JP5051999B2 - Treatment for inflammatory bone and cartilage diseases - Google Patents

Description

  The present invention relates to a novel anti-inflammatory agent comprising galactosaminoglycan as an active ingredient.

  Galactosaminoglycan is a glycosaminoglycan with a repeating structure of disaccharide units consisting of hexuronic acid residues and galactosamine residues as its basic skeleton, and representative examples include chondroitin, chondroitin sulfate, and dermatan sulfate. It has been. Chondroitin sulfate usually exists as a proteoglycan in living organisms such as animals, and as molecular species having different binding positions and numbers of sulfate groups in disaccharide units, chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, Chondroitin sulfate D, chondroitin sulfate E, chondroitin sulfate K and the like are known.

Chondroitin sulfate or derivatives thereof are used for various uses as pharmaceuticals and foods. Bone metabolism improving agent (Patent Document 1), anti-herpes virus agent (Patent Document 2), kallikrein-kinin inhibitor (Patent Document 3) , Pharmaceuticals, cosmetics, food additives, etc. (Patent Document 4), preventive and / or therapeutic agents for matrix metalloproteinase-related diseases (Patent Document 5), osteoclast formation inhibitors (Patent Document 6), immune regulators (Patent Document 5) Document 7), allergic disease treatment agents (Patent Document 8) and the like are disclosed, and Patent Document 2, Patent Document 6, and Patent Document 7 also disclose the use of chondroitin sulfate E. The chondroitin sulfates used in the above-mentioned applications are naturally derived chondroitin sulfate A and chondroitin sulfate C, and chondroitin Use of acid E is extremely limited, use as anti-inflammatory agents is not known.
JP-A-7-53388 Japanese Patent Laid-Open No. 9-202731 JP-A-11-147901 JP 2001-247602 A JP 2002-226380 A Japanese Patent Laid-Open No. 2004-210715 JP 2005-82491 A JP 2005-255678 A

  An object of the present invention is to provide a novel anti-inflammatory agent comprising galactosaminoglycan as an active ingredient.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have a specific galactosaminoglycan having an action of specifically suppressing an inflammatory reaction. By using such a substance as an active ingredient, a novel and It was found that an effective anti-inflammatory agent can be provided, and the present invention has been completed.

  That is, the present invention is a galactosaminoglycan having a repeating structure of a disaccharide unit consisting of a hexuronic acid residue and a galactosamine residue as a basic skeleton, and on average, about 50% to about 50% of the disaccharide unit in the molecule. 70%, preferably about 55% to about 65% (% is the mol% of disaccharide units in the molecule) hexuronic acid residues having no sulfate group and hydroxyl groups at the 4th and 6th positions are simultaneously sulfate esters Provided is an anti-inflammatory agent comprising, as an active ingredient, a galactosaminoglycan comprising a galactosamine residue that has been converted to galactosamine or a pharmacologically acceptable salt thereof.

  The galactosaminoglycan used in the anti-inflammatory agent of the present invention preferably has an average of preferably about 5% to about 25%, more preferably about 5% to about 20% of the disaccharide unit in the molecule, More preferably, about 10% to about 15% (% is the mol% of the disaccharide unit in the molecule), but only the hexuronic acid residue having no sulfate group and the hydroxyl group at the 6-position are sulfated. It preferably consists of a galactosamine residue.

  The galactosaminoglycan used in the anti-inflammatory agent of the present invention is obtained by adding chondroitinase ABC2.5U to 100 μL of a 1% solution of this galactosaminoglycan and reacting at pH 8.0 at 37 ° C. overnight. When digested, the average digestibility is preferably 80% to 100%, more preferably 90% to 100%, still more preferably 95% to 100% (% is hexuronic acid residue and galactosamine residue in the molecule) Galactosaminoglycan, which is a percentage of the number of cleaved bonds to the total number of bonds in between.

  Furthermore, the galactosaminoglycan used in the anti-inflammatory agent of the present invention is preferably about 1,000 to about 100,000 daltons (Da), more preferably about 10,000 to about 90,000 Da, and even more preferably about 30,000 to 80,000 Da. And most preferably has a weight average molecular weight of about 75,000 Da.

