WO2020177749A1 - 硫酸软骨素多糖，其半合成制备方法和应用 - Google Patents

硫酸软骨素多糖，其半合成制备方法和应用 Download PDF

Info

Publication number
WO2020177749A1
WO2020177749A1 PCT/CN2020/078075 CN2020078075W WO2020177749A1 WO 2020177749 A1 WO2020177749 A1 WO 2020177749A1 CN 2020078075 W CN2020078075 W CN 2020078075W WO 2020177749 A1 WO2020177749 A1 WO 2020177749A1
Authority
WO
WIPO (PCT)
Prior art keywords
chondroitin sulfate
polysaccharide
group
metal salt
sulfate polysaccharide
Prior art date
Application number
PCT/CN2020/078075
Other languages
English (en)
French (fr)
Inventor
赵哲辉
雷平生
吴练秋
杨爽
王文杰
张海婧
李想
Original Assignee
中国医学科学院药物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国医学科学院药物研究所 filed Critical 中国医学科学院药物研究所
Priority to US17/436,675 priority Critical patent/US20220143074A1/en
Priority to JP2021552974A priority patent/JP2022531825A/ja
Priority to CN202080023949.7A priority patent/CN113631584B/zh
Priority to KR1020217031157A priority patent/KR20210151798A/ko
Priority to EP20765896.4A priority patent/EP3936527A4/en
Publication of WO2020177749A1 publication Critical patent/WO2020177749A1/zh
Priority to JP2023196288A priority patent/JP2024020460A/ja

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of medicine, in particular to a chondroitin sulfate polysaccharide metal salt, and a preparation method and application thereof.
  • Chondroitin sulfate polysaccharide is a linear, sulfated glycosaminoglycan, which is formed by alternately connecting glucose and galactosamine with ⁇ (1 ⁇ 3) and ⁇ (1 ⁇ 4) glycosidic bonds.
  • the 2nd and 3rd positions of the acid and the 4th and 6th positions of the galactosamine undergo different degrees of sulfation, which are derived into 11 natural subtypes.
  • the specific structures are as follows:
  • Chondroitin sulfate polysaccharide is widely found in animals and is also an endogenous substance in humans. It plays an important role in physiological and pathological processes such as nervous system, cancer and inflammation by regulating the expression levels of various enzymes and factors.
  • chondroitin sulfate drugs and health products are all derived from cartilage tissue extracts of terrestrial animals, including type A and a small amount of type C.
  • the chondroitin sulfate polysaccharides derived from marine animals are different. Its main components are often subtypes such as type C (shark cartilage extract) and type E (squid cartilage extract).
  • type C shark cartilage extract
  • type E squid cartilage extract
  • the technical problem solved by the present invention is to provide a chondroitin sulfate polysaccharide metal salt and its preparation method and application.
  • the chondroitin sulfate polysaccharide metal salt provided by the present invention has excellent pharmacological activity.
  • the present invention provides a chondroitin sulfate polysaccharide metal salt (formula I), which is electrically neutral as a whole, and includes chondroitin sulfate polysaccharide anion and metal cation;
  • the average molecular weight of the chondroitin sulfate polysaccharide anion is 1,000 to 15,000 Da;
  • the range of the chondroitin sulfate polysaccharide anion n is 2 ⁇ n ⁇ 45.
  • the average molecular weight of the chondroitin sulfate polysaccharide anion is 4000 to 15000 Da.
  • the metal cation includes sodium ion and/or calcium ion/or potassium ion.
  • the range of n of the chondroitin sulfate polysaccharide anion is 6 ⁇ n ⁇ 20.
  • the chondroitin sulfate polysaccharide corresponding to the chondroitin sulfate polysaccharide anion mainly contains chondroitin sulfate polysaccharide C, chondroitin sulfate polysaccharide E and chondroitin sulfate polysaccharide A, and also contains the balance of other subtypes of chondroitin sulfate Polysaccharides.
  • the total weight content of the chondroitin sulfate polysaccharide C, chondroitin sulfate polysaccharide E and chondroitin sulfate polysaccharide A is preferably 60-92%; the chondroitin sulfate polysaccharide The weight content of C is preferably 0-30%, more preferably 10-25%; the weight content of the chondroitin sulfate polysaccharide E is preferably 0-80%, more preferably 10-60%; the chondroitin sulfate polysaccharide The weight content of A is preferably 0 to 90%, more preferably 0 to 70%.
