Fraction of certaincompanyshare and containing pharmaceutical preparation

 

(57) Abstract:

The pharmaceutical preparation contains a pharmaceutically acceptable carrier or diluent and, as active ingredient an effective amount of keratomalacia with two to five sharedname links, sulfated N-acetylglucosamine at its reducing end, with sulfated N-acetyllactosamine link, optionally containing sialic acid and/or fucose having at least two sulfated hydroxyl group in the molecule and/or its pharmaceutically acceptable salt. Preferably certaincompanyshare contains as an integral ingredient, at least, the disaccharide of formula Gal(6S) - GlcNAc(6S), where Gal represents galactose, GlcN is glucosamine, AC represents acetyl group and 6S is 6-O-sulfate ester. Pharmaceutical drug has anti-inflammatory, anti-allergic, immunological, inducing apoptosis and cell differentiation effects. The technical result consists in realization of the specified destination. 3 S. and 18 C.p. f-crystals, 11 tab., 22 Il.

The present invention relates to an anti-inflammatory agent, protivoballisticheskih, which can be used as the active ingredient in these pharmaceutical drugs.

Background of the invention

Keratinolytic is glycosaminoglycan containing N-acetyllactosamine as a basic structure in which the position of the C-6N-acetylglucosaminidase balance has O-sulfated hydroxyl group. In particular, it is known that vysokotarifitsirovannyh Kermanshah, except C-6N-acetylglucosamine residue contains sulfation hydroxyl group in the structural disaccharide glycosides link found in cartilaginous fish, such as sharks, and in the cartilage, bone and cornea mammals, such as whales and ruminants. Reported method of producing keratomalacia, which is the product of cleavage of keratomalacia, which includes the impact of splitting keratinolytic enzyme (keratinase II); published patent application Japan N 2-57182) derived from microorganisms belonging to Bacillus, keratinolytic.

In addition, there is a message (Biochemistry, 33, 4836-4846 (1994)), which gives an analysis of the 25 species of oligosaccharide fractions obtained by fractionation of the products rossalini tetrachlorophenol N-acetylglucosaminyltransferase, represented by the following formula (I) (referred to hereafter as the "createsunmaterial (I)"), resultatvindauge N-acetylglucosaminidase, represented by the following formula (II) (referred to hereafter as the "certaincompanyshare (II)"), desulfuromonas N-acetylglucosaminidase, represented by the following formula (III) (referred to hereafter as the "keratomalacia (III)"), and so forth:

Gal(6S) 1-4 GlcNAc(6S) 1-3 Gal(6S) 1-4 GlcNAc(6S).....(I)

NeuAc~Gal 1-4 GlcNAc(6S) 1-3 Gal(6S) 1-4 GlcNAc(6S)...(II)

Gal(6S) 1-4 GlcNAc(6S)... (III)

(In the above formulas, Gal is galactose, GlcN is glucosamine, Neu represents Narimanovo acid, Ac represents acetyl group, and 6S is 6-O-sulfate ester. "~" represents the connection 2, 3 or Association 2, 6).

To date, however, no messages relating to the effective mass production of clean keratomalacia, especially createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III), without contamination by impurities (e.g., endotoxins, nucleic acids, proteins, proteases, other glycosaminoglycans, excluding certaincompanyshare and so is ratesoriginally use as pharmaceuticals. In addition, the pharmacological action of certaincompanyshare (in particular, anti-inflammatory action, anti-allergic activity, immunomodulatory activity, inducing cell differentiation activity and inducing apoptosis of action) is not known.

Description of the invention

Based on the above carry out the present invention and its purpose is the use of keratomalacia essentially not containing impurities, as anti-inflammatory agent, an antiallergic drug, immunomodulator, an inducer of cellular differentiation (called here referred to as inducers of differentiation") or inducer of apoptosis.

In order to achieve the above objective, the applicants conducted a painstaking study of pharmacological activity of the oligosaccharide that contains from two to five sharidny links, in particular disaccharide, tetrasaccharide or pentasaccharide derived from cleavage products, which are obtained by splitting vysokomehanizirovannogo of createsurface splitting keratinolytic enzyme, and as a result found that the above oligosaccharide and its salts have prevoshodyashim cell differentiation activity and inducing apoptosis effect. Thus carry out the present invention.

Namely anti-inflammatory, antiallergic agent, an immunomodulator, an inducer of differentiation and inducer of apoptosis according to the present invention (referred to here as hereinafter collectively "pharmaceutical preparations of the present invention") contains certaincompanyshare and/or its pharmaceutically acceptable salt as an active ingredient. In the present description "certaincompanyshare" means the product of cleavage of keratomalacia, which can be obtained by splitting createsurface splitting keratinolytic enzyme type endo- -N-acetylglucosaminidase.

Examples of certaincompanyshare used for pharmaceutical preparations of the present invention, are certaincompanyshare, which contains sulfated N-acetyllactosamine link, optionally containing sialic acid and/or fucose, certaincompanyshare, which contains oligosaccharide with two to five sharedname links and has a sulfated N-acetylglucosamine at its reducing end, and in the molecule of which sulfotyrosine at least two hydroxyl the aqueous component, at least, a disaccharide represented by formula Gal(6S)-GlcNAc(6S) (where, Gal is galactose, GlcN is glucosamine. Ac represents acetyl group and 6S is 6-O-sulfate ester). Moreover, the preferred example of keratomalacia described above, is tetrachlorophenyl N-acetylglucosaminidase, represented by the following formula (I), trisulfonic N-acetylglucosaminidase represented by the formula (II), desulfuromonas N-acetylglucosaminidase represented by the formula (III), etc.:

Gal(6S) 1-4 GlcNAc(6S) 1-3 Gal(6S) 1-4 GlcNAc(6S)...(I)

NeuAc~Gal 1-4 GlcNAc(6S) 1-3 Gal(6S) 1-4 GlcNAc(6S)...(II)

Gal(6S) 1-4 GlcNAc(6S)... (III)

(In the above formulas, Gal is galactose, GlcN is glucosamine, Neu represents Narimanovo acid. Ac represents acetyl group and 6S is 6-O-sulfate ester. And ~ is the link 2, 3 or Association 2, 6).

The present invention also refers to the fraction of keratomalacia containing at least 99% of keratomalacia, which contains oligosaccharide with two to five sharedname links and sulfated N-acetylglucosamine at its reducing end, whose molecules sulfation the at does not contain endotoxin, and the content of nucleic acids, proteins and proteases in the faction less about the limits of detection; and

(b) fraction essentially does not contain hyaluronic acid, chondroitin sulfate, dermatooncology, heparansulfate and keratinolytic.

The present invention also refers to the fraction of keratomalacia containing at least 99% of keratomalacia, which contains as a structural component, at least a disaccharide represented by formula Gal (6S)-GlcNAc (6S), and having the properties (a) and (b) described above. Also preferred example of keratomalacia contained in the above oligosaccharide fractions is tetrachlorophenyl N-acetylglucosaminyltransferase represented by the formula (I), trisulfonic N-acetylglucosaminidase represented by the formula (II), desulfuromonas N-acetylglucosaminidase represented by the formula (III), etc. in Addition, certaincompanyshare includes certaincompanyshare obtained by fractionation of the products of cleavage vysokomehanizirovannogo of createsurface derived from cartilaginous fish, splitting keratinolytic enzyme type endo-athenslifehouse, including the stage of splitting createsurface splitting keratinolytic enzyme having the following physical and chemical properties:

(1) action:

digestive enzyme affects keratinolytic and hydrolyzes N-acetylglucosaminidase communication;

(2) the specificity of the substance:

digestive enzyme acts on keratinolytic I, keratinolytic II and keratinophilic and produces sulfated keratomalacia and sulfated createsunmaterial as the main cleavage products;

and preferably with the physical and chemical properties:

(3) optimum pH of the reaction:

digestive enzyme has an optimal pH of the reaction from 4.5 to 6 in 0.1 M acetate buffer or 10 mm Tris-acetate buffer at 37oC;

(4) pH stability:

digestive enzyme has a pH stability from 6 to 7, when the digestive enzyme remains in 0.1 M acetate buffer or 10 mm Tris-acetate buffer at 37oC for one hour;

(5) optimum temperature response:

digestive enzyme has an optimum reaction temperature from 50 to 60oC when splitting enzyme reacts in 0.1 M acetate buffer, pH 6,0, BP>C or below, when digestive enzyme remains in 0.1 M acetate buffer, pH 6,0, within one hour;

and fractionation of the products of the cleavage fractions keratomalacia with the following properties:

(A) fraction contains as a main ingredient certaincompanyshare, which contains sulfated N-acetyllactosamine as the main structural component;

(B) fraction essentially does not contain endotoxin, and the content of nucleic acids, protein and protease in the faction is a trace or less with respect to the limits of detection; and

(C) fraction essentially does not contain hyaluronic acid, chondroitin sulphate, dermatosurgery, heparan sulfate and keratomalacia.

In addition, when the method of obtaining the fraction of keratomalacia described above, for example the use of vysokomehanizirovannogo of createsurface as connection createsurface gives certaincompanyshare containing tetrachlorophenyl N-acetylglucosaminidase represented by the formula (I) described above, trisulfonic N-acetylglucosaminidase represented by the formula (II), desulfuromonas N-acetyllactosamine the sulfated N-acetylglucosaminidase, represented by formula (I) and desulfuromonas N-acetylglucosaminidase represented by the formula (III), as the main ingredients.