In addition, the anti-inflammatory agent of the present invention is preferably a therapeutic agent for inflammatory bone / cartilage disease, more preferably a therapeutic agent for arthritis or arthritis, and for rheumatoid arthritis or osteoarthritis. Most preferably, it is a treatment agent.
The anti-inflammatory agent of the present invention is preferably an injection or an oral administration agent.

  According to the present invention, by selecting a galactosaminoglycan having a specific structure and properties and using it as an active ingredient, it is possible to specifically suppress the inflammatory reaction of a living body, and also to use other galactosami A statistically significantly higher anti-inflammatory effect can be obtained as compared with noglycan. Therefore, among the anti-inflammatory agents containing galactosaminoglycan, which is a biological substance and safe for administration subjects such as animals, an extremely useful anti-inflammatory agent having a particularly high anti-inflammatory effect is available. Provided.

  First, the meanings of main abbreviations used in this specification are shown below.

ΔDi-0S: 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -D-galactose ΔDi-4S: 2- Acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose ΔDi-6S: 2- Acetamide-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-galactose ΔDi-diSD: 2-acetamido- 2-deoxy-3-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-
Galactose ΔDi-diSE: 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo- D-galactose ΔDi-diSB: 2-acetamido-2-deoxy-3-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -4- O-sulfo-D-
Glycose ΔDi-triS: 2-acetamido-2-deoxy-3-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -4,6- Bis-O-sulfo-D-glycose OA: osteoarthritis
RA: rheumatoid arthritis

  Hereinafter, embodiments of the present invention will be described in detail.

(1) Galactosaminoglycan used in the anti-inflammatory agent of the present invention Generally, “galactosaminoglycan” means a galactosamine residue (specific example: N-acetyl-D-galactosamine residue) and hexuronic acid. The basic structure is a disaccharide repeating structure composed of residues (specific examples: D-glucuronic acid residue or L-iduronic acid residue), and the galactosamine residue, which is a constituent sugar, is located at the 4th, 6th, 4th and 6th positions. Glycosaminoglycan having a sulfate group at the position or / and 2 position of hexuronic acid residue, usually means dermatan sulfate and chondroitin sulfate (Annu. Rev. Biochem., 60, 443-457, 1991) .

The galactosaminoglycan, which is an active ingredient of the anti-inflammatory agent of the present invention, has a repeating structure of disaccharide units consisting of hexuronic acid residues and galactosamine residues as a basic skeleton, and 50% to 70% of the molecule. A disaccharide unit is a galactosaminoglycan composed of a hexuron residue having no sulfate group and a galactosamine residue in which the hydroxyl groups at the 4th and 6th positions are simultaneously sulfated. As such a galactosaminoglycan, specifically, a disulfated disaccharide repeating unit {GlcAβ1-3GalNAc (4,6-bisSO ₄ ) or IdoAα1-3GalNAc (4,6-bisSO ₄ ) [GlcA is D- Glucuronic acid residue, IdoA is L-iduronic acid residue, GalNAc (4,6-bisSO ₄ ) is DN-acetylgalactosamine residue in which both hydroxyl groups at positions 4 and 6 are sulfated, β1- 3 represents a β1-3 bond, α1-3 represents an α1-3 bond]}, and the disulfated disaccharide repeating unit accounts for 50% to 70% of the molecule. It is done.

  Examples of such galactosaminoglycans include chondroitin sulfate E, chondroitin sulfate A (N-acetylgalactosamine-4-sulfate residue is a galactosamine residue) extracted and purified from squid cartilage such as mica, squid, squid and squid. Galactosaminoglycan in which the 6-position of galactosamine residue in galactosamine residue, which is the main part of galactosamine, is specifically sulfated, dermatan sulfate (N-acetylgalactosamine-4-sulfate residue is occupied by galactosamine residue) And galactosaminoglycan in which galactosamine residue 6-position of chondroitin sulfate whose hexuronic acid is mainly L-iduronic acid is specifically sulfated (http://www.glycoforum.gr.jp/ science / word / proteoglycan / PGA06J.html and http://hobab.fc2web.com/sub4-glycosaminoglucan.htm)

  The method for extracting and purifying chondroitin sulfate E from squid cartilage is not particularly limited and can be extracted and purified by a known method, but examples include the following methods. In other words, squid cartilage is hydrolyzed with protease to break down protein, the hydrolyzed solution is filtered, and the filtrate is precipitated with galactosaminoglycan fraction using alcohol such as ethanol in the presence of sodium chloride. E can be obtained. If necessary, the protein and peptide contents in the extract can be reduced by multiple protease treatments, aqueous sodium hydroxide treatment, alcohol precipitation, filtration, and desalting.