  • the weight content of -SO 3 - in the chondroitin sulfate polysaccharide anion is preferably 14-27%.
  • the weight content of uronic acid in the chondroitin sulfate polysaccharide anion is preferably 20 to 35%, and the weight content of hexosaminide is preferably 22 to 32%.
  • the metal cation preferably includes sodium ion and/or calcium ion and/or potassium ion, and more preferably sodium ion or calcium ion.
  • the second aspect of the technical solution of the present invention is to provide the method for preparing the chondroitin sulfate polysaccharide metal salt of the first aspect, which includes the following steps:
  • the sulfated product system is sequentially subjected to first sedimentation treatment, salt formation treatment, dialysis, second sedimentation treatment and gel column purification to obtain chondroitin sulfate polysaccharide metal salt;
  • the salt formation reagent used in the salt formation treatment is metal Hydroxide aqueous solution.
  • the polysaccharide chondroitin sulfate raw materials include chondroitin sulfate polysaccharide A and chondroitin sulfate polysaccharide C, wherein the weight content of chondroitin sulfate polysaccharide A is 70-90%, and the weight content of chondroitin sulfate polysaccharide C is 10-90%.
  • chondroitin sulfate polysaccharide material available from Yantai-East Pharmaceutical Group Co., chondroitin sulfate of the polysaccharide starting material average molecular weight 20000 ⁇ 23000Da, -SO 3 - and -COO -
  • the molar ratio is 1, the weight content of chondroitin sulfate polysaccharide A is 70-90%, and the weight content of chondroitin sulfate polysaccharide C is 10-30%, which meets the relevant requirements of the 2015 edition of the Chinese Pharmacopoeia.
  • the average molecular weight of the polysaccharide chondroitin sulfate raw material is 20,000-23,000 Da;
  • the molar ratio of -SO 3 - to -COO - in the polysaccharide chondroitin sulfate raw material is 0.9-1.1.
  • the sulfating reagent includes one or more of sulfur trioxide trimethylamine complex, sulfur trioxide pyridine complex, and sulfur trioxide triethylamine complex;
  • the equivalent ratio of the sulfation reagent to the repeating disaccharide fragments in the polysaccharide chondroitin sulfate raw material is (1-10):1. More preferably, it is (3-8):1.
  • the temperature of the sulfation reaction is 40 to 120° C., and the time is 2 to 36 hours; more preferably, 6 to 24 hours.
  • the sulfation reaction is preferably carried out under stirring conditions; the present invention has no special limitation on the stirring rate, and the stirring rate well known to those skilled in the art may be used. .
  • the salt-forming reagent is an aqueous sodium hydroxide solution and/or an aqueous potassium hydroxide solution; the concentration of the salt-forming reagent is 1 to 4 mol/L.
  • the first sedimentation reagent used in the first sedimentation treatment is an ethanol aqueous solution with a volume fraction of 90-95%;
  • the second sedimentation reagent used in the second sedimentation treatment is ethanol
  • the molecular weight cut-off of the dialysis bag used in the dialysis is 3000-12000 Da.
  • the time for the first sedimentation treatment is preferably 25 to 35 minutes, and the first sedimentation treatment is preferably performed at room temperature and under stirring conditions.
  • the present invention preferably filters the obtained system, dissolves the obtained solid material with water, and then mixes the obtained dissolved material with the salt-forming agent for salt-forming treatment.
  • the ratio of the amount of water used for dissolution to the solid material is preferably (0.8-1.2) mL:1g, more preferably 1mL:1g.
  • the salt-forming agent is preferably an aqueous sodium hydroxide solution and/or an aqueous potassium hydroxide solution, more preferably an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution; the concentration of the salt-forming agent is preferably 1 to 4 mol/ L is more preferably 2 to 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 refers to the total concentration of sodium hydroxide and potassium hydroxide.
  • the present invention has no special limitation on the addition amount of the salt-forming reagent, and it is preferable to ensure that the pH value of the system obtained after mixing the dissolved material and the salt-forming reagent is neutral, and the corresponding sodium salt and/or potassium salt can be obtained. .
  • the present invention After completing the salt-forming treatment, the present invention performs dialysis on the obtained solution, and the molecular weight cut-off of the dialysis bag used in the dialysis is preferably 3000 to 12000 Da, more preferably 8000 to 12000 Da.
  • the dialysis time is preferably 2 to 3 days, more preferably 2 days; the water is preferably changed every 12 hours during the dialysis process.
