Chondroitin sulfate polysaccharides, their semi-synthetic preparation method, and use

Abstract

The present invention provides a drug composition containing a metal salt of chondroitin sulfate polysaccharide. [Solution] A drug composition comprising a metal salt of chondroitin sulfate polysaccharide represented by the following general formula (I), wherein the metal salt of chondroitin sulfate polysaccharide has the following structure, TIFF2026071360000018.tif55166 R=H or SO3 - (I) The chondroitin sulfate polysaccharide is electrically neutral overall, contains a chondroitin sulfate polysaccharide anion and a metal cation, the average molecular weight of the anion is 1000 to 15000 Da, and the anion contains -SO3 - to-COO - A drug composition in which the molar ratio of is 1.46 to 2.73, and the range of the anion n is 2 ≤ n ≤ 45.

Description

[Technical Field]

[0001] This invention relates to the pharmaceutical field, and more specifically to chondroitin sulfate polysaccharide metal salts, as well as methods for preparing and using them. [Background technology]

[0002] Sulfate chondroitin polysaccharides (CS) are linear, sulfated glycosaminoglycans in which glucose and galactosamine alternately form β(1→3) and β(1→4) glycosidic bonds. Furthermore, different degrees of sulfation occur at positions 2 and 3 of glucuronic acid and positions 4 and 6 of galactosamine, resulting in 11 natural subtypes, the specific structures of which are as follows. [ka]

[0003] Chondroitin sulfate polysaccharides are widely present in animal bodies and are also endogenous substances in humans. They play an important role in physiological and pathological processes such as those involving the nervous system, cancer, and inflammation by regulating the expression levels of multiple enzymes and factors.

[0004] All currently available chondroitin sulfate pharmaceuticals and supplements are derived from cartilage tissue extracts of terrestrial animals and consist of type A and small amounts of type C. In contrast, chondroitin sulfate polysaccharides derived from marine animals differ in that their main component is often a subtype such as type C (shark cartilage extract) or type E (giant squid cartilage extract). Studies have shown that there are often significant differences in pharmacological activity among chondroitin sulfate polysaccharides with different compositions, and there remains a large gap in the development of pharmacologically active chondroitin sulfate polysaccharide drugs. [Overview of the Initiative]

[0005] The present invention aims to provide a chondroitin sulfate polysaccharide metal salt, a method for preparing the same, and its use. The chondroitin sulfate polysaccharide metal salt provided by the present invention has excellent pharmacological activity.

[0006] To solve the problems of the above invention, the present invention provides the following technical means.

[0007] A first aspect of the present invention provides a chondroitin sulfate polysaccharide metal salt (Formula I) which is electrically neutral overall and consists of a chondroitin sulfate polysaccharide anion and a metal cation. [ka]

[0008] The average molecular weight of the aforementioned chondroitin sulfate polysaccharide anion is 1000 to 15000 Da.

[0009] -SO3 in the chondroitin sulfate polysaccharide anion - to-COO - The molar ratio is 1.46 to 2.73; preferably -SO3 in the chondroitin sulfate polysaccharide anion. - to-COO - The molar ratio is 1.9 to 2.5.

[0010] The range of the chondroitin sulfate polysaccharide anion n is 2 ≤ n ≤ 45.

[0011] Preferably, the average molecular weight of the chondroitin sulfate polysaccharide anion is 4000 to 15000 Da.

[0012] Preferably, the metal cation comprises sodium ions and / or calcium ions and / or potassium ions.

[0013] Preferably, the range of n in the chondroitin sulfate polysaccharide anion is 6 ≤ n ≤ 20.

[0014] In the present invention, the chondroitin sulfate polysaccharides corresponding to the chondroitin sulfate polysaccharide anion mainly contain chondroitin sulfate polysaccharide C, chondroitin sulfate polysaccharide E, and chondroitin sulfate polysaccharide A, with the remainder further containing other subtypes of chondroitin sulfate polysaccharides. When the weight content of the chondroitin sulfate polysaccharides is taken as 100%, the total weight content of chondroitin sulfate polysaccharide C, chondroitin sulfate polysaccharide E, and chondroitin sulfate polysaccharide A is preferably 60-92%; the weight content of chondroitin sulfate polysaccharide C is preferably 0-30%, more preferably 10-25%; the weight content of chondroitin sulfate polysaccharide E is preferably 0-80%, more preferably 10-60%; and the weight content of chondroitin sulfate polysaccharide A is preferably 0-90%, more preferably 0-70%.

[0015] In the present invention, -SO3 in the chondroitin sulfate polysaccharide anion. - The weight content of is preferably 14-27%.

[0016] In the present invention, the weight content of alduronic acid in the chondroitin sulfate polysaccharide anion is preferably 20-35%, and the weight content of hexosamine is preferably 22-32%.

[0017] In the present invention, the metal cation preferably includes sodium ions and / or calcium ions and / or potassium ions, and more preferably sodium ions or calcium ions.

[0018] A second aspect of the present invention provides a method for preparing the chondroitin sulfate polysaccharide metal salt of the first aspect, comprising the following steps.

[0019] A process of mixing polysaccharide sulfate chondroitin raw material, a sulfation reagent, and an organic solvent, and carrying out a sulfation reaction to obtain a sulfation product system; A step of sequentially performing a first precipitation treatment, a salt formation treatment, dialysis, a second precipitation treatment, and column purification on the sulfated product system to obtain a chondroitin sulfate polysaccharide metal salt; the salt formation reagent used in the salt formation treatment is an aqueous metal hydroxide solution.