  The method for extracting and purifying galactosaminoglycan used in the anti-inflammatory agent of the present invention is not limited to the above method, and Japanese Patent Publication No. 60-9042, Japanese Patent Publication No. 61-212241, Japanese Patent Application Laid-Open No. Hei 9 It can also be obtained by extraction and purification according to a known method described in JP-202731.

  Moreover, the method of specifically sulfated specific positions of galactosamine residues of chondroitin sulfate A and dermatan sulfate is not particularly limited, and can be performed by a known chemical method. Chondroitin sulfate A and dermatan sulfate itself can be extracted and purified by a known method from natural materials known to contain chondroitin sulfate A or dermatan sulfate such as animal cartilage.

  The galactosaminoglycan used in the anti-inflammatory agent of the present invention may be in the form of a pharmacologically acceptable salt, and the type is particularly limited as long as it is a pharmacologically acceptable salt. Although not preferred, alkali metal salts and alkaline earth metal salts such as sodium salt, potassium salt, lithium salt, calcium salt and the like are preferable, and sodium salt is particularly preferable (hereinafter, unless otherwise specified, the term “galactosamino” "Glycan" is used in the sense including its pharmacologically acceptable salts).

  The weight average molecular weight of the galactosaminoglycan of the present invention is not particularly limited, but is preferably about 1,000 to about 100,000 Da, more preferably about 10,000 to about 90,000 Da, more preferably about 30,000 to 80,000 Da, and most preferably about 75,000 Da.

  In addition, the position and amount of the sulfate group in the galactosaminoglycan molecule can be analyzed by a known method. For example, Shinsei Kagaku Kogaku Koza 3, Carbohydrate II, p49-62 (1991, published by Tokyo Chemical Dojin, It can be analyzed by the method described in “Structural analysis combining 2 · 8 glycosaminoglycan degrading enzyme and HPLC”.

  More specifically, galactosaminoglycan is enzymatically treated with a lyase that acts on galactosaminoglycan to produce unsaturated disaccharide, and the galactosaminoglycan is formed to reflect the type and amount of the constituent disaccharide. Unsaturated disaccharides (unsaturated disaccharides indicated by the above abbreviations) can be identified and quantified by high performance liquid chromatography (HPLC).

  The lyase used in the analysis of unsaturated disaccharide is not particularly limited as long as it can be finally decomposed to unsaturated disaccharide, and can be appropriately selected by those skilled in the art depending on the galactosaminoglycan to be analyzed. For example, chondroitinase, hyaluronidase, etc. can be used.

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

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

  In the present invention, the “constitutive unsaturated disaccharide composition” of galactosaminoglycan refers to the produced unsaturated disaccharide obtained for each unsaturated disaccharide produced by decomposing galactosaminoglycan with lyase as described above. It means the ratio to the total sugar and is expressed in mol%. This value corresponds to the proportion of constituent disaccharides having sulfate groups at various positions in the galactosaminoglycan molecule before degradation with lyase. Therefore, when the galactosaminoglycan used in the anti-inflammatory agent of the present invention is analyzed by such a method, ΔDi-diSE is about 50 mol% to about 70 mol%, more preferably about 55 mol% to about 65 mol%. Mol%. Further, it is preferable that ΔDi-6S is about 5 mol% to about 25 mol%, more preferably about 5 mol% to about 20 mol%, and about 10 mol% to about 15 mol%. Further preferred. Further, ΔDi-4S is preferably about 5 mol% to about 30 mol%, more preferably about 10 mol% to about 25 mol%.