  • the present invention preferably rotates the obtained system, dissolves the obtained residue with sodium acetate aqueous solution (NaOAc aqueous solution), and then mixes the obtained dissolved material with ethanol and performs the second sedimentation treatment.
  • NaOAc aqueous solution sodium acetate aqueous solution
  • the mass concentration of the NaOAc aqueous solution is preferably 2 to 5%, more preferably 2 to 3%.
  • the use of the NaOAc aqueous solution to dissolve the residue in the present invention is to ensure that the residue is better soluble in ethanol and facilitate the subsequent second sedimentation treatment.
  • the dosage ratio of the residue, NaOAc aqueous solution and ethanol is preferably 1 g: (8-12) mL: (3.5-4.5) mL, more preferably 1 g: 10 mL: 4 mL.
  • the present invention preferably centrifuges the obtained system, and the obtained solid material is the crude chondroitin sulfate polysaccharide metal salt; in order to ensure that the final chondroitin sulfate polysaccharide metal salt has a higher purity, the present invention preferably The crude product of the chondroitin sulfate polysaccharide metal salt repeats the above-mentioned first sedimentation treatment, salt formation treatment, dialysis and second sedimentation treatment, and the obtained crude product is subjected to subsequent gel column purification.
  • the gel column used in the gel column purification is preferably a G25 gel column, and the eluent used in the gel column purification is preferably water.
  • the present invention preferably drying the obtained eluate to obtain the chondroitin sulfate polysaccharide metal salt.
  • the drying is preferably freeze-drying.
  • the present invention does not specifically limit the temperature and time of the freeze-drying, as long as the material can be fully dried.
  • the third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which contains the chondroitin sulfate polysaccharide metal salt described in the first aspect and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be prepared according to 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 adjuvants to prepare any dosage form suitable for human or animal use.
  • the weight content of the compound of the present invention in its pharmaceutical composition is usually 0.1-95%.
  • the compound of the present invention or a pharmaceutical composition containing it can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and Respiratory tract, skin, vagina, rectum, etc.
  • the dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w type, w/o type and double emulsion), suspensions, injections (including water injections, powder injections and infusions), eye drops Tablets, nasal drops, lotions and liniments;
  • solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, air (powder) sprays, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.
  • the compound of the present invention can be made into ordinary preparations, and can also be made into slow-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
  • the diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the humectant can be water, ethanol, iso Propanol, etc.
  • the binder can be starch syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.
  • the disintegrant can be dry starch, microcrystalline cellulose
  • the tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multilayer tablets.
  • the active ingredient of the compound of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard or soft capsule. It is also possible to prepare the compound of the present invention as an active ingredient with diluents, binders, and disintegrants into granules or pellets, and then place them in hard or soft capsules.
  • the various diluents, binders, wetting agents, disintegrants, and glidants used to prepare the compound tablets of the present invention can also be used to prepare the compound capsules of the present invention.
  • solubilizing agent or co-solvent can be poloxamer, lecithin, hydroxypropyl- ⁇ -cyclodextrin, etc.
  • the pH adjusting agent can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.
  • the osmotic pressure adjusting agent can be It is sodium chloride, mannitol, glucose, phosphate, acetate, etc.
  • mannitol, glucose, etc. can also be added as proppants.
  • coloring agents if necessary, coloring agents, preservatives, perfumes, flavoring agents or other additives can also be added to the pharmaceutical preparations.
  • the drug or pharmaceutical composition of the present invention can be administered by any known administration method.
  • the dosage of the compound pharmaceutical composition of the present invention can vary widely in accordance with the nature and severity of the disease to be prevented or treated, the individual conditions of the patient or animal, the route of administration, and the dosage form.
  • the appropriate daily dose range of the compound of the present invention is 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 can be administered in one dosage unit or divided into several dosage units, depending on the doctor's clinical experience and the dosage regimen including the use of other treatment methods.
  • the compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.
  • the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.
  • the fourth aspect of the technical solution of the present invention provides the application of the chondroitin sulfate polysaccharide metal in the first aspect of the present invention in the preparation of anti-inflammatory drugs.
  • the inflammatory diseases include osteoarthritis and rheumatoid arthritis.
  • the present invention provides a chondroitin sulfate polysaccharide metal salt.
  • the chondroitin sulfate polysaccharide metal salt provided by the present invention exerts an anti-inflammatory effect by inhibiting the NF- ⁇ B signaling pathway and simultaneously inhibiting the expression and function of inflammation-related factors and enzymes.