[0020] The polysaccharide chondroitin sulfate raw material contains chondroitin sulfate polysaccharide A and chondroitin sulfate polysaccharide C. Among them, the weight content of the chondroitin sulfate polysaccharide A is 70 - 90%, and the weight content of the chondroitin sulfate polysaccharide C is 10 - 30%; in the present invention, the polysaccharide chondroitin sulfate raw material used was purchased from Yantai Dongcheng Pharmaceutical Group Co., Ltd. The average molecular weight of the polysaccharide chondroitin sulfate raw material is 20,000 - 23,000 Da, and the molar ratio of -SO3 - and -COO - is 1, the weight content of chondroitin sulfate polysaccharide A is 70 - 90%, the weight content of chondroitin sulfate polysaccharide C is 10 - 30%, and it meets the relevant requirements of the "Chinese Pharmacopoeia" 2015 edition specification.

[0021] The average molecular weight of the polysaccharide chondroitin sulfate raw material is 20,000 - 23,000 Da; In the polysaccharide chondroitin sulfate raw material, the molar ratio of -SO3 - and -COO - is 0.9 - 1.1.

[0022] The sulfating reagent contains one or more of sulfur trioxide trimethylamine complex, sulfur trioxide pyridine complex, and sulfur trioxide triethylamine complex; The equivalent ratio of the sulfating reagent to the repeating disaccharide fragment in the polysaccharide chondroitin sulfate raw material is (1 - 10):1. More preferably, it is (3 - 8):1.

[0023] The temperature of the sulfation reaction is 40 to 120°C, and the duration is 2 to 36 hours; more preferably 6 to 24 hours. In the present invention, the sulfation reaction is preferably carried out under stirring conditions; however, there are no particular limitations on the stirring speed in the present invention, and any stirring speed well known to those skilled in the art is acceptable.

[0024] The salt-forming reagent is an aqueous solution of sodium hydroxide and / or an aqueous solution of potassium hydroxide; the concentration of the salt-forming reagent is 1 to 4 mol / L.

[0025] The primary precipitation reagent used in the aforementioned primary precipitation treatment is an aqueous ethanol solution with a volume fraction of 90-95%; The secondary precipitation reagent used in the aforementioned secondary precipitation treatment is ethanol; The cutoff molecular weight of the dialysis bag used in the aforementioned dialysis is 3000 to 12000 Da.

[0026] In the present invention, there are no particular limitations on the method of adding the first precipitation reagent, the rate of addition, or the amount used, as long as the sulfation product in the sulfation product system is completely precipitated. In the present invention, the time for the first precipitation treatment is preferably 25 to 35 minutes, and the first precipitation treatment is preferably carried out at room temperature and under stirring conditions.

[0027] After the completion of the first sedimentation treatment, the present invention preferably filters the obtained system, dissolves the obtained solid by adding water, and performs salt formation treatment by mixing the obtained solution with a salt-forming reagent. In the present invention, the ratio of the amount of water used for dissolution to the amount of solid is preferably (0.8~1.2) mL:1 g, and more preferably 1 mL:1 g. In the present invention, the salt-forming reagent is preferably an aqueous sodium hydroxide solution and / or an aqueous potassium hydroxide solution, and more preferably an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution; the concentration of the salt-forming reagent is preferably 1~4 mol / L, and more preferably 2~3 mol / L; when the salt-forming reagent is an aqueous sodium hydroxide solution and an aqueous potassium hydroxide solution, the concentration of the salt-forming reagent is the sum of the concentrations of sodium hydroxide and potassium hydroxide. In the present invention, there are no particular limitations on the amount of salt-forming reagent added, but preferably, it is sufficient to ensure that the pH value of the obtained system becomes neutral after mixing the dissolved substance and the salt-forming reagent, and that a corresponding sodium salt and / or potassium salt is obtained.

[0028] After the salt formation process is completed, in this invention, the obtained solution is subjected to dialysis, and the cutoff molecular weight of the dialysis bag used in the dialysis is preferably 3000 to 12000 Da, more preferably 8000 to 12000 Da. In this invention, the dialysis time is preferably 2 to 3 days, more preferably 2 days; it is desirable to change the water every 12 hours during the dialysis process.

[0029] After the completion of the dialysis, it is preferable in the present invention to evaporate the obtained system, dissolve the resulting residue by adding an aqueous sodium acetate solution (NaOAc aqueous solution), and then mix the resulting dissolved substance with ethanol and perform a second precipitation treatment. In the present invention, the mass concentration of the NaOAc aqueous solution is preferably 2-5%, and more preferably 2-3%. In the present invention, the reason for using an aqueous NaOAc aqueous solution to dissolve the residue is to ensure that the residue becomes more soluble in ethanol, and to facilitate the subsequent second precipitation treatment. In the present invention, the ratio of the amount of residue, aqueous NaOAc aqueous solution, and ethanol used is preferably 1g:(8-12)mL:(3.5-4.5)mL, and more preferably 1g:10mL:4mL.