  In the present invention, the galactosaminoglycan is administered to a mammal as an anti-inflammatory agent, as described later, and is thus purified to a high purity and substantially free of substances that are not allowed to be mixed as pharmaceuticals or foods. Those are preferred. That is, for example, the heavy metal content is preferably 20 ppm or less, more preferably 10 ppm or less, the arsenic content is preferably 2 ppm or less, more preferably 1.5 ppm or less, and the general viable count is preferably 1000 cells / g or less, more preferably 300 cells. Satisfies conditions such as / g or less. Moreover, when using the anti-inflammatory agent of this invention as an injection, what does not contain an endotoxin and an anticoagulant substance further is preferable. Specifically, for example, the endotoxin content is 0.03 EU (endotoxin unit) or less (see Japanese Industrial Standard, official regulations such as biochemical reagent general rules K8008 "Endotoxin test"), heparin content (analysis method by HPLC is WO95 / 09188) Is preferably 0.15% or less.

(2) Anti-inflammatory agent The anti-inflammatory agent of the present invention comprises the above-described galactosaminoglycan as an active ingredient, and is a pharmaceutical and functional food (specifically) for administration / ingestion to mammals including humans. It is used as food and drink such as food for insurance. Examples of mammals other than humans include monkeys, dogs, cats, horses, cows, pigs, mice, rats, and guinea pigs.

  The anti-inflammatory agent of the present invention is used for the purpose of prevention, maintenance (prevention of deterioration), reduction (symptom improvement), treatment, etc. of inflammatory diseases. Here, the inflammatory disease refers to a disease caused by a local or systemic inflammatory reaction in mammalian tissues and / or organs, and the anti-inflammatory agent of the present invention is preferably inflammatory. Used as a treatment for bone and cartilage diseases. Examples of the bone / cartilage disease include arthritis or arthritis, and more typically, rheumatoid arthritis, which is a systemic inflammatory disease, and osteoarthritis due to a local inflammatory reaction in a joint.

  By the way, midkine is a kind of heparin-binding growth / differentiation factor. In recent years, both midrhine arthritis (RA) and osteoarthritis (OA) have increased midkine levels in joint fluid. High levels of midkine have been reported in inflammatory conditions involving deep neutrophils (Takeda, T. et al, J. Biochem., 122, 453-458, 1997) and midkine antagonists A therapeutic agent for inflammatory diseases due to the effect of inhibiting the migration of neutrophils has also been proposed (WO99 / 03493). The anti-inflammatory agent of the present invention is not limited by the mechanism of action, but the action of the galactosaminoglycan of the present invention on midkine is also expected (http://www.glycoforum.gr.jp). /science/glycogenes/09/09J.html and http://www.midkine.org/p01-j.html), there is also a possibility of exerting an anti-inflammatory effect through such a mechanism of action.

  As long as the anti-inflammatory agent of the present invention contains galactosaminoglycan as described above as an active ingredient and is a therapeutic agent for inflammatory diseases, its purpose of administration, administration subject, administration route, administration method, dosage form, effective There are also no particular limitations on the amount of components, the dose, the dose interval, etc.

  The administration route of the anti-inflammatory agent of the present invention is not particularly limited as long as the above object is achieved. Examples of the administration method include injection (intra-articular cavity, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal). , Intrathoracic, intrathecal, etc.), drip, oral, transdermal, inhalation, enema, transrectal, nasal, ophthalmic, nasal, and administration to mucous membrane. The administration method can be appropriately selected depending on the site where treatment or the like is desired, and direct administration to a specific site by injection, oral administration, and other administration methods described above can be appropriately selected.

  Examples of the dosage form of the anti-inflammatory agent of the present invention include solution preparations (solutions, suspensions, emulsions, solid preparations for dissolution at the time of use), dispersion preparations, semi-solid preparations, powder preparations, molded preparations, and leaching. Specific examples include tablets, coated tablets, dragees, pills, troches, hard capsules, microcapsules, liposizing agents, implants, powders, powders, granules, fine granules , Injection, liquid, elixir, emulsion, syrup, water, emulsion, suspension, liniment, lotion, aerosol, spray, inhalant, spray, ointment, plaster, patch , Pasta, cataplasm, tape, gel, cream, oil, suppository, tincture, skin solution, eye drops, nasal drop, ear drops, coating agent, infusion solution, lyophilized formulation, injection Powders for functional agents, functional foods and the like.

  The liquid preparation can be produced, for example, by dissolving galactosaminoglycan as an active ingredient in a suitable aqueous solvent or a solvent commonly used in the pharmaceutical field. Examples of such a solvent include distilled water, water for injection, buffer solution, physiological saline, water containing a water-miscible organic solvent, and the like.