  • the results of the examples show that the chondroitin sulfate polysaccharide metal salt provided by the present invention has an anti-inflammatory effect and can be applied to prepare anti-inflammatory drugs.
  • the present invention provides a chondroitin sulfate polysaccharide metal salt.
  • the chondroitin sulfate polysaccharide metal salt provided by the present invention not only has anti-inflammatory activity, but also has the effect of improving bone density.
  • the results of the examples show that the chondroitin sulfate polysaccharide metal salt provided by the present invention has anti-inflammatory and bone protective effects, and can be applied to the preparation of anti-rheumatoid arthritis drugs.
  • the present invention provides a chondroitin sulfate polysaccharide metal salt.
  • the chondroitin sulfate polysaccharide metal salt provided by the present invention can increase the water content of cartilage and has a cartilage protection effect.
  • the results of the examples show that the chondroitin sulfate polysaccharide metal salt provided by the present invention has anti-inflammatory and chondroprotective effects, and can be applied to the preparation of anti-osteoarthritis drugs.
  • the compound of the present invention has outstanding anti-inflammatory activity in vivo, which exceeds that of natural source CS-E.
  • the sulfation degree of the compound in the present invention has an obvious structure-activity relationship with the anti-inflammatory activity in the body.
  • the compound of the present invention also shows outstanding biological activity and has obvious therapeutic effects.
  • the compounds of the present invention show anti-type I collagen-induced toe swelling (RA) animal models in mice and papain-induced arthritis (OA) models in rats. Highlight biological activity and have obvious therapeutic effects.
  • the present invention provides a preparation method of the chondroitin sulfate polysaccharide metal salt.
  • the present invention can obtain chondroitin sulfate polysaccharide metal salt with different sulfation degrees through semi-synthetic means, and the method is simple to operate and suitable for large-scale production.
  • Figure 1 is a high performance liquid chromatogram of polysaccharide 1 prepared in Example 1;
  • Figure 2 is a high performance liquid chromatogram of polysaccharide 2 prepared in Example 2;
  • Figure 3 is a high performance liquid chromatogram of polysaccharide 3 prepared in Example 3;
  • Figure 4 is a high performance liquid chromatogram of polysaccharide 4 prepared in Example 4.
  • Figure 5 is a comparison chart of weight changes of UC mice in different groups in Experimental Example 3. Note: ## p ⁇ 0.01vs.Con;*p ⁇ 0.05vs.Mod;
  • Figure 6 is a comparison diagram of colon length of UC mice in different groups in Experimental Example 3. Note: ## p ⁇ 0.01vs.Con;*p ⁇ 0.05vs.Mod;
  • Figure 7 is a comparison diagram of the DAI scores of mice in different groups in Experimental Example 3. Note: ## p ⁇ 0.01vs.Con;**p ⁇ 0.01,*p ⁇ 0.05vs.Mod.
  • Figure 8 shows the MRI images of the knee joints of rats in different groups in Experimental Example 5.
  • the joint space is normal, the articular cartilage surface is intact and undamaged, the subchondral bone is normal, the cartilage thickness is normal, and the amount of subpatellar fat is normal.
  • Joint synovium and a little joint synovial fluid MOD group showed narrowing of joint space, loss of articular cartilage surface, incomplete outer edge of cartilage, unclear cartilage shape, and reduced cartilage thickness
  • 5H group showed clear joint shape and joint space Back to normal, there is a little joint synovial fluid.
  • Figure 9 shows the CT images of the knee joints of rats in different groups in Test Example 5. Note: Figure 9 shows the morphology of the knee joints of normal rats in the CON group; in the MOD model group, the subchondral bones of the knee joints showed bone destruction, severe erosion, and joints Obvious cavities are formed in the area, and the bone and joints can be deformed; the POS positive drug group and the 5L group have less bone destruction and erosion than the model group; the 5M and 5H groups have bone destruction and erosion compared to the model group Obviously improved.
  • the saccharide chondroitin sulfate polysaccharide was prepared according to the steps of Example 1, except that in step (1), 6.7 g of the commercial polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and sulfur trioxide trimethylamine was added. Complex 8g (4eq.); react under stirring at 60°C for 24 hours. Finally, 4.4 g of chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 2) was obtained.