[0030] After the completion of the second precipitation treatment, in the present invention, it is preferable to centrifuge the obtained system, and the obtained solid is the crude product of chondroitin sulfate polysaccharide metal salt; in order to ensure that the final obtained chondroitin sulfate polysaccharide metal salt has high purity, in the present invention, it is preferable to repeatedly perform the first precipitation treatment, salt formation treatment, dialysis and second precipitation treatment on the crude product of chondroitin sulfate polysaccharide metal salt, and then perform subsequent column purification on the obtained crude product. In the present invention, the gel column used in the column purification is preferably a G25 gel column, and the elution solvent used in the column purification is preferably water.

[0031] After the column purification is complete, the eluate is preferably dried to obtain a chondroitin sulfate polysaccharide metal salt. In this invention, the drying is preferably freeze-drying. In this invention, there are no particular limitations on the temperature and time of the freeze-drying, as long as the substance is sufficiently dried.

[0032] A third aspect of the present invention provides a drug composition comprising the aforementioned chondroitin sulfate polysaccharide metal salt and a pharmaceutically acceptable carrier as described in the first aspect. The drug composition can be prepared by methods known in the art. The compound of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or auxiliaries to form any dosage form suitable for use in humans or animals. The compound of the present invention is typically present in a weight content of 0.1-95% in the drug composition.

[0033] The compound of the present invention or a drug composition containing the same can be administered in the form of a unit dose, and the method of administration may be gastrointestinal or non-gastrointestinal, for example, by oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and airways, skin, vagina, rectum, etc.

[0034] The drug may be in the form of a liquid, solid, or semi-solid preparation. Liquid preparations may include solutions (including true solutions and colloidal solutions), emulsions (including o / w type, w / o type, and double emulsions), suspensions, injections (including liquid injections, powder injections, and intravenous infusions), eye drops, nasal drops, lotions, and topical preparations; solid preparations may include tablets (general tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, pills, suppositories, membranes, patches, aerosols (powder), and sprays; semi-solid preparations may include ointments, gels, and pastes.

[0035] The compounds of the present invention may be used as general formulations, as well as as sustained-release formulations, release-controlled agents, targeting formulations, and various particulate drug delivery systems.

[0036] To form the compound of the present invention into tablets, various excipients commonly known in the art may be used, specifically diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents may include starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; Wetting agents may include water, ethanol, isopropanol, etc.; Binders may include starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic slurry, gelatin paste, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyglycol, etc.; Disintegrants may include dried starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; Lubricants and fluidity aids may include talc powder, silica, stearate, tartaric acid, liquid paraffin, polyglycol, etc.

[0037] Furthermore, the tablets may be coated tablets, such as sugar-coated tablets, thin-film coated tablets, enteric-coated tablets, or two-layer tablets and multi-layer tablets.

[0038] To form a capsule for the medication unit, the active ingredient, the compound of the present invention, may be mixed with a diluent and a flow aid, and the mixture may be placed directly into a hard capsule or soft capsule. Alternatively, the active ingredient, the compound of the present invention, may be first prepared with a diluent, binder, and disintegrant in the form of granules or micro-pills, and then placed into a hard capsule or soft capsule. The types of diluents, binders, wetting agents, disintegrants, and flow aids used in the preparation of tablets of the compound of the present invention may also be applied to the preparation of capsules of the compound of the present invention.

[0039] To prepare the compound of the present invention as an injectable preparation, water, ethanol, isopropanol, propylene glycol, or a mixture thereof may be used as a solvent, and appropriate amounts of commonly used solubilizers, solubilizers, pH adjusters, and osmotic pressure adjusters may also be added. The solubilizer or solubilizer may be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjuster may be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When preparing a lyophilized powder injectable preparation, mannitol, glucose, etc. may also be added as propane.

[0040] Furthermore, if necessary, colorants, preservatives, fragrances, flavoring agents, or other additives may be added to the pharmaceutical formulation.

[0041] To achieve the purpose of drug administration and enhance the therapeutic effect, the drug or drug composition of the present invention may be administered by any known method of administration.

[0042] The dosage of the compound drug composition of the present invention may vary over a wide range depending on the nature and progression of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally, the preferred daily dosage range of the compound of the present invention may be 0.001-150 mg / kg body weight, preferably 0.1-100 mg / kg body weight, more preferably 1-60 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The above dosage may be administered in one dose unit or divided into multiple dose units, in which case it should be appropriately determined in accordance with the physician's clinical experience and a drug administration plan that includes other treatment means.

[0043] The compounds or compositions of the present invention may be taken alone or in combination with other therapeutic agents or symptom-controlling agents. When the compounds of the present invention have a synergistic effect with other therapeutic agents, the dosage should be adjusted according to the actual situation.

[0044] A fourth aspect of the present invention is the use of the chondroitin sulfate polysaccharide metal salt of the first aspect of the present invention in the preparation of an anti-inflammatory disease agent. The inflammatory disease includes osteoarthritis and rheumatoid arthritis.

[0045] This invention provides metal salts of chondroitin sulfate polysaccharides. The metal salts of chondroitin sulfate provided by this invention exert anti-inflammatory effects by inhibiting the NF-κB signaling pathway and simultaneously suppressing the expression and function of inflammation-related factors and enzymes. As can be seen from the results of the examples, the metal salts of chondroitin sulfate provided by this invention have anti-inflammatory effects and can be used in the preparation of anti-inflammatory disease drugs.