In the present invention the preferred certainaithousholder, which contains oligosaccharide with two to five sharedname links, is usually an oligosaccharide, which is sulfated in two to four positions. In addition, preferably, cartonconveyance containing sialic acid, which can be used according to the present invention, the sialic acid comprises N-acetylneuraminic acid and N-glycolylneuraminic acid and N-acetylneuraminic acid is preferred. Certaincompanyshare, which can be used according to the present invention, described above, contains oligosaccharides that bind sialic acid 2.3 communication and 2.6 communication and, preferably, binding of sialic acid 2.3 communication.

The present invention is described next.

(1) Certaincompanyshare and the fraction of keratomalacia used in the present invention

Kermanshah, which is the starting product for keratomalacia, spolzli galactose-6-sulfate and N-acetylglucosamine-6-sulfate, the content of sulfate which varies depending on the species, tissue, etc. and are usually produced from feedstocks such as cartilage, bone and cornea cartilaginous fish such as sharks and marine mammals such as whales and ruminants.

Used as initial substances keratinolytic is usually available material and has no special limitation, but preferred vysokotarifitsirovannyh keratinolytic (vysokotarifitsirovannyh keratinolytic containing from 1.5 to 2 molecules of sulfate group on structural disaccharide, referred to here as keratinophilic), which sulfated included in the structure of monosaccharides, such as galactose residue. Sulfate group, preferably located in position 6 on the galactose residue. This vysokotarifitsirovannyh keratinolytic can be obtained, for example, of the proteoglycan of cartilage cartilaginous fish such as sharks. Commercially available keratinolytic can also be used.

Certaincompanyshare of the present invention can be obtained by acting on keratinolytic splitting keratinolytic enzyme type endo- -N-acetylglucosaminidase, for example, keratinase II, received and what his keratinolytic enzyme, discovered by the authors of the present invention the microorganism belonging to the genus Bacillus, in a buffer solution and splitting it, and subsequent fractionation of the products of cleavage. This cleavage reaction is conducted, for example, in a buffer solution containing keratinolytic in concentrations of from 1.0 to 100 mg/ml at a pH of from 6.0 to 7.0 at 25 to 40oC for from 1 to 72 hours When the reaction concentration of the buffer is typically in the range from 0.01 to 0.2 M Type of buffer is not particularly limited if its pH can be set in the above range, and the number of such buffers include, for example, acetate buffer, phosphate buffer and Tris buffer. The amount of enzyme used in the cleavage reaction is, for example, in the range from 0.1 to 1.0 units per 1 g of keratomalacia. Here, one unit is the amount of enzyme that produces the reducing end, corresponding to 1 μmol of N-acetylglucosamine per minute.

New above splitting keratinolytic enzyme is produced by a microorganism belonging to Bacillus circulans, for example Bacillus circulans KsT202, was selected by the applicants, and it has excellent heat resistance. This enzyme is produced by culturing Bacillus circulans the cops. Bacillus circulans KsT202 deposited in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan) September 5, 1994 under inventory number FERM P-14516 and then transferred to international Deposit under the Budapest Treaty, November 6, 1995 , and deposited under inventory number FERM BP-5285.

Physical and chemical properties of the new splitting keratinolytic enzyme described above is described next.

(1) Action

Digestive enzyme acts on keratinolytic and hydrolyzes N-acetylglucosaminidase link.

(2) the Specificity of the substrate

Digestive enzyme acts on keratinolytic I, keratinolytic II and keratinophilic and produces sulfated keratomalacia and createsunmaterial as the main products of the cleavage. Confirmed that digestive enzyme produces sulfated createsolidbrush.

(3) Optimum pH of the reaction:

Digestive enzyme has an optimal pH of the reaction from 4.5 to 6 in 0.1 M acetate buffer or 10 mm Tris-acetate buffer at 37oC.

(4) pH Stability:

Digestive enzyme on tatom buffer at 37oC for one hour.

(5) Optimum temperature response:

Digestive enzyme has an optimum reaction temperature from 50 to 60oC when splitting enzyme reacts in 0.1 M acetate buffer, pH to 6.0, for 10 minutes

(6) thermal stability:

Digestive enzyme is stable at least at 45oC or below, when digestive enzyme remains in 0.1 M acetate buffer, pH 6,0, within hours.

Fraction of keratomalacia of the present invention can be obtained also by reacting the immobilized enzyme that breaks down Kermanshah, including this new splitting keratinolytic enzyme and keratinase II, above, are immobilized in the usual way.

In the cleavage reaction by an enzyme, as described above, keratinolytic into oligosaccharide.

Then thus obtained oligosaccharide is separated and purified by conventional means, such as precipitation with ethanol and various types of chromatography, and you can get the desired certaincompanyshare. This cleanup will be described in detail when describing the purification of keratomalacia from disaccharide, tetrasaccharide the filtration (interval fractionated molecular weight of from 100 to 10000), so roughly to fractionate crude certaincompanyshare from disaccharide, tetrasaccharide and pentasaccharide. Then of coarse fraction and share fractionary anion-exchange chromatography, respectively, to obtain essentially pure disaccharide, tetrasaccharide and pentasaccharide, which essentially does not contain endotoxin, and the content of nucleic acids, proteins and proteases in less than limits of detection and which essentially does not contain hyaluronic acid, chondroitin sulphate, dermatosurgery, heparan sulfate and keratomalacia. In the present invention essentially do not contain" means such content, which can be detected by sensitive methods of detection, but does not affect the pharmacological functions of keratomalacia, such as anti-inflammatory function, antiallergic function, immune function, function, induction of cellular differentiation and function of inducing apoptosis.

Certaincompanyshare of the present invention is obtained from createsurface as the original product by splitting createsurface endo- -N-acetylglucosaminidase and fractionation of oligosaccharide from disaccharide is the Rial.

Certaincompanyshare can also be obtained from the products of cleavage of createsurface cleaved by a chemical method, which is preferable, or specifically destroyed the relationship between galactose or galactose-6-sulfate and N-acetylglucosamine-6 - sulfate, which are keratinolytic.

Certaincompanyshare, especially tetrachlorophenyl N-acetylglucosaminyltransferase represented by the formula (I) described above, trisulfonic N-acetylglucosaminidase represented by the formula (II), desulfuromonas N-acetylglucosaminidase represented by the formula (III), and so on, receive, as described above. In the following examples are given spectra nuclear magnetic resonance (1H-NMR and13C-NMR) and the results of mass spectrometric analysis by fast atom bombardment substances represented by formulae (I), (II) and (III).

Certaincompanyshare the present invention also includes ionized substance, protonated substance and a pharmaceutically acceptable salt of a number of salts with inorganic bases, such as alkali metals (sodium, potassium, lithium, etc), alkaline earth metals (calcium, etc ), and the l amino acids.

Pharmaceutical composition containing certaincompanyshare and/or its salt used in the present invention, carriers, diluents and other additives commonly used in pharmaceutical preparations, is also new and can be entered with the purpose of anti-inflammatory, anti-allergic, immune-modulating activity, with the intention of inducing cellular differentiation, induction of apoptosis, etc.

Fraction of keratomalacia of the present invention contains at least 99% of keratomalacia, which is obtained from the products of the enzymatic cleavage of keratomalacia, removing from them endotoxin, nucleic acid, protein, protease and other glycosaminoglycans, except the right keratomalacia, especially from the products of cleavage vysokomehanizirovannogo of createsurface by following the above method.

(2) the Pharmaceutical composition according to the present invention

Certaincompanyshare and/or its pharmaceutically acceptable salt described above can be widely applied as anti-inflammatory agent, an antiallergic drug, immunomodulator, inductor kleptoparasitism tool of the present invention is effective for many diseases, associated with inflammation, and specific indications for which include chronic rheumatoid arthritis, systemic lupus erythematosus, spondylitis changes, osteoarthritis, lumbago, remission of inflammation and hypertrophy after operations and traumas, nodosa blades, temporal-niechlujny arthritis, tendonitis, enveloping inflammation of the tendons, inflammation of condyle of humerus (tennis elbow), myalgia, keratoconjunctivitis, and such inflammation. Anti-inflammatory agent of the present invention has anti-inflammatory effects, such as analgesic effect, anti-inflammatory and antipyretic effects, diseases and symptoms, which operates certaincompanyshare and/or its pharmaceutically acceptable salt.

Anti-inflammatory agent of the present invention is effective for any disease that is associated with allergies, and specific indications for which include allergic rhinitis, allergic keratoconjunctivitis, vernal conjunctivitis, eczema, dermatitis, urticaria, atopic dermatitis and similar diseases.

Immunomodulator of the present invention is effective for any disease related syndrome person (Cell 81, 935-946 (1995), Science 268, 1347-1349 (1995)), lymphoproliferative disorder (the Leukemia and Lymphoma 16, 363-368 (1995), angioimmunoblastic lymphadenopathy (Blood 85(10), 2862-2869 (1995)), immunoblastic lymphadenopathy (The American Journal of Medicine 63, 849-851 (1977)), chronic rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, dermatomyositis (dermatobia), scleroderma, mixed diffuse connective tissue disease, chronic thyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia, the combination of pneumopathy with severe anemia, acute progressive glomerulonephritis, myasthenia gravis, common bladderwort, bullous pemphigoid, non-insulin-dependent diabetes mellitus, juvenile diabetes, a disease Adisson, atrophic gastritis, male sterility, menopause precox (precox), phacogenic uveitis, sympathetic anghit, multiple sclerosis, progressive systemic sclerosis, inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.,), primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenic purpura, Sjogren syndrome, syndrome antiphospholipid antibodies and similar to the NII, caused by deficiency of physiological cellular differentiation, immune disorder, malignant tumor, etc., and the specific indications for which are autoimmune lymphoproliferative syndrome human lymphoproliferative violation, angioimmunoblastic lymphadenopathy, immunoblastic lymphadenopathy, chronic rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc), progressive systemic sclerosis, dermatomyositis (dermatobia), Sjogren syndrome, carcinoma, leukemia, lymphoma, delay carcinophaga metastasis, prevention of prostatic (treatment against psoriasis, etc), the healing of wounds, syndrome osteomielofibros, scleroderma and similar diseases.