  When provided as an injection, the form may be any of a solution, a frozen product, a lyophilized product, and the like. This can be filled and sealed in an appropriate container such as an ampoule, vial, or syringe for injection, and can be distributed or stored as it is to be administered as an injection.

  Known methods can be used for the preparation of the anti-inflammatory agent of the present invention. In addition, other pharmaceutical active ingredients (for example, anti-inflammatory agents, analgesics, vitamins, antibacterial agents, growth factors, adhesion factors, etc.) are used as long as they do not adversely affect the anti-inflammatory effects of galactosaminoglycans. And stabilizers, emulsifiers, osmotic pressure adjusting agents, pH adjusting agents, buffering agents, isotonic agents, preservatives, soothing agents, coloring agents, excipients, binders, lubricants, disintegrating agents, etc. Ingredients usually used in the pharmaceutical field can be used.

  The compounding amount of galactosaminoglycan in the preparation, dosage per administration, administration interval, etc., depends on the purpose of administration, administration subject, administration route, administration method, dosage form, patient specific symptoms, age, sex, body weight, etc. It is a matter to be determined individually depending on the case, but it is not particularly limited. In the case of oral administration, about 1 mg to 5 g per adult, and in the case of parenteral administration, 0.1 mg to 1 g per adult The degree is illustrated.

  In addition, the treatment agent of the present invention may be administered once or multiple times. When administered multiple times, it may be administered every day or at intervals of about 1 to 7 days. Moreover, it may be administered once a day, or may be divided into 2 to 3 times a day.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to the following Example. In the examples, unless otherwise specified, “molecular weight” and “average molecular weight” mean “weight average molecular weight”. Further, “%” in “constitutive unsaturated disaccharide composition” means “mol%”.

[Example 1] Inhibition of inflammation in arthritis model mice
Balb / C mice (6 weeks old) were intraperitoneally administered on day 1 with 0.125 ml of a solution containing 0.5 mg of an arthritis-expressing monoclonal antibody mixture (sold by Iwai Chemical Co., Ltd.) and the same on day 2. The solution was administered in the same volume and route. Next, 0.1 ml of a phosphate buffered saline (PBS) solution containing 50 μg of lipopolysaccharide was intraperitoneally administered on the fourth day to induce arthritis, and an arthritis model mouse was prepared.

  Two hours after lipopolysaccharide administration, the galactosaminoglycan fraction 1 prepared in Reference Example 1 described later as a drug administration group (CS-E / constitutive unsaturated disaccharide composition: ΔDi-0S = 6.3%, ΔDi-4S = 25 ml of ΔDi-6S = 11.2%, ΔDi-diSD = 0%, ΔDi-diSE = 57.2% / average molecular weight: 75,000 Da) PBS solution containing 5 mg / ml 0.2 ml of the peritoneal cavity of arthritis model mice (8 mice) Administered. As a positive control group, chondroitin sulfate A (CS-A; manufactured by Seikagaku Corporation) in the same amount as galactosaminoglycan was placed in the abdominal cavity of the same mouse (8 mice), and 0.2 ml of PBS solution as a negative control group. Were administered intraperitoneally to the mice (7 animals). On the 8th day, the severity of arthritis in each foot of each mouse was evaluated based on the following 5 criteria (arthritis score: 0 to 4), and the values of the limbs were summed. The results are shown in FIG.

  As is clear from the results shown in FIG. 1, in the drug administration group to which the galactosaminoglycan of the present invention was administered, the sulfated galactosaminoglycan was sulfated in the same manner as the galactosaminoglycan of the present invention. The joint inflammation was statistically significantly suppressed as compared with the positive control group and the negative control group to which chondroitin sulfate A was administered at different esterified positions.

[Arthritis score]
0: Normal 1: Although slight, the ankle and wrist are clearly swollen and red.
2: The ankles and wrists were moderately swollen and red.
3: The entire foot is swollen and red.
4: The feet and joints were swollen to the highest degree.
[Formulation Example 1] (Tablet)

  Take 0.2g of galactosaminoglycan fraction 1 and add 52g of lactose, 22.8g of corn starch, a small amount of disintegrant (5g) and 20g of cellulose to make a total powder of 100g. 200 mg tablets.