[0046] This invention provides metal salts of chondroitin sulfate polysaccharides. The metal salts of chondroitin sulfate provided by this invention not only have anti-inflammatory activity but also have the effect of increasing bone density. As can be seen from the results of the examples, the metal salts of chondroitin sulfate provided by this invention have anti-inflammatory and bone-protective effects and can be used in the preparation of anti-rheumatic arthritis drugs.

[0047] This invention provides a metal salt of chondroitin sulfate polysaccharide. The metal salt of chondroitin sulfate provided by this invention can increase the water content of cartilage and has a cartilage-protecting effect. As can be seen from the results of the examples, the metal salt of chondroitin sulfate provided by this invention has anti-inflammatory and cartilage-protecting effects and can be used in the preparation of anti-osteoarthritis drugs.

[0048] Beneficial technical effects: When the degree of sulfate of the compound in the present invention is high (the ratio of sulfate ions to carboxylate ions is within a specific range), it exhibits outstanding in vivo anti-inflammatory activity, exceeding the anti-inflammatory activity of naturally derived CS-E. There is a clear structure-activity relationship between the degree of sulfate of the compound in the present invention and its in vivo anti-inflammatory activity. In an animal model of acute ulcerative proctitis, the compound in the present invention also exhibits outstanding biological activity and has a remarkable therapeutic effect. Compared with naturally derived CS-A and CS-E, the compound in the present invention exhibits outstanding biological activity and has a remarkable therapeutic effect in both an anti-type I collagen-induced mouse toe swelling (RA) animal model and a papain-induced rat arthritis (OA) model.

[0049] The present invention provides a method for preparing the aforementioned chondroitin sulfate polysaccharide metal salts. This method allows for the production of chondroitin sulfate polysaccharide metal salts with different degrees of sulfatedness from a semi-synthetic approach, and the method is simple to operate and suitable for scale production. [Brief explanation of the drawing]

[0050] [Figure 1] This is the HPLC spectrum of polysaccharide 1 prepared in Example 1; [Figure 2] This is the HPLC spectrum of polysaccharide 2 prepared in Example 2; [Figure 3] This is the HPLC spectrum of polysaccharide 3 prepared in Example 3; [Figure 4] This is the HPLC spectrum of polysaccharide 4 prepared in Example 4; [Figure 5] This is a comparison chart of body weight changes in different groups of UC mice in Experimental Example 3. Here, p<0.01 vs. Con; p<0.05 vs. Mod; [Figure 6] This is a comparison chart of colon lengths in different groups of UC mice in Experimental Example 3. Here, p<0.01 vs. Con; p<0.05 vs. Mod; [Figure 7]This is a comparison chart of DAI scores in different groups of mice in Experiment Example 3. Here, ##p<0.01 vs. Con; **p<0.01, *p<0.05 vs. Mod. [Figure 8] These are MRI images of rat knee joints from different groups in Test Example 5. As shown in Figure 8, in the CON group, the joint space was normal, the articular cartilage surface was complete and undamaged, the subchondral bone was normal, the cartilage thickness was normal, there was a moderate amount of infrapatellar fat, and the synovial membrane and a small amount of synovial fluid were observed. In the MOD group, the joint space was narrowed, the articular cartilage surface was damaged and missing, the outer edge of the cartilage was incomplete, the cartilage shape was unclear, and the cartilage thickness was reduced. In the 5H group, the joint shape was clear, the joint space had recovered to normal, and there was a small amount of synovial fluid. [Figure 9] These are CT images of rat knee joints from different groups in Test Example 5. From Figure 9, the CON group showed a rat knee joint shape; the MOD model group showed bone destruction in the bone beneath the knee articular cartilage, significant erosion, clear cavities in the joint, and deformation of the bone and joint; the POS-positive drug group and the 5L group showed relatively milder bone destruction and erosion compared to the model group; and the 5M and 5H groups showed clear improvement in bone destruction and erosion compared to the model group. [Modes for carrying out the invention]

[0051] Example 1 (1) 26.8 g of commercially available polysaccharide sulfate chondroitin A (Yantai Dongcheng Pharmaceutical Group Co., Ltd., NO. CSJ1170701) was dissolved in 360 mL of dimethyl sulfoxide, 24 g (3 eq.) of sulfur trioxide trimethylamine complex was added, and the mixture was stirred and reacted at 50°C for 6 hours;

[0052] (2) The reaction system obtained was cooled to room temperature, 400 mL of 95% volume fraction ethanol aqueous solution was added, stirred for 30 minutes, filtered, and water was added to dissolve the solid (the ratio of water to solid used was 1 mL:1 g). The pH value was adjusted to 7.0 with 2 mol / L NaOH aqueous solution, and the obtained solution was placed in a dialysis bag (cutoff molecular weight 12000 Da). After dialysis for 2 days, the solid was obtained by evaporation.

[0053] (3) The obtained solid was dissolved in a 2% NaOAc aqueous solution, then anhydrous ethanol was added, and after sufficient precipitation, the solution was centrifuged to obtain the crude product; the ratio of the solid, NaOAc aqueous solution, and anhydrous ethanol used was 1 g:10 mL:40 mL;

[0054] (4) The crude product obtained was repeatedly subjected to steps (2) and (3), and the obtained substance was then purified using a G25 gel column (the eluent used was H2O), and finally the obtained eluate was freeze-dried to obtain 21.3 g of chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 1).