Inducer of apoptosis according to the present invention is effective for any disease caused by a deficiency of physiological apoptosis, immune disorder, malignant tumor, etc., and the specific indications for which are autoimmune lymphoproliferative syndrome human lymphoproliferative violation, angioimmunoblastic lymphadenopathy, immunoblastic lymphadenopathy, chronic rawmat is h, dermatomyositis (dermatobia), Sjogren syndrome, carcinoma, leukemia, lymphoma, delay carcinophaga metastasis, prevention of prostatic (treatment against psoriasis, etc), syndrome osteomielofibros, scleroderma, apoptosis inducing abnormal mesangial cells (treatment against glomerulonephritis) and similar diseases.

Effects of weight reduction lymph nodes, inducing differentiation and induction of apoptosis was observed in mice MRL-lpr/lpr. The pathogenesis of autoimmune lymphoproliferative syndrome person is the same violation of the Fas gene, as in mice MRL-lpr/lpr, and pathology of the syndrome has remarkable similarities with the pathology of mice MRL-lpr/lpr, as for example, in relation to the swelling of the lymph nodes. Mice MRL-lpr/lpr can be taken as a suitable model of autoimmune lymphoproliferative syndrome person. Therefore, the preferred reading is the use of pharmaceutical drugs immunomodulator, the inducer of differentiation and inducer of apoptosis of the present invention is an autoimmune lymphoproliferative syndrome person.

The pharmaceutical preparations of the present invention can be designed as a way of introduction, the intraperitoneal and so on), instillation in the eye, instillation, transcutaneous introduction, oral administration and inhalation. Dosage forms include forms for injections (solutions, suspensions, emulsions, solid preparations for injection, soluble before use, etc), tablets, capsules, granules, powders, solutions, liposomal inclusion, ointments, gels, powders for external use, sprays, powders for inhalation, eye drops, eye ointments, and the like. Upon receipt of the pharmaceutical preparations can be applied ingredients commonly used in medicines, such as traditional carriers, binders, lubricants, dyes and leavening agents. In the pharmaceutical preparations of the present invention certaincompanyshare can be used simultaneously with other anti-inflammatory ingredients, hypoallergenic ingredients, immunomodulatory ingredients, ingredients that induce differentiation, ingredients, inducing apoptosis, etc.

In table. 1 shows the preferred routes of administration and dosage forms anti-inflammatory and anti-allergic medicines. In table. 2 shows the preferred routes of administration and dosage form, and tivovospalitiona means and an antiallergic drug of the present invention is from 30 to 300 mg/patient/day of keratomalacia when introduced into the body and from 1 to 10 mg/patient/day when the outer introduction. Effective dose immunomodulator, inductor differentiation and inducer of apoptosis is in the range of from 30 to 6000 mg/patient/day of keratomalacia.

In Fig. 1 shows the chromatogram of gel filtration using HPLC tetrachlorophenol N-acetylglucosaminyltransferase (createsunmaterial (I)), obtained in example 1.

In Fig. 2 shows the chromatogram of gel filtration using HPLC resultatvindauge N-acetylglucosaminidase (createsolidbrush (II)) obtained in example 1.

In Fig. 3 shows the chromatogram of gel filtration using HPLC desulfuromonas N-acetylglucosaminidase (keratomalacia (III) obtained in example 1.

Fig. 4 shows the range of1H-NMR at 400 MHz certaincompanyshare (II) obtained in example 1.

Fig. 5 is a graph showing the volume of synovial fluid in rabbit models of arthritis, called papain, which was introduced createsunmaterial (I), createsolidbrush (II) or keratan sulfatgehalt (III).

Fig. 6 is graphs showing the change in time of the development of edema denene administered to rats for five minutes before induction of inflammation.

Fig. 7 is graphs showing the time change of swelling stop in rats, which caused the inflammation, when createsunmaterial (I) or of different test compounds administered to rats for three hours before induction of inflammation.

In Fig. 8 is a diagram that shows the volume of pleural effusion in rats models carragenine (carrageenin) of pleurisy, which was introduced createsunmaterial (I) or dexamethasone.

In Fig. 9 provides a graph showing the number of cells in the pleural effusion in rats models carragenine pleurisy, which was introduced createsunmaterial (I) or dexamethasone.

In Fig. 10 presents a graph showing the inhibitory effect on the generation of active oxygen (O-2) createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III) for neutrophils Guinea pigs, which is stimulated by N-formyl-Met-Leu-Phe (FMLP).

In Fig. 11 provides a graph showing the evaluation of the degree of development of conjunctivitis in models of allergic conjunctivitis in Guinea pigs, which impose or not impose createsunmaterial (I).

Fig. 13 is a graph showing the evaluation of the degree of development of conjunctivitis in models of allergic conjunctivitis in Guinea pigs, which impose createsunmaterial (I) in various concentrations.

In Fig. 14 is a chart showing the weight of the submandibular lymph nodes in mice MRL, which is a model of autoimmune disease, which within 28 days re-injected different doses of keratomalacia (III).

In Fig. 15 is a chart showing the weight of mesenteric lymph nodes in mice MRL, which within 56 days re-injected different doses of keratomalacia (III).

Fig. 16 is a graph showing the weight of the submandibular lymph nodes in mice MRL, which within 56 days re-injected different doses of keratomalacia (III).

Fig. 17 is a chart on which are given the results of the analysis of the concentrations staining samples of sections of mesenteric lymph nodes (NON-staining) in mice MRL, the OI are given the results of the analysis of the concentrations of the coloring of the sample slices submandibular lymph nodes (Non - staining) in MRL mice, which were injected with varying doses of keratomalacia (III).

Fig. 19 is a chart showing the proportion (%) of CD3 and CD4 positive cells for lymphocytes in MRL mice, which were injected with varying doses of keratomalacia (III).

Fig. 20 is a chart showing the proportion (%) of CD3 and CD8a positive cells for lymphocytes in MRL mice, which were injected with varying doses of keratomalacia (III).

Fig. 21 is a chart showing the proportion (%) of CD3 and 220V positive cells for lymphocytes in MRL mice, which were injected with varying doses of keratomalacia (III).

Fig. 22 is a diagram showing the number of apoptotic cells in the submandibular lymph nodes of MRL mice, which were injected with varying doses of keratomalacia (III).

Preferred embodiments of the invention

The present invention is detailed below using the following examples. In the production example describes the production of new split keratinolytic enzyme derived from a microorganism belonging to the genus Bacillus. Example 1 describes how to obtain fractions of createsortspecification. In addition, in examples 3-6 are examples of pharmaceutical preparations.

An example of the production. The production of the enzyme that breaks down keratinolytic

(1) Isolation of Bacillus circulans KsT202

A small amount of soil added to 5 ml of a liquid medium containing a nitrogen source, inorganic salts and keratinolytic and cultured with shaking at 45oC for three days. After cultivation, 10 μl of the supernatant culture medium is placed on a filter paper, and also in the same way put on filter paper 10 μl of a liquid medium (control). After drying in air filter paper moistened in a solution of toluidine blue and consequently washed with diluted acetic acid solution and the color on the side of the drops of the supernatant culture medium compared to the color control patches. So toluidine blue connects with keratomalacia with the formation of blue color, less intense staining than the control sample, confirms that the sample is acquiring keratinolytic microorganism. Acquiring keratinolytic microorganism isolated in pure form from a solution of the conventional culture method using the absorption createsurface using a liquid medium as well as described above, and receive the desired microorganism to assimilate keratinolytic. Studies of the morphology, growth and physiological properties of the strain in question, the microorganism identified as Bacillus circulans. The specified strain is a new strain that can be distinguished from known strains, as it absorbs keratinolytic. KsT202 Bacillus circulans deposited in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology September 5, 1994 under inventory number FERM P-14516, and he transferred to an international Deposit based on Budapest Treaty, November 6, 1995, and deposited under inventory number FERM BP-5285.

(2) Obtaining an enzyme that breaks down keratinolytic

In a fermenter with a capacity of 30 l load 20 l of a medium (pH 8.0) containing 1.5% of peptone (Kyokuto Seiyaku Kogyo), 0,75% of yeast extract (Nippon Seiyaku), 0,75% keratoprosthesis derived from shark cartilage (Seikagaku Corporation), 0.5% of K2HPO4, 0,02% MgSO47H2O, 0.5% of NaCl and 0,0015% protivovspenivayushchie substances Adekanol (trade name, Asahi Denka Kogyo), autoclave at 121oC for 20 min, inoculant filled 1 l (5%) solution culture strain KsT202 pre-cultured with shaking in the same medium at 37oC is 00 rpm). Treated with 20 l of a solution culture on the centrifuge with continuous action to remove the microbial cells, and receive approximately 20 liters of extracellular fluid.