  • the saccharide chondroitin sulfate polysaccharide was prepared according to the steps of Example 1, except that in step (1), 6.7 g of the commercial polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and sulfur trioxide trimethylamine was added. Complex 10g (5eq.); reacted for 24 hours under stirring and 60°C conditions, the molecular weight cut-off of the dialysis bag was 7000Da, and finally 4.1g chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 3) was obtained.
  • the saccharide chondroitin sulfate polysaccharide was prepared according to the steps of Example 1, except that in step (1), 6.7 g of the commercial polysaccharide chondroitin sulfate A was dissolved in 90 mL of dimethyl sulfoxide, and sulfur trioxide trimethylamine was added. Complex 12g (6eq.); reacted for 20 hours under stirring and 70°C conditions, the molecular weight cut-off of the dialysis bag was 5000 Da, and finally 3.9g chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 4) was obtained.
  • the saccharide chondroitin sulfate polysaccharide was prepared according to the steps of Example 1, except that in step (1), 1.0 g of the commercial polysaccharide chondroitin sulfate A was dissolved in 13 mL of dimethyl sulfoxide, and sulfur trioxide trimethylamine was added. Complex 2.4g (8eq.); reacted for 36 hours at 100°C under stirring, the molecular weight cut-off of the dialysis bag was 3500Da, and finally 1.0g chondroitin sulfate polysaccharide sodium salt (denoted as polysaccharide 5) was obtained.
  • composition of the polysaccharides 1 to 5 prepared in Examples 1 to 5 was analyzed, including average molecular weight (Da), -SO 3 - / - COO-(molar ratio), -SO 3 - content, uronic acid content, amino hexyl Sugar content, anticoagulant titer (IU), degradable polysaccharide content and the ratio of each subtype component (use chondroitin sulfate ABC enzyme to degrade the polysaccharides 1 to 5 prepared in Examples 1 to 5, and test them by high performance liquid chromatography
  • the contained components of each subtype specifically refer to the ratio of polysaccharide chondroitin sulfate A, polysaccharide chondroitin sulfate C, and polysaccharide chondroitin sulfate E, wherein the polysaccharides 1 to 5 also contain the balance of other subtypes of chondroitin sulfate Polysaccharides are not further limited here; the high performance liquid chromatograms of
  • Test method Evaluation experiment of pharmacodynamic activity of CS compound in croton oil-induced acute swelling and inflammation of mouse ears
  • mice Balb/c mice, male (20-22g); 8 mice per group.
  • polysaccharides 2 to 5 showed good anti-inflammatory effects on mouse ear swelling models, which were better than natural source CS-E polysaccharides.
  • polysaccharide 3 exhibits obvious anti-inflammatory effects in the range of 50-200 mg/kg, and there is an obvious dose-effect relationship.
  • control group 0.5% sodium carboxymethyl cellulose by gavage.
  • SASP positive drug sulfasalazine group: Shanghai Xinyi Pharmaceutical Co., Ltd. (batch number: 036151102); 500 mg/kg, prepared with 0.5% sodium carboxymethyl cellulose, stored at 4°C, once a day Gavage.
  • SEMI5-50 group 50mg/kg, prepared with distilled water. Store at 4°C and give it by intragastric administration once a day.
  • SEMI5-150 group 150mg/kg, prepared with distilled water. Store at 4°C and give it by intragastric administration once a day.
  • mice C57BL/6J mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK ( ⁇ )2014-0023) for one week, and they were randomly divided into 5 groups according to the above scheme.
  • the mice in the UC model group and the administration group were modeled daily with DSS (MP, CA9011-18-1, US) according to the established laboratory model of ulcerative colitis.
  • the normal control group (control group) and the UC model group (model group) were intragastrically administered with 0.5% sodium carboxymethylcellulose by mass concentration, once a day.
  • the SASP group, SEMI5-50 group and SEMI5-150 group were administered intragastrically according to the experimental protocol in the "Experimental Grouping" section, once a day.
  • the animals in the UC model group showed typical UC lesions, such as fatigue, reduced activity, weight loss, loose stools, and blood in the stool.
  • the experiment was terminated, the animals in each group were sacrificed, and various related evaluation indexes of colitis were detected (see the follow-up experimental results section), and the anti-UC pharmacodynamic activity of each compound was comprehensively evaluated.
  • the animal disease comprehensive index DAI of the UC model group increased significantly, and the statistical difference was very significant, indicating that the model was successful.
  • the SEMI5-50 group and the SEMI5-150 group can significantly reduce the score of the experimental animal disease comprehensive index DAI, and the statistical difference is very significant.