[0055] Example 2 Chondroitin sulfate polysaccharides were prepared according to the procedure of Example 1. The difference was that in step (1), 6.7 g of commercially available polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and 8 g (4 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted at 60°C for 24 hours. Finally, 4.4 g of chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 2) was obtained.

[0056] Example 3 Chondroitin sulfate polysaccharides were prepared according to the procedure of Example 1. The difference is that in step (1), 6.7 g of commercially available polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and 10 g (5 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted for 24 hours at 60°C, with a cutoff molecular weight of 7000 Da on the dialysis bag. Finally, 4.1 g of chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 3) was obtained.

[0057] Example 4 Chondroitin sulfate polysaccharides were prepared according to the procedure of Example 1. The difference is that in step (1), 6.7 g of commercially available polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and 12 g (6 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted at 70°C for 20 hours, with a cutoff molecular weight of 5000 Da on the dialysis bag. Finally, 3.9 g of chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 4) was obtained.

[0058] Example 5 Chondroitin sulfate polysaccharides were prepared according to the procedure of Example 1. The difference is that in step (1), 1.0 g of commercially available polysaccharide chondroitin sulfate A was dissolved in 13 mL of dimethyl sulfoxide, and 2.4 g (8 eq.) of sulfur trioxide trimethylamine complex was added; the mixture was stirred and reacted at 100°C for 36 hours, with a cutoff molecular weight of 3500 Da on the dialysis bag. Finally, 1.0 g of chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 5) was obtained.

[0059] Pharmacology experiments Experimental Example 1 The composition of polysaccharides 1-5 prepared in Examples 1-5 was analyzed. Specifically, the average molecular weight (Da) and -SO3 were analyzed. - COO - (Molar ratio), -SO3 -The data includes content, alduronic acid content, hexosamine content, anticoagulant activity (IU), degradable polysaccharide content, and the proportion of each subtype component (polysaccharides 1-5 prepared in Examples 1-5 were degraded with chondroitin sulfate ABC enzymes, and the proportion of each subtype component, specifically chondroitin sulfate A, chondroitin sulfate C, and chondroitin sulfate E, was measured using HPLC. Note that polysaccharides 1-5 also contain other extra subtypes of chondroitin sulfate polysaccharides, so this is not further limited here; the HPLC spectra of polysaccharides 1-4 are shown in Figures 1-4), and the specific results are shown in Table 1.

[0060] [Table 1]

[0061] Experimental Example 2 The anti-inflammatory effects of polysaccharides 1-5 prepared in Examples 1-5 were evaluated, and the details are as follows:

[0062] (1) Test method: Experiment to evaluate the pharmacodynamic activity of CS compound in a mouse ear acute swelling and inflammation model induced by snake oil. Animals: Balb / c mice, male (20-22g); 8 individuals / group.

[0063] Grouping: The subjects were divided into a model group, a positive drug indomethacin group (5 mg / kg), a natural extract CS-E group (200 mg / kg), and a polysaccharide group (200 mg / kg, 100 mg / kg, 50 mg / kg). Both the positive drug and the CS compounds were prepared with 0.5% sodium carboxymethylcellulose by mass and stored in a refrigerator at 4°C.

[0064] Administration and Measurement: The drug was administered once daily for three days. After the last dose, the percentage of mouse ear swelling induced by the compound and the percentage of drug suppression of ear swelling were calculated.

[0065] Statistical Analysis: Experimental results are presented as "mean ± standard deviation". Statistical differences between the two groups were calculated using t-analysis. * indicates p<0.05, and ** indicates p<0.01.

[0066] (2) The anti-inflammatory effects of polysaccharides are shown in Tables 2-4.

[0067] [Table 2]

[0068] [Table 3]

[0069] [Table 4] As can be seen from Tables 2-4, a structure-activity relationship exists between the degree of sulfated polysaccharides 1-5 and their anti-inflammatory activity. Polysaccharides 2-5 exhibited excellent anti-inflammatory effects in a mouse ear swelling model, outperforming naturally derived CS-E polysaccharides. In particular, polysaccharide 3 showed remarkable anti-inflammatory effects in the range of 50-200 mg / kg, demonstrating a clear dose-response relationship.

[0070] Experimental Example 3 The polysaccharide 3 (denoted as SEMI5) prepared in Example 3 was subjected to a pharmacodynamic evaluation of an anti-DSS model. Details are as follows:

[0071] 1. Materials and Methods 1. Laboratory animals C57BL / 6J mice, male (18~20g), 6 / group. Purchased from Beijing Huafukang Biological Technology Co., Ltd.; Permit number: SCXK (King) 2014-0004.

[0072] 2. Experimental group division (1) Normal control group: 0.5% sodium carboxymethylcellulose was administered orally.

[0073] (2) UC (Ulcerative Colitis) model group: Sodium carboxymethyl cellulose with a mass concentration of 0.5% was force-fed.

[0074] (3) SASP (positive drug sulfasalazine) group: Shanghai Xinyi Pharmaceutical Factory Co., Ltd. (lot number: 036151102); Prepared with 500 mg / kg of sodium carboxymethyl cellulose with a mass concentration of 0.5%, stored at 4°C, and force-fed once a day.