This extracellular fluid add ammonium sulfate to 70% saturation and centrifugation remove loose sediment, and dissolved it in 2.5 l of 10 mm Tris-acetate buffer (pH 7.5). To this solution add ammonium sulfate to 35% saturation, the precipitation is removed by centrifugation, and then add ammonium sulfate to 55% saturation, and the precipitation is removed by centrifugation.

The residue is dissolved in 2.5 l of 10 mm Tris-acetate buffer (pH 7.5) and passed through a column (5.2 x 24 cm) of DEAE-cellulose (DE52, Whatman Co.), which pre-balance with the same buffer, and allow the enzyme to be adsorbed. Then the column was washed with 1.5 l of the same buffer and the enzyme elute with a linear increasing concentration of sodium chloride from zero to 0.3 M in the same buffer.

The active fractions are collected, add ammonium sulfate to 55% saturation, and then precipitated precipitate is removed by centrifugation and dissolved in a small amount of 10 mm Tris-acetate buffer (pH 7.5). Then the solution is loaded onto a column sephacryl S-300 (Pharmacia) (3,4 the tx2">

The active fraction is concentrated by ultrafiltration using membranes UK-10 (Advantec Toyo) and cialiswhat against approximately 100-fold amount of 10 mm Tris-acetate buffer (pH 7.5). Cialisovernight internal solution passed through a column of DEAE-Toyopearl (Tosoh Corporation) (2.2 x 15 cm), which is pre-balance with the same buffer, and allow the enzyme to be adsorbed. Then the column was washed with 150 ml of the same buffer containing 0.1 M sodium chloride, and the enzyme elute with a linear increase in the concentration of NaCl from 0.1 to 0.2 M in the same buffer.

The active fraction is concentrated by ultrafiltration, loaded onto a column sephacryl S-300 (2.2 x 101 cm), and subjected to gel filtration.

Once added to the active fraction of sodium chloride to a concentration of 4 M solution passed through the column with phenyltetrazol (Pharmacia) (1.6 x 15 cm), which balance Tris-acetate buffer (pH 7.5) containing 4 M sodium chloride, and then elute the enzyme in a linear decrease in the concentration of sodium chloride from 4 M to zero in the same buffer. Get 29 units of enzyme, and its specific activity is 2.09 units/mg (calculated on the weight of bovine serum albumin). The purified enzyme is not sodergren has the following properties:

(1) Action

Digestive enzyme affects keratinolytic and hydrolyzes N-acetylglucosaminidase link.

(2) Substrate specificity

Digestive enzyme affects keratinolytic I, keratinolytic II and keratinophilic and produces sulfated keratomalacia and sulfated createsunmaterial as the main cleavage products. Also confirmed that digestive enzyme produces sulfated createsolidbrush.

(3) Optimum pH of the reaction

Digestive enzyme has an optimal pH of the reaction from 4.5 to 6 in 0.1 M acetate buffer or VMM Tris-acetate buffer at 37oC.

(4) pH Stability

Digestive enzyme has a pH stability from 6 to 7, when the digestive enzyme remains in 0.1 M acetate buffer or 10 mm Tris-acetate buffer at 37oC for one hour.

(5) the Optimal reaction temperature

The optimum reaction temperature splitting enzyme ranges from 50 to 60oC when splitting enzyme reacts in 0.1 M acetate buffer, pH to 6.0, for 10 minutes

(6) heat

Digestive enzyme resistant to at measures"ptx2">

Splitting keratinolytic enzyme obtained as described above, is used in the following examples. However, the present invention is not limited to this enzyme, and in the present invention can use other digestive keratinolytic enzymes type endo- -N-acetylglucosaminidase, such as keratinase II.

Example 1

Dissolve 50 g vysokomehanizirovannogo of createsurface derived from shark cartilage, 300 ml of 0.1 M acetate buffer (pH 6,0). To this solution add 25 units splitting

keratinolytic enzyme described above, and vysokotarifitsirovannyh keratinolytic split at 37oC within 24 hours After the cleavage reaction, to the reaction mixture add double (by volume, and more) amount of ethanol and the mixture is stirred and allowed to stand overnight at room temperature. The next day, the mixture was separated by centrifugation (4000 rpm, 15 min) the supernatant and precipitate, and the supernatant concentrated under reduced pressure, and get the concentrate (called hereafter "A supernatant"). On the other hand, the residue is dissolved in 300 ml of distilled water, mixed with triple the amount of ethanol and stirred. Then the MCA is mutant and the precipitate and the supernatant concentrated under reduced pressure, and get the concentrate (called hereafter "the supernatant B").

The supernatant dissolved in A small amount of distilled water, subjected to gel chromatography using columns with Bio-Gel P-2 (Bio-Rad) (3.6 x 134 cm) and distilled water as a solvent, and then subjected to ion-exchange chromatography, and get the fractions containing createsunmaterial (I), createsolidbrush (II) and keratomalacia (III), respectively. Each of the fractions are dried by freezing.

Each of these certainaithousholder fractions dissolved in a small amount of distilled water, and optionally purified by anion exchange chromatography using a column with Muromac (Muromachi Depending Kogyo) (4.3 x 35 cm), which is pre-balance distilled water. As eluent using a physiological solution containing a linearly increasing amount of sodium chloride from zero to a concentration of 0.3 M, and elute createsunmaterial (I), createsolidbrush (II) and keratomalacia (III), respectively.

Each of the obtained fractions createsunmaterial (I), certaincompanyshare (II) and createsurface the key with cellulating GCL-25 (Seikagaku Corporation) (3,2 x 125 cm) and dried by freezing.

Process also supernatant B in the same way described above, and receive createsunmaterial (I), createsolidbrush (II) and keratomalacia (III).

Chromatogram, obtained by gel filtration using HPLC (high performance liquid chromatography), createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III) obtained above are shown in Fig. 1-3, respectively.

50 g of createsurface gain of 7.8 g (15.6 per cent) createsunmaterial (I), 1.3 g (2.6 per cent) certaincompanyshare (II) and 10.4 g (20.8 per cent) keratomalacia (III), and none of them contain endotoxins, nucleic acids, proteins, proteases and other glycosaminoglycans.

Spectra1H-NMR and13C-NMR createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III), obtained above, is determined using a spectrometer JNM-EX400 (400 MHz, JEOL Ltd.) in the case of spectra1H-NMR and spectrometer JNM-EX400 (100 MHz, JEOL Ltd.) in the case of spectra13C-NMR, respectively, with sodium 3-(trimethylsilyl)propionate-D4as a standard substance. The chemical shift and the constant interaction present (M. D.) and Hz is P (D2O 40oC): 4,757 (1H, d), 4,565 (1H, d), 4,561 (1H, d), 4,402 (1H, DD), 4,342 (2H, DD), 3,711 (1H, DD), 3,626 (1H, DD), 3,555 (1H, DD), 2,069 (3H, s), 2,047 (3H, s)

13C-NMR (D2O, 25oC): 177,81, 177,63, 177,30, 105,87, 105,69, 97,84, 93,34, 84,98, 82,41, 81,91, 81,25, 75,55, 75,44, 75,20, 75,13, 75,02, 73,70, 72,68, 72,07, 71,10, 71,01, 70,61, 69,82, 69,82, 69,59, 69,29, 58,92, 57,94, 56,42, 25,09, 24,76

Createsolidbrush (II)

1H-NMR (D2O, 25oC): the spectrum is given in Fig. 4

13C-NMR (D2O, 25oC): 177,80, 177,32, 176,86, 105,92, 105,78, 104,94, 102,60, 97,86, 93,32, 85,05, 82,55, 82,04, 79,75, 78,16, 77,93, 75,69, 75,59, 75,48, 75,24, 74,98, 74,36, 72,68, 72,33, 72,07, 71,38, 71,01, 70,65, 70,35, 69,62, 69,13, 65,38, 63,94, 58,92, 57,99, 56,42, 54,57, 42,47, 25,07, 24,93, 24,76

Keratomalacia (III)

1H-NMR (D2O 40oC): 5,235 (0,64 H, d, J=1,46 Hz), 4,766 (0,41 H, d, J= 4,88 Hz) 4,562 (1,15 H, d, J=of 7.82 Hz), of 4.44 (0,15 H, OSiR.), 4,42 (0,22 H, usher. ), 4,357 (1,30 H, d, J=3.42 Hz), 4,313 (0,22 H, d, J=4,88 Hz) 4,286 (0,15 H, d, J=3,90 Hz) 4,213 (2,37 H, d, J=lower than the 5.37 Hz), 4,183 (0,37 H, t, J=3,42, with 2.93 Hz), 4,01-3,97 (2.06 to H, m) 4,006 (d, J=2,44 Hz) 3,927 (1,27 H, d, J= lower than the 5.37 Hz), 3,86-3,83 (0,37 H, OSiR.), of 3.78 at 3.69 (2,78 H, m), 3,59 of 3.56 (1,04 H, m) 2,052 (3,00 H, m)

13C-NMR (D2O, 25oC): 177,61, 177,30, 105,80, 105,63, 97,82, 93,38, 82,02, 81,55, 75,60, 75,47, 75,15, 75,09, 73,70, 72,04, 71,12, 71,07, 69,93, 69,51, 59,00, 56,44, 25,05, 24,76

Createsunmaterial (I), createsolidbrush (II) and keratomalacia (III) obtained above, analyze mass spectrometry with bombardero the s atoms)

Createsunmaterial (I), createsolidbrush (II) and keratomalacia (III) is dissolved in water and get water solutions with concentrations of 25, 40 and 50 nmol/ál, respectively, and every 0.1 µl of aqueous solutions mixed with 1.0 μl of diglycerin (used as matrix) and used for determining. The determination is performed using a three-phase quadrupole mass spectrometer finnigan MAT TSQ700 with xenon (8 kV) as the source of bombarding atoms. The results are given in table. 3. The numbers in parentheses in the table represent the relative intensity of the peaks (%).