  • the SASP group improved to a certain extent, and there were differences in statistical detection.
  • the DAI score is evaluated from the animal's weight loss, stool characteristics, blood in the stool and other indicators. The lower the DAI score, the closer the animal is to the normal physiological state. See Table 8 for the DAI scoring criteria.
  • 1Normal stool formed stool
  • 2Loose stool mushy, semi-formed stool that does not adhere to the anus
  • 3Large stool watery stool.
  • the polysaccharide 3 (indicated by SEMI5) prepared in Example 3 was evaluated against the model of anti-type I collagen-induced toe swelling in mice, as follows:
  • the measurement range is the bone density in the 1.00mm area of the joint.
  • mice in Table 9 According to the comparison of the end weight results of the mice in Table 9, it can be seen that the SEMI5 drug has a significant recovery effect on the weight loss of the mice.
  • Table 10 shows that mouse joints usually develop swelling at 4 weeks, from toes to soles and even ankle joints, reaching the peak of lesions at 5-6 weeks, and the mold rate can reach 100%. SEMI5 drugs can significantly improve joint swelling.
  • Table 11 indicates that SEMI5 drugs have a significant improvement effect on bone mineral density, and are better than positive drugs. The above results suggest that the semi-synthetic SEMI5 drug has anti-RA activity.
  • the polysaccharide 3 (represented by SEMI5) prepared in Example 3 was evaluated against papain-induced OA model, as follows:
  • Model group DDW gavage.
  • Positive drug celecoxib group purchased from Pfizer (W47055); prepared with DDW, stored at 4°C, and administered by intragastric administration once a day.
  • Compound semi5 low-dose group 50 mg/kg, prepared with DDW, stored at 4°C, and administered once a day by intragastric administration.
  • Compound semi5 medium-dose group 100 mg/kg, prepared with DDW, stored at 4°C, and administered once a day by intragastric administration.
  • Using a vernier caliper to measure the width of the skinned and fat-free knee joint can visually detect the degree of joint swelling.
  • CT can detect the subchondral bone of the rat knee joint and judge the remodeling of the subchondral bone.
  • the SEMI5-50 group can effectively reduce the weight loss of model animals, and show certain anti-UC activity in UC model animals' loose stools, blood in the stool and lower DAI scores, with statistically significant differences.
  • the above results indicate that SEMI5-50 has a significant therapeutic effect on DSS-induced UC animal models under this test system.
  • the SEMI5-150 group failed to reduce the weight loss of UC model animals. There was no statistically significant difference, but it could improve colon contracture. There was a statistically significant difference. In the UC model animals, loose stools, blood in the stool and decreased DAI and tissue scores all showed anti-UC activity, with statistically significant differences. The above results indicate that SEMI5-150 has a certain therapeutic effect on DSS-induced UC animal models under this test system.
  • SEMI5 can significantly improve joint swelling.
  • SEMI5 has a significant improvement effect on joint bone density, and it is better than positive drugs.
  • the above results suggest that semi-synthetic SEMI5 has anti-RA activity.
  • the joint space was normal, the articular cartilage surface was intact, the subchondral bone was normal, the cartilage thickness was normal, the amount of subpatellar fat, the joint synovium and a little joint synovial fluid were seen;
  • the MOD group showed that the joint space was narrowed and the joints The 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.
  • the joint shape was clear, the joint space returned to normal, and there was a little joint synovial fluid.