[0075] (4) SEMI5-50 group: Prepared with 50 mg / kg in distilled water. Stored at 4°C and force-fed once a day.

[0076] (5) SEMI5-150 group: Prepared with 150 mg / kg in distilled water. Stored at 4°C and force-fed once a day.

[0077] 3. Experimental method After C57BL / 6J mice were adaptively fed in an SPF-class animal house (experimental animal use permit number: SYXK (Beijing) 2014-0023) for one week, they were randomly divided into 5 groups according to the above protocol. For the UC model group and the dosing group mice, a UC inflammatory model was established daily in the laboratory using DSS (MP, CA9011-18-1, US) by a well-established ulcerative colitis modeling method. The normal control group (control group) and the UC model group (model group) were force-fed sodium carboxymethyl cellulose with a mass concentration of 0.5% once a day. The SASP group, the SEMI5-50 group, and the SEMI5-150 group were force-fed once a day according to the experimental protocol in the "Experiment Group Division" section. Seven days after modeling, the UC model group animals showed typical UC lesions such as lethargy, reduced activity, weight loss, loose stools, and bloody stools. At the end of the experiment, the animals in each group were euthanized, various parameters related to various colonic inflammations were measured (as shown in the experimental results section), and the anti-UC pharmacodynamic activities of each compound were comprehensively evaluated.

[0078] II. Experimental results 1. Effects of various SEMI5 concentrations on body weight in DSS-induced UC model mice (Table 5 and Figure 5).

[0079] [Table 5]

[0080] As can be seen from Table 5 and Figure 5, the body weight of the UC model group mice was significantly lower than that of the normal control group, and the statistically significant difference suggests that UC modeling was successful. In the SASP group and the SEMI5-150 group, DSS-induced acute UC was not effectively alleviated, and the body weight of the C57 BL / 6J mice decreased. On the other hand, when comparing the SEMI5-50 group and the UC model group, weight loss was effectively mitigated, and statistically significant differences were observed.

[0081] 2. Effects of various SEMI5 concentrations on colonic contracture in UC mice (Table 6 and Figure 6).

[0082] [Table 6]

[0083] As can be seen from Table 6 and Figure 6, in the DSS-induced C57 BL / 6J mouse acute UC animal model, the colon contracture of the UC model mice was significantly shorter compared to the normal control group, showing a statistically very significant difference. The SASP group did not effectively improve colon contracture in the model animals. On the other hand, when the SEMI5-50 group and SEMI5-150 group were compared to the UC model group, the improvement in colon contracture was effective and statistically significant.

[0084] 3. Effects of each SEMI5 concentration on the UC mouse disease overall index (DAI) score (Table 7 and Figure 7).

[0085] [Table 7]

[0086] As can be seen from Table 7 and Figure 7, the DAI (Disease Index) of the UC model group animals was clearly increased compared to the normal control group, showing a statistically significant difference and suggesting the success of the modeling. Compared to the UC model group, the SEMI5-50 group and the SEMI5-150 group were able to significantly reduce their DAI scores, showing a statistically significant difference. The SASP group showed some improvement, but the difference was statistically significant. The DAI score was evaluated using indicators such as the degree of weight loss, stool characteristics, and blood in the stool. A lower DAI score suggests that the animal is closer to a normal physiological state. The DAI score criteria are shown in Table 8.

[0087] [Table 8]

[0088] Experimental Example 4 The polysaccharide 3 (denoted as SEMI5) prepared in Example 3 was evaluated in an anti-type I collagen-induced mouse toe swelling model. Details are as follows:

[0089] 1. Materials and Methods 1 animal: DBAI mouse, male (20-22g); 7 mice / group.

[0090] The subjects were divided into two groups: a blank control group, a model group, and groups receiving the positive drug indomethacin (5 mg / kg), CSA (200 mg / kg), CSE (200 mg / kg), CS-E-semi3 (200 mg / kg), and CS-Semi-5 (200 mg / kg). Both the positive drug and the CS compounds were prepared in 0.5% carboxymethylcellulose sodium and stored in a refrigerator at 4°C.

[0091] 3. Dosage frequency: Once daily for 3 days.

[0092] 4. Experimental Method: Following established laboratory methods, the body weight of mice was measured weekly after the initial immunization. After a second shock immunization on day 21 of the experiment, the degree of joint swelling in each group of mice was observed weekly, and the joint swelling index score was evaluated.

[0093] 5 Statistical analysis: The experimental results were expressed as "mean ± standard deviation". The statistical differences between the two groups were calculated and analyzed using the t-test method. * indicates p < 0.05, and ** indicates p < 0.01.

[0094] II Experimental results

Table 9

[0095]

Table 10

[0096]

Table 11

[0097] As can be seen from the comparison of the mouse final body weight results in Table 9, the SEMI5 drug has an obvious recovery effect on the weight loss of mice. From Table 10, it can be seen that the mouse joints usually show swelling at 4 weeks, which develops from the toes to the soles of the feet and further to the ankle joints. The lesion reaches its peak at 5 - 6 weeks, and the modeling success rate reaches 100%. The SEMI5 drug has an obvious improvement effect on joint swelling. Table 11 suggests that the SEMI5 drug has an obvious improvement effect on bone density, and it was found that it is superior to the improvement effect of the positive drug. From the above results, it was suggested that the semi-synthetic SEMI5 drug has anti-RA activity.