(2) Anionic FABMS (anionic mass spectrometry with fast atom bombardment)

Createsunmaterial (I), createsolidbrush (II) and keratomalacia (III) is dissolved in water and get water solutions with concentrations of 25, 40 and 50 nmol/ál, respectively, and 1.0, with 1.0 and 0.5 μl of an aqueous solution of keratomalacia obtained as described above, respectively, is mixed with 1.0 μl of diglycerin (used as matrix) and used for determining. The determination is performed using a three-phase quadrupole mass spectrometer finnigan MAT TSQ700 with xenon (8 kV) as the source of bombardirashe peaks (%).

In addition, determine the content of endotoxins, nucleic acids, protein and protease treated createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III), obtained as described above. The results are given in table. 5.

The content of glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate, dermatosurgery, heparansulfate, createsurface etc., treated createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III) is determined using electrophoresis with cellulose acetate membrane (Cepallax, Fuji Photo Film) (buffer 0.1 M pyridinylamino acid; pH 3.0; a current of 0.5 mA/cm; the time of electrophoresis for 30 min; the dye 0.5% solution of ellenboro blue). As a result none of these compounds were not detected in any of certaincompanyshare (less than detection limit).

Example 2. Createsunmaterial (I), createsolidbrush (II) and keratomalacia (III) obtained as described above, the test for acute toxicity and subjected to various pharmacological tests.

"The test for acute toxicity"

Peeled certaincompanyshare, taste five weeks. Solutions createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III), respectively, in SFR (phosphate buffered saline) was injected intravenously at a dose of 1000 or 2000 mg/kg and then observe the General appearance and mortality, and within 14 days measure the body weight of experimental mice. After that kill animals and cut.

The result found that none of the animal does not die and there is no abnormal appearance, body weight and at autopsy.

Based on these results conclude that the minimum lethal dose of the above-described certaincompanyshare even with intravenous exceeds 2000 mg/kg

"Anti-test"

(1) anti-Inflammatory tests on rabbit model induced by papain arthritis

Anti-inflammatory effect of keratomalacia rabbit model of papain-induced arthritis test, using as an indicator the amount of synovial fluid.

(1-1) an anti-Inflammatory effect of createsunmaterial on the model of bilateral arthritis intra-articular introduction

In the a (1%) in physiological solution, and get the model of arthritis. One day after injection of papain in the articular cavity of the left knee is injected with 150 μl of a solution of purified createsunmaterial (I) in SFR (1% solution, 1.5 mg/joint), which is obtained in example 1 (hereinafter referred to here means "stop-treated createsunmaterial (I)"), and in the first joint is injected with 150 μl SFR (hereinafter referred to here means "stop, raw createsunmaterial (I)"). There are rabbits, which were introduced createsunmaterial (I) are called the "treated group". Rabbits that received only an injection of papain (hereinafter called the control group), and rabbits, which were not given injection of papain (hereinafter called normal group), also check in the same way, which is described next.

Seven days after injection of the articular arteries take the blood sample and separate the plasma containing heparin. After sampling the blood of rabbits dissect and separate both knees. The articular cavity was washed with 2 ml of saline solution three times to remove synovial fluid. Determine the content of calcium in the plasma and in the extracted synovial fluid, and calculate the volume of synovial fluid following poderzhanie calcium in plasma (µg/ml)

The results obtained in the above test are given in table. 6. In the table "n" indicates the number of rabbits in each group used in the experiment.

As can be seen from these results, the amount of synovial fluid in the group treated with createsunmaterial (I) of the present invention, significantly less than in the control group, and therefore confirmed the function of createsunmaterial (I) to improve the condition of arthritis.

(1-2) anti-Inflammatory effect of createsunmaterial on the model of bilateral arthritis in intramuscular

The model of arthritis get in the way described in (1-1), and a day after the injection of papain in the left thigh muscle injected with 150 μl of solution in SFR of createsunmaterial (I) (1% solution; 0.5 mg/kg of body weight) obtained in example 1 (denoted here as "group treated with createsunmaterial (I)"). As a control model of arthritis is injected with 150 μl SFR instead of the solution of createsunmaterial (I) in SFR, and these rabbits hereinafter referred to as the control group. Rabbits, which were not given an injection of papain (hereinafter called normal group), then also the Prov matter (1-1).

As can be seen from these results, the amount of synovial fluid in the group treated with createsunmaterial (I) of the present invention, significantly less than in the control group, and therefore confirmed the function of createsunmaterial (I) to improve the condition of arthritis.

(1-3) an anti-Inflammatory effect of createsunmaterial on the model of unilateral arthritis in intramuscular

In the articular cavity of the left joint white Japanese rabbits (females) mass 3 kg is injected with 150 μl of papain (1%) in physiological solution, and the right joint process and get the model of unilateral arthritis. One day after injection of papain in the left thigh muscle injected with 150 μl of a solution of purified createsunmaterial (I) in SFR (2, 1 and 0.5% solution of 1.0, 0.5 and 0.25 mg/kg, respectively), which was obtained in example 1 (hereinafter, this group is called here "the group treated with createsunmaterial (I)"). As a control model of arthritis is injected with 150 μl SFR instead of the solution of createsunmaterial (I) in SFR (hereinafter, this group is called here "the group, raw createsunmaterial (I)"). Rabbits, which were not given an injection of the popes who functions calculate the volume of synovial fluid in the same way, as in section (1-1).

The results obtained in the above test are given in table. 8. In the table "n" indicates the number of rabbits used in the experiment in each group.

As can be seen from these results, the amount of synovial fluid in the group treated with createsunmaterial (I) of the present invention, significantly less than in the control group, and therefore confirmed the function of createsunmaterial (I) to improve the condition of arthritis.

(1-4) anti-Inflammatory effect of different certaincompanyshare on the model of unilateral arthritis in intramuscular

Checks carried out in the same manner as in (1-3), except that they use 150 ál solutions of purified createsunmaterial (I), certaincompanyshare (II) and keratomalacia (III) (1% solution; 0.5 mg/kg) obtained in example 1 in SFR. The results are shown in Fig. 5. This figure*,**and***imagine that there is a significant difference with a p<0.05 to p<0.01 and p<0,001 respectively. In each group to check take 10 rabbits.

According to these results, the amount of synovial fluid in louboutinsale.com (III), much less than in the control group, and therefore confirmed the function of improving the condition of arthritis for each of the above certaincompanyshare.

(2) anti-Inflammatory tests with a modified passive reaction artusa swelling of the foot in rats

The effect of certaincompanyshare check modified passive reaction artusa swelling of the foot in rats, which is a model of allergic inflammation type III. For this rabbit antialbumin serum injected subcutaneously in the soles of the feet rats, which previously administered the test substance, then enter ovalbumin through an injection in the caudal vein to cause inflammation (swelling), and check inhibitory effect on edema of the test substances.

(Introduction of the test substances)

Male rats, Crj: SD at the age of five weeks withstand about 17 hours without food after a week of quarantine and before induction of inflammation is administered as the test substances purified createsunmaterial (I) obtained in example 1, indomethacin (Sigma; series N 19F0018) or dexamethasone (Banyu Pharmaceutical; injection Decadron; series 8D307P), or saline (Otsuka Pharmaceutical; seriological solution, respectively, and indomethacin dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na; Wako Pure Chemical Industries; series N PTN1418). The amount of dose is 0.5 ml per 100 g weight for each test. As way of introduction, createsunmaterial (I), saline and dexamethasone was administered via the caudal vein, and indomethacin administered orally. Substances, with the exception of indomethacin administered covertly (blind method).

Schedule introduction of each test substance is given below. Each group consists of five animals.

(I) Introduction five minutes before the induction of inflammation

(1) saline (negative control)

(2) Createsunmaterial (I) 1 mg/kg

(3) Createsunmaterial (I) 3 mg/kg

(4) Createsunmaterial (I) 10 mg/kg

(II) Injection 30 min before the induction of inflammation

(5) Indomethacin (positive control) 5 mg/kg

(III) the Introduction of three hours before induction of inflammation

(6) Createsunmaterial (I) 1 mg/kg

(7) Createsunmaterial (I) 3 mg/kg

(8) Createsunmaterial (I) 10 mg/kg

(9) Dexamethasone (positive control), 1 mg/kg

(Induction of inflammation)

Each of the rats described above, enter the enter ovalbumin, to induce the inflammatory swelling of the feet. Use anticigarette obtained as follows. Rabbits in areas on the back is injected intradermally 1 ml mixed in equal parts of solution (emulsion) physiological solution containing 2% of ovalbumin (10 mg / animal, egg albumin 5 x Cryst, series R; Seikagaku Corporation) and complete adjuvant's adjuvant (FCA) once a week just three times for their sensitization. After 34 days after the last sensitization away the blood and get anticigarette. The antibody titer of the obtained antisera determined by the method of the double layer. Namely, determination is performed with the use of reaction "white" precipitation and antisera, diluted with saline, and saline solution containing 0.1% ovalbumin (1 mg/ml) as an indicator. As a result, the titer of the antisera is x 27.