  • the MOD model group showed bone destruction of the subchondral bone of the knee joint, severe erosion, obvious cavity formation at the joint, and deformation of the bone and joint; POS positive drug group and SEMI5L group Compared with the model group, the bone destruction and erosion phenomenon is lighter; the SEMI5M group and the SEMI5H group have significantly improved bone destruction and erosion phenomenon compared with the model group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Dermatology (AREA)
  • Polymers & Plastics (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Materials Engineering (AREA)
  • Biochemistry (AREA)
  • Rheumatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pain & Pain Management (AREA)
  • Immunology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

本发明涉及医药技术领域，具体涉及硫酸软骨素多糖，其半合成制备方法和应用。本发明提供的硫酸软骨素多糖金属盐具有抗炎效果，能够应用于制备抗炎症疾病药物。具体而言,本发明提供的硫酸软骨素多糖金属盐具有抗炎、骨保护功效，能够应用于制备抗类风湿性关节炎疾病药物和制备抗骨关节炎疾病药物。本发明提供了所述硫酸软骨素多糖金属盐的制备方法，本发明通过半合成的手段，能够获得不同硫酸化程度的硫酸软骨素多糖金属盐，方法操作简单、适宜规模化生产。

Description

硫酸软骨素多糖，其半合成制备方法和应用 技术领域
本发明涉及医药技术领域，具体涉及一种硫酸软骨素多糖金属盐及其制备方法和应用。
背景技术
硫酸软骨素多糖(CS)是直链型、硫酸化的糖胺聚糖，由葡萄糖和半乳糖胺以β(1→3)和β(1→4)糖苷键交替连接形成，并在葡萄糖醛酸的2位和3位、半乳糖胺的4位和6位，发生不同程度的硫酸化，从而衍生为11种天然亚型，具体结构如下所示：
Figure PCTCN2020078075-appb-000001
硫酸软骨素多糖广泛存在于动物体内，也是一种人类内源物质，通过调控多种酶、因子的表达水平，在神经系统、癌症和炎症等生理和病理过程中发挥着重要作用。
目前上市的硫酸软骨素药品和保健品都来自于陆生动物的软骨组织提取物，包含A型和少量C型。海洋动物来源的硫酸软骨素多糖与之不同，其主成分常常为C型(鲨鱼软骨提取物)、E型(巨鱿软骨提取物)等亚型。研究表明，不同组成的硫酸软骨素多糖之间，常常表现出显著的药理活性差异，高药理活性的硫酸软骨素多糖药物的开发仍存在很大空白。
发明内容
本发明解决的技术问题在于提供一种硫酸软骨素多糖金属盐及其制备方法和应用，本发明提供的硫酸软骨素多糖金属盐具有优异的药理活性。
为了实现上述发明的技术问题，本发明提供以下技术方案：
本发明提供了一种硫酸软骨素多糖金属盐(式I)，整体呈电中性，包括硫酸软骨素多糖阴离子和金属阳离子；
Figure PCTCN2020078075-appb-000002
所述硫酸软骨素多糖阴离子的平均分子量为1000～15000Da；
所述硫酸软骨素多糖阴离子中-SO 3 -与-COO -的摩尔比值为1.46～2.73；优选的硫酸软骨素多糖阴离子中-SO 3 -与-COO -的摩尔比值为1.9～2.5。
所述硫酸软骨素多糖阴离子n的范围为2≤n≤45。
优选地，所述硫酸软骨素多糖阴离子的平均分子量为4000～15000Da。
优选地，所述金属阳离子包括钠离子和/或钙离子/或钾离子。
优选地，所述硫酸软骨素多糖阴离子的n的范围为6≤n≤20。
在本发明中，所述硫酸软骨素多糖阴离子对应的硫酸软骨素多糖中主要含有硫酸软骨素多糖C、硫酸软骨素多糖E和硫酸软骨素多糖A，还含有余量其它亚型的硫酸软骨素多糖。以所述硫酸软骨素多糖重量含量为100％计，所述硫酸软骨素多糖C、硫酸软骨素多糖E和硫酸软骨素多糖A的总重量含量优选为60～92％；所述硫酸软骨素多糖C的重量含量优选为0～30％，更优选为10～25％；所述硫酸软骨素多糖E的重量含量优选为0～80％，更优选为10～60％；所述硫酸软骨素多糖A的重量含量优选为0～90％，更优选为0～70％。