[Formulation Example 2] (Capsule)
Galactosaminoglycan fraction 2 150g
Calcium chloride 50g
Corn starch 150g
Talc 80g
Magnesium stearate 30g

  The above is mixed and the particle size is adjusted by passing through a 60-mesh wire mesh, and then filled into 1000 gelatin capsules. This capsule is intended for oral administration of 1 to 3 capsules per day to humans.

[Formulation Example 3] (Injection)
Galactosaminoglycan fraction 1 200mg
Calcium chloride 200mg
Polyoxyethylene hydrogenated castor oil 500mg
Distilled water for injection

  According to the above formulation, an injection is prepared by a conventional method, and 2 ml of 1 ampule is filled.

[Reference Example 1] Preparation of Galactosaminoglycan Fraction 1 750 g of cartilage obtained from Japanese squid was shredded, boiled for 10 minutes, and then cooled to 50 ° C. After adjusting to pH 11 using sodium hydroxide (NaOH), extraction was performed overnight under conditions of pH 10 and 50 ° C. using 150 g of Esperase (Novo Novartis).

  3.75 g of sodium carbonate was added to the obtained extract and stirred for 1 hour at pH 10 and 50 ° C., followed by filtration, and the filtrate was concentrated to 750 ml.

  16 g of NaOH was added to the obtained concentrated liquid, reacted at 35 ° C. for 2 hours, and then centrifuged. The obtained supernatant was fractionated three times with 1105 ml of ethanol, 600 ml of ethanol + 3% sodium acetate (pH 4.8) and 612 ml of ethanol + 3% sodium acetate (pH 4.8), and then 1500 ml of ethanol was added to obtain crude galactosamino. 18.8 g of glycan was obtained.

  10 g of the obtained crude galactosaminoglycan was dissolved in 0.1 M Tris-HCl (pH 8.0), actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) was added, and the protein was further degraded overnight at 37 ° C.

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

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

  An equal amount of ethanol was added to the obtained filtrate to make it white turbid, and the white turbid solution was added to the same amount of ethanol as previously added to cause precipitation. The resulting precipitate was collected and washed with alcohol, and then the precipitate was separated. The precipitate was dried using a vacuum dryer, and purified galactosaminoglycan fraction 1 (average molecular weight: about 75,000 Da, constituent unsaturated disaccharide composition: ΔDi-0S = 6.3%, ΔDi-4S = 25.3% ΔDi-6S = 11.2%, ΔDi-diSD = 0%, ΔDi-diSE = 57.2%; chondroitinase ABC digestibility: 100%) was obtained at 6.8 g.

[Reference Example 2] Preparation of galactosaminoglycan fraction 2 1.5 g of galactosaminoglycan fraction 1 prepared in Reference Example 1 was collected and dissolved in PBS (pH 5.3). To this solution, 6,000 U of sheep-derived testicular hyaluronidase (manufactured by SIGMA) was added and reacted at 37 ° C. A part of the reaction solution was taken over time, and the degree of low molecular weight was examined by GPC-HPLC.

  When the molecular weight reached about 30 kDa, the reaction solution was boiled to stop the reaction. Activated carbon was added to the obtained reaction solution and reacted at 50 ° C. for 1 hour. The reaction solution was filtered, and sodium acetate was added to the filtrate so as to be 3%. Four times the amount of ethanol was added to this solution to obtain a precipitate. The precipitate is washed with ethanol, dried and purified (galactosaminoglycan fraction 2 / average molecular weight: about 30,000 Da, constituent unsaturated disaccharide composition: ΔDi-0S = 6.1%, ΔDi-4S = 18.8% ΔDi-6S = 17.1%, ΔDi-diSD = 0%, ΔDi-diSE = 58.0%; chondroitinase ABC digestibility: 100%) was obtained 1.3 g.

[Reference Example 3] Analysis of galactosaminoglycan The weight average molecular weight, digestibility by chondroitinase ABC, and constituent unsaturated disaccharide composition of galactosaminoglycan fractions 1 and 2 described in Reference Examples 1 and 2 are as follows: It measured by the method shown in.
(1) Measurement of weight average molecular weight The weight average molecular weight of each galactosaminoglycan was measured by the method of Arai et al. (Biochim.
Biophy. Acta, 1117, 60-70, 1992).