Through the span of time that is set for each group, rats who were administered each of the tested substances injected into the sole of the left rear stop 0.1 ml of the diluted six times antisera. Then in the caudal vein to induce inflammation injected with saline containing 0.5% ovalbumin (25 Tannoy foot before how to cause inflammation and through one, two, three and four hours after induction of inflammation, and contrast volume to induce edema receive the degree of swelling of the foot, and calculates the degree of inhibition of edema development in the groups that were administered the test substance, relative to the control group. Each received the degree of swelling of the difference between the mean values of the test using the criterion of minimum significant difference. The degree of swelling of the foot and the degree of inhibition of edema development in each treated group is shown in table. 9, and the degree of swelling of the foot in the treated groups, which the substance was injected in five minutes and three hours before induction of inflammation, shown in Fig. 6 and in Fig. 7, respectively. These figures*and**show that there is a significant difference with a p<0.05 and p<0,001, respectively.

The results show that the degree of swelling after an hour in the group treated with saline solution, which is a negative control, is of 40.9%, while in the groups treated with createsunmaterial (I), when its introduction five minutes before the induction of inflammation, this ratio is avisimas dose-response not observed during and one hour after induction of inflammation, the degree of swelling of the foot in the group treated with 3 mg/kg, and in the group treated with 10 mg/kg, is 37,2 and 35.4%, respectively, which is quite small even after four hours. In the groups treated with createsunmaterial (I) three hours before induction of inflammation, although significant differences were not observed one hour after induction of inflammation, significantly lower values observed in the group treated with 3 mg/kg, two hours later, in the group treated with 10 mg/kg, in three hours and in the group treated with 1 mg/kg, four hours, and these values are 35,3, 41,5% and 38% respectively. In the group treated with indomethacin, significantly lower values observed after two hours and four hours. In the group treated with dexamethasone, significantly lower values see anywhere in the definitions between one and four hours after induction of inflammation.

The degree of inhibition of edema development in the group treated with indomethacin, is from 9 to 17.5% after one hour and four hours, while it is estimated that in the groups treated with createsunmaterial (I) with the introduction ahead on five minutes, these figures are from 8.6 to 36.5%. In the groups treated with Kero is 24.4%. On the other hand, in the group treated with dexamethasone, the degree of inhibition of swelling of the foot reaches 31,7 at 58.1%.

(3) anti-Inflammatory test model with pleurisy was induced in rats by carragenine

The effect of certaincompanyshare check on rat model carragenine pleurisy, which is usually used for evaluation of anti-inflammatory drugs.

When this test is used female rats S. D. (n=37) maccol 150-170, Dissolve carrageenin (Sigma) in saline to a concentration of 2%, and the solution is filtered through a 0.8 μm filter. The resulting solution carragenine enter intraorale in the amount of 100 μl/animal, and get the model of pleurisy.

Rats models of pleurisy (n=19), described above, is injected subcutaneously (s.c.) createsunmaterial (I) dissolved in SFR, at doses of 20 or 10 mg/kg as a positive control eight rats, described above, is injected subcutaneously steroid (dexamethasone; Banyu Pharmaceutical) in an amount of 150 μg/kg, which is the clinical dose. As a negative control 10 rats injected subcutaneously SFR.

Each introduction is carried out immediately after the introduction carrageenin. The treated groups are indicated in the table. 10.

*and**imagine that there is a significant difference with a p<0.05 and p<0.01 respectively (where "p" means the significance level criterion Duncan).

As shown in Fig. 8, the volume of pleural effusion in the group treated with 10 mg/kg of createsunmaterial (I), significantly less than in the negative control, but almost the same as in the group treated with 20 mg/kg of createsunmaterial (I) and, therefore, is not observed, depending on the dose. The amount of pleural effusion in the group treated with dexametasone, significantly lower than the number in the group of negative control. The number of cells in the pleural effusion in each of the groups treated with 10 mg/kg and 20 mg/kg of createsunmaterial (I), significantly less than the number of cells in the group of negative control and hexametaphosphates, significantly lower number of cells in the control group (see Fig. 9).

From the above results, it follows that confirmed the anti-inflammatory effect of createsunmaterial (I) in the case of a rat model of pleurisy induced by carragenine.

(4) Inhibitory effect on the generation of active oxygen (O2-in the Guinea-pig neutrophils

Female Hartley Guinea pigs at the age of five weeks (purchased from Japan SLC) administered intraperitoneally injected with 20 ml of 0.2% aqueous solution of glycogen (type II; Oyster; Sigma) dissolved in saline solution, which is pre-sterilized in the autoclave. Guinea pigs killed by krovoisliania 16 hours after treatment and injected into the abdominal cavity 20 ml of saline containing heparin at a concentration of 10 u/ml to extract peritoneal bleed and have exudate. All further operations are carried out while cooling the mixture with ice water, except for incubation. The extracted bleed and have exudate centrifuged at 1000 rpm for 10 min, the precipitate hemolysate purified water for 30 seconds and return to isotonic state solution Hanks if you double the concentration (does not contain phenol red is NCSA and centrifuged, and the operation is repeated again to obtain neutrophils. Collected neutrophils Guinea pigs are suspended in Hanks solution, determine the number of cells in suspension using a cytometer (system K-2000; Toa lyo Denshi Co. and the suspension is diluted with Hanks solution to a concentration of 2 x 106cells/ml, used as a cell suspension. After mixing, 1 ml of the cell suspension and 10 μl of purified keratomalacia (createsunmaterial (I), certaincompanyshare (II) or keratomalacia (III) obtained in the above-described example 1, the mixture is pre-incubated at 37oC for one hour. Then to the mixture is added 50 μl of 1.6 mm cytochrome C (type III; Horse Heart; Sigma) and 10 μl of 100 μm N-formyl-Met-Leu-Phe (hereinafter sometimes called "FMLP", Sigma) in that order and stirred. The mixture is then incubated at 37oC for 10 min, cooled on ice to stop the reaction and centrifuged at 3000 rpm for five minutes.

The supernatant, obtained by centrifugation, separates and determine its absorption at 550 nm, and estimate the amount of reducing cytochrome C in 2 x 106cells after incubation for 10 min. Quantity bastantes with the addition of recombinant human SOD (superoxiddismutase) to a final concentration of 20 μg/ml used in the quality control. When the experiment used independently of six Guinea pigs. The proportion of the amount of reducing cytochrome C in each group, received certaincompanyshare, calculated by taking the number of reducing cytochrome C in the control (without treatment certainaithousholder) for 100% and calculate the difference from the number in the control group as the degree of inhibition (%) generation of active oxygen (O-2and, in addition, receive the average values in each experiment. The results are presented in Fig. 10. The concentration of keratomalacia this figure indicates the final concentration.

The results show that createsunmaterial (I) at concentrations of 0.01, 0.1 and 1.0 mg/ml significantly inhibited the generation of active oxygen (O-2depending on concentration and this fact confirms the presence of createsunmaterial (I) anti-inflammatory functions. Also to a small extent observed the function of inhibiting the generation of active oxygen (O-2of certaincompanyshare (II) or keratomalacia (III). These results suggested that certaincompanyshare show protivovospalitel">

(Antiallergic function)

Various certaincompanyshare instilled into the eyes of Guinea pigs, which have caused allergic conjunctivitis, to test their effect.

(1) the Effect of createsunmaterial (I) for allergic conjunctivitis

Colorvision (referred to hereafter TDI) dissolved in ethyl acetate to a concentration of 10%. In bilateral nasal vestibule 10 female Hartley Guinea pigs, weighing approximately 900-1000 g, served once a day for five days 10% of TDI (10 µl/animal), obtained as described above, and receive model, TDI sensitized. Solution in SFR of createsunmaterial (I) obtained in example 1 (100 mg/ml), buried sensitized TDI models in the left eye and the right eye buried SFR as a control. Dose instillation is one drop (484,6 ál (STD., S. D.)) at each instillation in the eye. After 10 minutes in both eyes buried and 6.5 ál of 10% of the TDI to cause conjunctivitis. After soaking for five minutes in the left eye again buried the solution in SFR of createsunmaterial (I) (100 mg/ml), and the right eye buried SFR. After 15 minutes check both eyes. Assess the degree of development of conjunctivitis by three indicators: the ISU and and standard deviation, shown in Fig. 11. This figure *means that there is a significant difference with a p<0,05 (where "p" means the significance level criterion2).

From the results it follows that createsunmaterial (I) has the ability to demonstrate inhibition of all three phenomena: hyperemia, edema and slezootdelenia and, in particular, is significantly reduced swelling compared to control animals.

(2) the Effect of different certaincompanyshare for allergic conjunctivitis

Solutions in SFR obtained in the above-described example 1, purified keratomalacia (III), createsunmaterial (I) and certaincompanyshare (II) (6.0 mg/ml, respectively) as the test substance, respectively buried in the left eye sensitized TDI models (10 animals in each group), which is obtained in the same manner as described above in (1), and in the right eye as a control buried SFR. The development of conjunctivitis also be appreciated, as described above in (1). The result of this test get the average values for the points and the standard deviation, shown in Fig. 12. This figure*means that there is a significant difference with a p<0,05 (where "p" means the level of snatched (III), createsunmaterial (I) and certaincompanyshare (II) has the ability to show the weakening of all signs of redness, swelling and slezootdelenia and, in particular, confirmed that createsunmaterial (I) detects a significant inhibitory effect on edema.