[0098] Experimental Example 5 The polysaccharide 3 (denoted as SEMI5) prepared in Example 3 was evaluated against the papain-induced OA model. The details are as follows:

[0099] I Materials and methods: 1 Animals: SD rats, male (180 - 220 g); 6 rats / group; purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; license number: SCXK (Beijing) 2012 - 0001.

[0100] Divided into two groups: (1) Normal control group (Con group): Forced oral administration of DDW.

[0101] (2) Model group (Mod group): Forced oral administration of DDW.

[0102] (3) Positive drug celecoxib group (Pos group): Purchased from Pfizer (W47055); prepared in DDW, stored at 4°C, and administered orally once daily.

[0103] (4) Compound semi5 low dose group (5L group): 50 mg / kg, prepared in DDW, stored at 4°C, and administered orally once daily.

[0104] (5) Compound semi5 medium dose group (5M group): 100 mg / kg, prepared in DDW, stored at 4°C, and administered orally once daily.

[0105] (6) Compound semi5 high-dose group (5H group): 200 mg / kg, prepared in DDW, stored at 4°C, and administered orally once daily.

[0106] 2. Experimental Results 1. Knee joint actual width (net width) Using calipers, the width of the knee joint after removing skin and fat is measured, and the degree of joint swelling is intuitively assessed.

[0107] [Table 12]

[0108] 2. Nuclear Magnetic Resonance Results The degree of damage to rat knee articular cartilage was assessed using nuclear magnetic resonance (as shown in Figure 8), and at the same time, data on changes in cartilage calcification levels measured under computationally appropriate conditions were calculated. This data reflects the water content of the articular cartilage in each group, suggesting that the higher the water content, the closer the articular cartilage is to a normal physiological state (see Table 13).

[0109] [Table 13]

[0110] As can be seen from Table 13, the calcification levels of the model group (Mod) animals showed a statistically significant difference compared to the normal control group (Con), strongly suggesting the success of the modeling of cartilage destruction; the calcification levels of the high-dose group animals also showed a statistically significant difference compared to the model group (Mod). These results suggest that while the cartilage of the model group was significantly destroyed, the treatment group showed a clear improvement effect. **p<0.01vsCon;##p<0.01vsMod.

[0111] 3 CT examination results CT scans allow us to examine the subchondral bone of the rat knee joint and determine the state of subchondral bone reconstruction.

[0112] III. Experimental Conclusions 1. Pharmacodynamic evaluation of an anti-DSS-induced acute ulcerative colitis model. 1) The UC model group animals showed clear weight loss, bloody stools, diarrhea, colonic contracture, and elevated DAI total index scores and colonic tissue pathology scores, suggesting successful modeling of the DSS-induced mouse ulcerative colitis model. This indicates that the system is reliable and suitable for evaluating the activity of anti-UC compounds.

[0113] 2) In experiments with the SASP group, while it was not able to effectively reduce weight loss in UC model animals, it was able to improve phenomena such as diarrhea, bloody stools, and colonic contracture in UC model animals, suggesting that SASP has some anti-UC activity.

[0114] 3) The SEMI5-50 group effectively reduced weight loss in the model animals and also exhibited a certain level of anti-UC activity in terms of reducing diarrhea, bloody stools, and DAI scores in the UC model animals, with statistically significant differences observed. Based on these results, it was found that under this measurement system, SEMI5-50 has a clear therapeutic effect on DSS-induced UC animal models.

[0115] 4) The SEMI5-150 group was unable to reduce weight loss in UC model animals, and the difference was not statistically significant. However, it was able to improve colonic contracture, and the difference was statistically significant. Furthermore, it exhibited anti-UC activity in all aspects, including diarrhea, bloody stools, and DAI reduction, as well as tissue scores, in UC model animals, and the difference was statistically significant. From these results, it was found that under this measurement system, SEMI5-150 has a certain therapeutic effect on DSS-induced UC animal models.

[0116] 2. Pharmacodynamic evaluation of a mouse toe swelling model induced by anti-type I protein. 1) As can be seen from the comparison of the end-of-study body weight results of the mice, SEMI5 was found to have a clear restorative effect on weight loss in arthritis mice.

[0117] 2) SEMI5 has a clear ameliorative effect on joint swelling.

[0118] 3) SEMI5 has a clear improving effect on joint bone mineral density, and its improving effect is superior to that of positive drugs. These results suggest that semi-synthetic SEMI5 has anti-RA activity.

[0119] 3. Pharmacodynamic evaluation of an antipapain-induced rat osteoarthritis model. 1) The actual knee joint width of the model group (Mod) animals was significantly increased compared to the normal control group (Con), showing a statistically very significant difference, suggesting clear swelling in the joints of the model group animals; there was no statistically significant difference in the positive drug group (Pos) compared to the model group; the actual knee joint width of each treatment group of animals showed a statistically significant difference compared to the model group (Mod), suggesting that SEMI5 has relatively superior anti-inflammatory activity.

[0120] 2) In the CON group, the joint space was normal, the articular cartilage surface was intact without damage, the subchondral bone was normal, the cartilage thickness was normal, there was a moderate amount of infrapatellar fat, and the synovial membrane and a small amount of synovial fluid were observed. In the MOD group, the joint space was narrowed, the articular cartilage surface was missing due to damage, the outer edge of the cartilage was incomplete, the cartilage shape was unclear, and the cartilage thickness was reduced. In the SEMI5H group, the joint shape was clear, the joint space had recovered to normal, and there was a small amount of synovial fluid.