That is, chondroitin sulfate (weight average molecular weight 52,200, 31,400, 20,000, 10,200, 6,570, 459) with a known molecular weight and sodium hyaluronate (weight average molecular weight 104,000, 62,000) as standard products and gel filtration using high performance liquid chromatography ( GPC-HPLC) was determined by elution time. Column is TSK gel
G4000PWXL, G3000PWXL, G2500PWXL (each φ7.8 × 300 mm, manufactured by Tosoh Corporation) were used. The solvent is a 0.2 mol / L sodium chloride solution, the flow rate is 0.6 mL / min, and the detector is a differential refractive index detector (RI-8100, manufactured by Tosoh Corporation) and an ultraviolet-visible detector (UV-8010, Tosoh). (Made by Co., Ltd.) was used.

(2) Measurement of digestibility by chondroitinase ABC Add Tris-HCl buffer (pH 8.0) and 2.5 U chondroitinase ABC (Seikagaku Corporation) to 100 μL of each galactosaminoglycan 1% solution. And digested overnight at 37 ° C. The reaction was stopped by heating in a boiling water bath for 3 minutes, the digestion equivalent to 50 μg was analyzed by GPC-HPLC, and the digestibility was calculated by comparing with the enzyme undigested galactosaminoglycan.

(3) Constitutive unsaturated disaccharide composition analysis The sulfate group position of each galactosaminoglycan can be analyzed by a known method [New Chemistry Experiment Course 3, Carbohydrate II, p49-62 (1991, published by Tokyo Chemical Doujin) As described in “2. 8 Structural analysis combining glycosaminoglycan-degrading enzyme and HPLC”.

  That is, galactosaminoglycan is completely digested into disaccharides by chondroitinase ABC (manufactured by Seikagaku Corporation), and the resulting disaccharides (unsaturated disaccharides) containing unsaturated bonds are anion-exchanged. Analyzed by HPLC.

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

(I) Digestion with chondro-6-sulfatase In contrast to 100 μg of digestion product with chondroitinase ABC, buffer C (0.02
mol / L Tris-AcOH, pH7.0) Add 0.25 U chondro-6-sulfatase (Seikagaku Co., Ltd.) dissolved in 50 μL, digest at 37 ° C for 24 hours, and centrifuge for insoluble matter Was removed.

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

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

The result of having evaluated the seriousness of arthritis by the arthritis score by administering the galactosaminoglycan (CS-E), positive control (CS-A), and negative control (PBS) of this invention to an arthritis model mouse is shown.

Claims (7)

A hexuronic acid residue having a repeating structure of a disaccharide unit composed of a hexuronic acid residue and a galactosamine residue as a basic skeleton , and 50% to 70% of the disaccharide unit in the molecule does not have a sulfate group, and positions 4 and 6 A therapeutic agent for inflammatory bone and cartilage diseases comprising , as an active ingredient, a galactosaminoglycan comprising a galactosamine residue in which a hydroxyl group at the position is simultaneously sulfated, or a pharmacologically acceptable salt thereof . The galactosaminoglycan is composed of 5% to 25% of the disaccharide unit in the molecule of a hexuronic acid residue having no sulfate group and a galactosamine residue in which only the hydroxyl group at the 6-position is sulfated. The therapeutic agent for inflammatory bone / cartilage disease according to claim 1, which is galactosaminoglycan . The galactosaminoglycan is a galactosaminoglycan having a digestibility of 80% to 100% when chondroitinase ABC2.5U is added to 100 μL of the 1% solution and reacted overnight at 37 ° C. at pH 8.0. The therapeutic agent for inflammatory bone / cartilage disease according to claim 1 or 2, wherein: The therapeutic agent for inflammatory bone / cartilage disease according to any one of claims 1 to 3, wherein the galactosaminoglycan is a galactosaminoglycan having a weight average molecular weight of 1000 to 100,000 daltons . The therapeutic agent for inflammatory bone / cartilage disease according to any one of claims 1 to 4 , which is a therapeutic agent for arthritis or arthritis . The therapeutic agent for inflammatory bone / cartilage disease according to claim 5 , wherein the arthropathy or arthritis is rheumatoid arthritis or osteoarthritis . The therapeutic agent for inflammatory bone / cartilage disease according to any one of claims 1 to 6 , which is an injection or an oral administration agent.

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