(3) the Effect of createsunmaterial (I) in various concentrations for allergic conjunctivitis

Solution in SFR containing purified createsunmaterial (I) obtained in the above-described example 1 at various concentrations (6, 3, 1.5 or 0.75 mg/ml) buried sensitized TDI models 10 animals in each group, which are prepared in the same manner as described above in (1) respectively in the left eye and the right eye buried SFR as a control. The development of conjunctivitis also be appreciated, as described above in (1). The result of this test get the average values for the points and the standard deviation, shown in Fig. 13. This figure*means that there is a significant difference with a p<0,05 (where "p" means the significance level criterion2).

The result reveals that the solutions createsunmaterial (I) in SFR at all concentrations tend Ei concentration 6, 3 and 1.5 mg/ml detect inhibitory effect on edema.

"Immunomodulatory function of inducing cellular differentiation and function of inducing apoptosis"

The effect of certaincompanyshare check on the MRL mice, which represent a murine model of autoimmune disease.

(1) inhibitory effect on the mass of lymph nodes

(1-1) Tests with repeated intramuscular injection of createsunmaterial (I) the MRL mice for four weeks

Solution in SFR purified createsunmaterial (I) (100 mg/piece), which is obtained in the above-described example 1, at a dose of 10 mg/kg of body weight (this is called hereafter the treated group) and SFR as control injected intramuscularly in the thigh mice MRL-lpr/lpr five times per week for four weeks. After that, mice reveal and determine the mass of the spleen and mesenteric lymph nodes, and get the average value of the mass. Results and standard deviations are given in table. 11. In this table, "n" indicates the number used mice.

The result is that the group that were injected with createsunmaterial (I), there is a tendency noise reduct is errday together with immunomodulating function createsunmaterial (I).

(1-2) Tests with repeated intramuscular injection of keratomalacia (III) the MRL mice for 28 days.

Solution in SFR purified keratomalacia (III) obtained in the above-described example 1, at a dose of 1, 5 or 25 mg/kg of body weight (called hereafter the group treated with 1 mg/kg, group treated with 5 mg/kg, and the group treated with 25 mg/kg, respectively) and SFR as control injected intramuscularly in the thigh mice MRL-lpr/lpr seven times per week for four weeks. After that, mice reveal and determine the mass of the submandibular lymph nodes, and get the average value of the mass. Results and standard deviations are given in Fig. 14. In each group using six mice. In addition, this figure*shows that there is a significant difference with a p<0,05 (where "p" means the significance level for multiple comparison Bonferroni criterion).

The result is that in the groups treated with keratomalacia (III), is observed when compared with the control group, the effect of suppressing increase in mass submandibular lymph nodes at any dose, especially when injected dose of 5 mg/kg and, therefore, this in-depth and repeated intramuscular injection of keratomalacia (III) mice MRL for 56 days.

Solution in SFR purified keratomalacia (III) obtained in the above-described example 1, at a dose of 2.5, 5 or 10 mg/kg of body weight (called hereafter the group treated with 2.5 mg/kg, group treated with 5 mg/kg, and a group treated with 10 mg/kg, respectively) and SFR as control (sometimes called here the control group) injected intramuscularly in the thigh mice MRL-lpr/lpr seven times per week for eight weeks. After that, mice reveal and determine the weight of mesenteric lymph nodes and submandibular lymph nodes, and get the average value of mass, respectively. Results related to mesenteric lymph nodes and submandibular lymph nodes, are given in Fig. 15 and 16, respectively, together with standard deviations. In each group use the seven mice.

As a result, in the group treated with 2.5 mg/ml keratomalacia (III), observe the effect of suppressing the increase in weight of the mesenteric lymph nodes, and in the group treated with 10 mg/kg, see suppression of increase in weight of the mesenteric and submandibular lymph nodes and, therefore, these facts confirm immunodeficiency is nzioki

(2-1) the Analysis of the induction of cellular differentiation using as an indicator of the concentration of the staining of cells

Solution in SFR purified keratomalacia (III) obtained in the above-described example 1, at a dose of 1, 5 or 25 mg/kg of body weight (called hereafter the group treated with 1 mg/kg, group treated with 5 mg/kg, and the group treated with 25 mg/kg, respectively) and SFR as control (sometimes called here the control group) injected intramuscularly in the thigh mice MRL-lpr/lpr seven times per week for four weeks. After that, mice reveal and prepare samples slicers (Non - staining), mesenteric lymph nodes and submandibular lymph nodes. Analyze the concentration of staining per unit area of the samples using an image analyzer (PIAS). In the case of undifferentiated cells, the concentration of staining per unit area is small because of the high proportion of cytoplasm and nuclei stained badly. In the case of differentiated cells in a concentration per unit area is high due to high proportion of nuclei and dense staining.

The results of the analysis of mesenteric lymph nodes and submandibular, limfaticheskie with p<0,01 (where "p" means the significance level for multiple comparison Bonferroni criterion). Each group used six mice.

As a result, in the group treated with keratomalacia (III), observe the increasing concentration of staining per unit area (decrease in relative light) at any dose, and in particular in the group treated with 5 mg/kg, and in the group treated with 25 mg/kg, the concentration of the staining significantly increased when compared with the control group. This fact indicates an increase in the number of differentiated cells by keratomalacia (III), and assume keratomalacia (III) the function of inducing cellular differentiation.

(2-2) analysis of the induction of cellular differentiation using as an indicator limfozitah surface antigens lymph nodes

Solution in SFR purified keratomalacia (III) obtained in the above-described example 1, at a dose of 2.5, 5 or 10 mg/kg of body weight (called hereafter the group treated with 2.5 mg/kg, group treated with 5 mg/kg, and a group treated with 10 mg/kg, respectively) and SFR as control (sometimes called here the control group) injected intramuscularly in the thigh mice MNA cell filter (Falcon 2350), to get lymphocytes. Lymphocytes obtained from each group, are subjected to double colouring immune method with antibody against CD3 (Seikagaku Corporation) and an antibody against CD4 (Pharminjen), antibody against CD3 and antibody against CD8a (Pharminjen), and antibody against CD3 and antibody against 220V (Pharminjen), respectively. The surface antigens of the cells CD3, CD4 and CD8a are cell surface antigens that are expressed on T-cells and 220V expressed on b cells. It is known that no expression of these cell surface antigens is not observed for the zero-lymphocytes.

Proportion (%) of positive cells CD3 and CD4 (hereinafter sometimes referred to as "CD3+CD4+cells"; mainly helper T-cells) from all of lymphocytes and standard deviations are shown in Fig. 19, the percentage of CD3 positive cells and CD8a (hereinafter sometimes referred to as "CD3+CD8a+cells"; mainly, suppressor T cells and cytotoxic T cells) and standard deviations shown in Fig. 20, and the proportion of CD3 positive cells and 220V (hereinafter sometimes referred to as "CD3+220V+cells; abnormal T-cells with the surface antigen of b-cells) and standard deviations shown in Fig. 21. These figures *indicates that there is a significant difference with a p<0,05 (where "R" on the I CD3+CD4+cells in the group, treated with a 10 mg/kg keratomalacia (III) increases significantly relative to the control group, the group treated with 2.5 mg/kg and the group treated with 5 mg/kg This indicates differentiation zero-lymphocytes CD3+CD4+cells by introduction of an adequate number of keratomalacia (III).

As can be seen from Fig. 20, the proportion of CD3+CD8+cells in the group treated with 10 mg/kg of keratomalacia (III) increases relative to the control group, the group treated with 2.5 mg/kg, and the group treated with 5 mg/kg This indicates differentiation zero-lymphocytes CD3+, CD8a+cells by introduction of an adequate number of keratomalacia (III).

As can be seen from Fig. 21, the proportion of CD3+220V+cells in the group treated with 10 mg/kg of keratomalacia (III) is reduced with respect to the control group, the group treated with 2.5 mg/kg, and the group treated with 5 mg/kg This indicates the disappearance of CD3+220V+cells that are abnormal, with the introduction of an adequate number of keratomalacia (III) and confirms the induction of differentiation in normal lymphocytes. These results suggest that the function of inducing cellular differentiation in the number of keratomalacia (III).

In addition, prepare samples slicers (Non - staining) and submaxillary lymph nodes of mice in each group and in the light microscope to observe the presence of apoptotic bodies in the groups treated with keratomalacia (III) at any dose.

These results suggest that the function of inducing apoptosis in keratomalacia (III).

The above results confirm together with immunomodulating function, function, induction of cellular differentiation and function of inducing apoptosis certaincompanyshare.

Example 3. Ointment

Following the traditional method, purified createsunmaterial (I) obtained in example 1 dissolved in a hydrophilic ointment base, the Pharmacopoeia of Japan, at a concentration of 10 mg/ml and get the ointment. This ointment can be used as anti-inflammatory agent and as an antiallergic drug.

Example 4. Eye drops

Following the traditional method, purified createsunmaterial (I) obtained in example 1, and sodium hyaluronate solution is get eye drops. These eye drops can be used as anti-inflammatory agents and anti-allergic medicines.

Example 5. The drug with liposomal incorporation

Purified createsunmaterial (I), obtained in example a was dissolved at a concentration of 10 mg/ml liposomal inclusion (Aquasome LA; Nikko Chemicals) containing lecithin, and exposed to ultrasound, to obtain a clathrate. This liposomal incorporation can be used as any funds from a number of anti-inflammatory agent, an antiallergic drug, immunomodulator, an inducer of cellular differentiation and inducer of apoptosis.

