by weight, The antipruritic according to claim 1, characterized in that: 3. The antipruritic according to claim 1, wherein the galactosaminoglycan is derived from a bird tissue or organ. 4. The antipruritic according to claim 1, wherein the weight average molecular weight of the galactosaminoglycan is about 1,600 Da to about 100,000 Da. 5. The antipruritic according to claim 4, wherein the weight average molecular weight of the galactosaminoglycan is about 23 kDa to about 45 kDa. 6. A disaccharide composition which is digested with chondroitinase ABC and analyzed by high performance liquid chromatography and has a composition of 2-acetamido-2-deoxy-3-O- (4 The antipruritic according to claim 1, wherein the content of (-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -D-galactose is about 2 to about 15%. 7. Prevention of atopic dermatitis, hives, eczema, pruritus cutaneous, prurigo, or pruritus associated with psoriasis vulgaris.
The antipruritic according to claim 1, which is used for treatment.