[0121] 3) The calcification levels of the model group (Mod) animals were statistically significantly different from those of the normal control group (Con), suggesting successful modeling of significant cartilage destruction; the calcification levels of the high-dose SEMI5 group animals were statistically significantly different from those of the model group (Mod). From these results, while cartilage destruction was significant in the model group, a clear improvement was observed in the treatment group.

[0122] 4) Compared to the normal knee joint shape of rats in the CON group, the MOD model group showed bone quality destruction in the bone beneath the knee articular cartilage, significant erosion, clear cavities in the joint, and deformation of the bone and joint; the POS-positive drug group and the SEMI5L group showed relatively milder bone quality destruction and erosion compared to the model group; the SEMI5M and SEMI5H groups showed clear improvement in bone quality destruction and erosion compared to the model group.

[0123] These results suggest that semi-synthetic SEMI5 possesses anti-OA activity.

[0124] The above are merely preferred embodiments of the present invention, and it goes without saying that any improvements and modifications made by those skilled in the art, provided they do not deviate from the principles of the present invention, should also be considered within the scope of protection of the present invention.

Claims

1. A chondroitin sulfate polysaccharide metal salt represented by the following general formula (I), having the following structural general formula, 【Chemistry 1】 Overall, it is electrically neutral and contains chondroitin sulfate polysaccharide anions and metal cations. The average molecular weight of the aforementioned chondroitin sulfate polysaccharide anion is 1,000 to 15,000 Da. The aforementioned chondroitin sulfate polysaccharide anion contains -SO ３ － to-COO － The molar ratio is 1.46 to 2.

73. The range of the chondroitin sulfate polysaccharide anion n is 2 ≤ n ≤ 45, and this is a metal salt of chondroitin sulfate polysaccharide.

2. The average molecular weight of the chondroitin sulfate polysaccharide anion is 4000 to 15000 Da, and the chondroitin sulfate polysaccharide anion contains -SO ３ － to-COO － The chondroitin sulfate polysaccharide metal salt according to claim 1, characterized in that the molar ratio is 1.9 to 2.

5.

3. The metal salt of chondroitin sulfate polysaccharide according to claim 1 or 2, characterized in that the metal cation comprises sodium ions and / or calcium ions and / or potassium ions.

4. The process involves mixing a polysaccharide sulfate chondroitin raw material, a sulfation reagent, and an organic solvent to carry out a sulfation reaction and obtain a sulfation product system. The process includes sequentially performing a first precipitation treatment, a salt formation treatment, dialysis, a second precipitation treatment, and column purification on the aforementioned sulfation product system to obtain a metal salt of chondroitin sulfate polysaccharide. A method for preparing a metal salt of chondroitin sulfate polysaccharide according to any one of claims 1 to 3, characterized in that the salt-forming reagent used in the salt-forming treatment is an aqueous solution of a metal hydroxide.

5. The aforementioned polysaccharide chondroitin sulfate raw material contains chondroitin sulfate polysaccharide A and chondroitin sulfate polysaccharide C, of ​​which the weight content of chondroitin sulfate polysaccharide A is 70-90%, and the weight content of chondroitin sulfate polysaccharide C is 10-30%. The average molecular weight of the aforementioned polysaccharide sulfate chondroitin raw material is 20,000 to 23,000 Da. In the polysaccharide chondroitin sulfate raw material, -SO ３ － and -COO － The preparation method according to claim 4, characterized in that the molar ratio is 0.9 to 1.

1.

6. The sulfation reagent comprises one or more of the following: sulfur trioxide trimethylamine complex, sulfur trioxide pyridine complex, and sulfur triethylamine complex. The preparation method according to claim 4 or 5, characterized in that the equivalent ratio of the sulfation reagent to the repeating disaccharide fragment in the polysaccharide sulfate chondroitin raw material is (1 to 10):

1.

7. The preparation method according to claim 6, characterized in that the temperature of the sulfation reaction is 40 to 120°C, the duration is 2 to 36 hours, and the equivalent ratio of the sulfation reagent to the repeating disaccharide fragment in the polysaccharide sulfate chondroitin raw material is (3-8):

1.

8. The preparation method according to claim 4, characterized in that the salt-forming reagent is an aqueous solution of sodium hydroxide and / or an aqueous solution of potassium hydroxide, and the concentration of the salt-forming reagent is 1 to 4 mol / L.

9. The primary precipitation reagent used in the aforementioned primary precipitation treatment was an ethanol aqueous solution with a volume fraction of 90-95%. The second precipitation reagent used in the aforementioned second precipitation treatment was ethanol. The preparation method according to claim 4, characterized in that the cutoff molecular weight of the dialysis bag used in the dialysis is 3,000 to 12,000 Da.

10. A drug composition characterized by comprising a chondroitin sulfate polysaccharide metal salt according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

11. Use of the chondroitin sulfate polysaccharide metal salt according to any one of claims 1 to 3 in the preparation of an anti-inflammatory disease drug.

12. The use according to claim 11, characterized in that the inflammatory disease includes ulcerative colitis, osteoarthritis, and rheumatoid arthritis.

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