Example 6

Injection

Following the traditional method, purified createsunmaterial (I) obtained in example 1 dissolved in a physiological solution with a pH of 6.8 to 7.6, installed phosphate, at a concentration of 10 mg/ml and the solution is filtered aseptically through a 0.22 μm filter and get the solution for injection. This solution for injection can be used as any of a number of anti-inflammatory agent, an antiallergic drug, immunomodulator, an inducer of cellular differentiation and inducer of apoptosis.

Industrial use which have a high degree of purification and does not contain essentially endotoxin, nucleic acid, protein, protease and other glycosaminoglycans, except as described above, oligosaccharides, and can therefore be used as pharmaceuticals, for example, new anti-inflammatory drugs.

1. Pharmaceutical composition having anti-inflammatory, anti-allergic, immunological, inducing apoptosis and cell differentiation activity, containing a pharmaceutically acceptable carrier or diluent and, as active ingredient an effective amount of keratomalacia with two to five sharedname links, sulfated N-acetylglucosamine at its reducing end, with sulfated N-acetyllactosamine link, optionally containing sialic acid and/or fucose having at least two sulfated hydroxyl group in the molecule and/or its pharmaceutically acceptable salt.

2. The pharmaceutical composition under item 1, which has anti-inflammatory action.

3. The pharmaceutical composition under item 1 or 2, where the specified certaincompanyshare contains as an integral ingredient, at least, a disaccharide of the formula

Gal (the second group;

6S is 6-O-sulfate ester.

4. The pharmaceutical composition according to p. 3, where the specified certaincompanyshare selected from the group comprising tetrachlorophenyl N-acetylglucosaminidase represented by formula I trisulfonic N-acetylglucosaminidase represented by formula II, and desulfuromonas N-acetylglucosaminidase represented by formula III

Gal(6S)1-4GlcNAc(6S)l-3Gal(6S)1-4GlcNAc(6S) (I)

NeuAc~Gal1-4GlcNAc(6S)l-3Gal(6S)1-4GlcNAc(6S) (II)

Gal(6S)1-4GlcNAc(6S) (III),

where Gal represents galactose,

GlcN is glucosamine,

Neu is Narimanovo acid,

AC represents acetyl group,

6S is 6-O-sulfate ester,

~ means a connection 2, 3 or Association of 2.6.

5. The pharmaceutical composition under item 1, with anti-allergic effect.

6. The pharmaceutical composition under item 5, where the specified certaincompanyshare contains as an integral ingredient, at least, a disaccharide of the formula

Gal(6S)-GlcNAc(6S),

where Gal represents galactose;

GlcN is glucosamine;

AC represents acetyl group;

6S is 6-O-sulfate ester.

7. Pharmaceutical coy N-acetylglucosaminidase, represented by formula I, trisulfonic N-acetylglucosaminidase represented by formula II, and desulfuromonas N-acetylglucosaminidase represented by formula III

Gal(6S)1-4GlcNA(6S)1-3Gal(6S)1-4GlcNAc(6S) (I)

NeuAc~Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) (II)

Gal(6S)1-4GlcNAc(6S) (III),

where Gal represents galactose,

GlcN is glucosamine,

Neu is Narimanovo acid,

AC represents acetyl group,

6S is 6-O-sulfate ester,

~ means a connection 2, 3 or Association of 2.6.

8. The pharmaceutical composition under item 1, which has an immunomodulatory effect.

9. The pharmaceutical composition according to p. 8, where the specified certaincompanyshare contains as an integral ingredient, at least, a disaccharide of the formula

Gal(6S)-GlcNAc(6S),

where Gal represents galactose;

GlcN is glucosamine;

AC represents acetyl group;

6S is 6-O-sulfate ester.

10. The pharmaceutical composition according to p. 9, where the specified certaincompanyshare selected from the group comprising tetrachlorophenyl N-acetylglucosaminidase represented by formula I, trisulfonic N-acetyllactosamine the Loy III

Gal(6S)1-4GlcNA(6S)1-3Gal(6S)1-4GlcNAc(6S) (I)

NeuAc~Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) (II)

Gal(6S)1-4GlcNAc(6S) (III),

where Gal represents galactose,

GlcN is glucosamine,

Neu is Narimanovo acid,

AC represents acetyl group,

6S is 6-O-sulfate ester,

~ means a connection 2, 3 or Association of 2.6.

11. The pharmaceutical composition under item 1, with inducing cellular differentiation effect.

12. The pharmaceutical composition according to p. 11, where the specified certaincompanyshare contains as an integral ingredient, at least, a disaccharide of the formula

Gal(6S)-GlcNAc(6S),

where Gal represents galactose;

GlcN is glucosamine;

AC represents acetyl group;

6S is 6-O-sulfate ester.

13. The pharmaceutical composition according to p. 12, where the specified certaincompanyshare selected from the group comprising tetrachlorophenyl N-acetylglucosaminidase represented by formula I, trisulfonic N-acetylglucosaminidase represented by formula II, and desulfuromonas N-acetylglucosaminidase represented by formula III

Gal(6S)1-4GlcNA(6S)1-3Gal(6S)1-4GlcNAc(6S) (I)

NeuAc~Gal1-4GlcNAc(6S)1-3GBR> Neu is Narimanovo acid,

AC represents acetyl group,

6S is 6-O-sulfate ester,

~ means a connection 2, 3 or Association of 2.6.

14. The pharmaceutical composition under item 1, with inducing apoptosis effect.

15. The pharmaceutical composition according to p. 14, where the specified certaincompanyshare contains as an integral ingredient, at least, a disaccharide of the formula

Gal(6S)-GlcNAc(6S),

where Gal represents galactose;

GlcN is glucosamine;

AC represents acetyl group;

6S is 6-O-sulfate ester.

16. The pharmaceutical composition according to p. 15, where the specified certaincompanyshare selected from the group comprising tetrachlorophenyl N-acetylglucosaminidase represented by formula I, trisulfonic N-acetylglucosaminidase represented by formula II, and desulfuromonas N-acetylglucosaminidase represented by formula III

Gal(6S)1-4GlcNA(6S)1-3Gal(6S)1-4GlcNAc(6S) (I)

NeuAc~Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) (II)

Gal(6S)1-4GlcNAc(6S) (III),

where Gal represents galactose,

GlcN is glucosamine,

Neu is Narimanovo acid,

AC is acetylen consultationmeridia fraction, containing not less than 99% of keratomalacia with two to five sharedname links, sulfated N-acetylglucosamine at its reducing end, with sulfated N-acetyllactosamine link, optionally containing sialic acid and/or fucose having at least two sulfated hydroxyl group in the molecule, with keratomalacia fraction is essentially free of endotoxin, hyaluronic acid, chondroitin sulphate, dermatosurgery, heparan sulfate and keratomalacia, and the content of nucleic acids, protein and protease in fractions, respectively, less than the limits of detection.

18. Keratomalacia fraction on p. 17, where the specified certaincompanyshare contains as an integral ingredient, at least, the disaccharide of formula Gal(6S)-GlcNAc(6S),

where Gal represents galactose;

GlcN is glucosamine;

AC represents acetyl group;

6S is 6-O-sulfate ester.

19. Keratomalacia fraction under item 17 or 18, where the specified certaincompanyshare selected from the group comprising tetrachlorophenyl N-acetylglucosaminidase, to depict liftirovaniyu N-acetylglucosaminidase, represented by formula III

Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) (I)

NeuAc~Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) (II)

Gal(6S)1-4GlcNAc(6S) (III),

where Gal represents galactose,

GlcN is glucosamine,

Neu is Narimanovo acid,

AC represents acetyl group,

6S is 6-O-sulfate ester,

~ means a connection 2, 3 or Association of 2.6.

20. The method of obtaining certainaithousholder fraction described in any of paragraphs.17 - 19, including the stage of splitting createsurface splitting enzyme, which is carried out in a buffer solution containing keratinolytic concentrations of 1.0 - 100 mg/ml, at pH 6,0 - 7,0, the reaction temperature 25 - 40oC for 1 to 72 h, and where the specified enzyme affects keratinolytic representing keratinolytic I, keratinolytic II or keratinophilic, and it hydrolyzes glycosidic bond of N-acetylglucosamine with the formation of sulfated keratomalacia, createsunmaterial and certaincompanyshare as the main products of cleavage, with the specified digestive enzyme has the following physical and chemical properties: has optimal activity at pH 4.5 - 6.0 in 0.1 M acetals in 0.1 M acetate buffer or 10 mm Tris-acetate buffer at 37oC for one hour; has an optimum activity at 50 - 60oC, when it reacts in 0.1 M acetate buffer at pH 6.0 for 10 min; maintains stability at 45oC or below, when left in 0.1 M acetate buffer at pH 6.0 in an hour; and stage fractionation of purified certainaithousholder fraction of the fission products, containing not less than 99% of keratomalacia.

21. The method of obtaining certainaithousholder faction on p. 20, where the specified keratinolytic is keratomalacia and certaincompanyshare selected from the group comprising tetrachlorophenyl N-acetylglucosaminidase represented by formula I, trisulfonic N-acetylglucosaminidase represented by formula II, and desulfuromonas N-acetylglucosaminidase represented by formula III

Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) (I)

NeuAc~Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) (II)

Gal(6S)1-4GlcNAc(6S) (III),

where Gal represents galactose,

GlcN is glucosamine,

Neu is Narimanovo acid,

AC represents acetyl group,

6S is 6-O-sulfate ester,

~ means a connection 2, 3 or Association of 2.6.

 

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