在本发明中，所述硫酸软骨素多糖阴离子中-SO 3 -的重量含量优选为14～27％。
在本发明中，硫酸软骨素多糖阴离子中糖醛酸的重量含量优选为20～35％，氨基己糖的重量含量优选为22～32％。
在本发明中，所述金属阳离子优选包括钠离子和/或钙离子和/或钾离子，更优选为钠离子或钙离子。
本发明技术方案的第二方面是提供了第一方面所述硫酸软骨素多糖金属盐的制备方法，包括以下步骤：
将多糖硫酸软骨素原料、硫酸化试剂和有机溶剂混合，进行硫酸化反应，得到硫酸化产物体系；
将所述硫酸化产物体系依次进行第一沉降处理、成盐处理、透析、第二沉降处理和凝胶柱纯化，得到硫酸软骨素多糖金属盐；所述成盐处理采用的成盐试剂为金属氢氧化物水溶液。
所述多糖硫酸软骨素原料中包括硫酸软骨素多糖A和硫酸软骨素多糖C，其中，所述硫酸软骨素多糖A的重量含量为70～90％，硫酸软骨素多糖C的重量含量为10～30％；在本发明中，采用的多糖硫酸软骨素原料购自烟台东诚药业集团股份有限公司，所述多糖硫酸软骨素原料的平均分子量是20000～23000Da，-SO 3 -与-COO -的摩尔比值为1，硫酸软骨素多糖A的重量含量为70～90％，硫酸软骨素多糖C的重量含量为10～30％，符合《中国药典》2015年版标准的相关要求。
所述多糖硫酸软骨素原料的平均分子量为20000～23000Da；
所述多糖硫酸软骨素原料中-SO 3 -与-COO -的摩尔比值为0.9～1.1。
所述硫酸化试剂包括三氧化硫三甲胺络合物、三氧化硫吡啶络合物和三氧化硫三乙胺络合物中的一种或几种；
所述硫酸化试剂与多糖硫酸软骨素原料中重复二糖片段的当量比为(1～10)：1。更优选为(3～8)：1。
所述硫酸化反应的温度为40～120℃，时间为2～36小时；更优选为6～24小时。在本发明中，所述硫酸化反应优选在搅拌条件下进行；本发明对于所述搅拌的速率没有特殊的限定，采用本领域技术人员熟知的搅拌速率即可。。
所述成盐试剂为氢氧化钠水溶液和/或氢氧化钾水溶液；所述成盐试剂的浓度为1～4mol/L。
所述第一沉降处理所采用的第一沉降试剂为体积分数为90～95％的乙醇水溶液；
所述第二沉降处理所采用的第二沉降试剂为乙醇；
所述透析所采用的透析袋的截留分子量为3000～12000Da。
本发明对于所述第一沉降试剂的添加方式、添加速度以及用量没有特殊的限定，能够将所述硫酸化产物体系中硫酸化产物完全沉降即可。在本发明中，所述第一沉降处理的时间优选为25～35分钟，所述第一沉降处理优选在室温、搅拌条件下进行。
完成所述第一沉降处理后，本发明优选将所得体系过滤，将所得固体物料加水溶解，然后将所得溶解物料与成盐试剂混合后进行成盐处理。在本发明中，所述溶解所用水与所述固体物料的用量比优选为(0.8～1.2)mL：1g，更优选为1mL：1g。在本发明中，所述成盐试剂优选为氢氧化钠水溶液和/或氢氧化钾水溶液，更优选为氢氧化钠水溶液或氢氧化钾水溶液；所述成盐试剂的浓度优选为1～4mol/L，更优选为2～3mol/L；当所述成盐试剂为氢氧化钠水溶液和氢氧化钾水溶液时，所述成盐试剂的浓度是指氢氧化钠和氢氧化钾的总浓度。本发明对于所述成盐试剂的添加量没有特殊的限定，优选能够保证所述溶解物料与成盐试剂混合后所得体系的pH值为中性，得到相应的钠盐和/或钾盐即可。
完成所述成盐处理后，本发明将所得溶液进行透析，所述透析所采用的透析袋的截留分子量优选为3000～12000Da，更优选为8000～12000Da。在本发明中，所述透析的时间优选为2～3天，更优选为2天；所述透析的过程中优选每隔12小时换一次水。
完成所述透析后，本发明优选将所得体系旋蒸，将所得剩余物加醋酸钠水溶液(NaOAc水溶液)溶解，然后将所得溶解物料与乙醇混合后进行第二沉降处理。在本发明中，所述NaOAc水溶液的质量浓度优选为2～5％，更优选为2～3％。本发明采用NaOAc水溶液溶解所述剩余物是为了保证所述剩余物更好地溶于乙醇，便于进行后续第二沉降处理。在本发明中，所述剩余物、NaOAc水溶液与乙醇的用量比优选为1g：(8～12)mL：(3.5～4.5)mL，更优选为1g：10mL：4mL。
完成所述第二沉降处理后，本发明优选将所得体系离心，所得固体物料即为硫酸软骨素多糖金属盐粗品；为了保证最终所得硫酸软骨素多糖金属盐具有较高的纯度，本发明优选将所述硫酸软骨素多糖金属盐粗品重复上述第一沉降处理、成盐处理、透析和第二沉降处理，将所得粗品进行后续凝胶柱纯化。在本发明中，所述凝胶柱纯化所采 用的凝胶柱优选为G25凝胶柱，所述凝胶柱纯化所采用的洗脱剂优选为水。
完成所述凝胶柱纯化后，本发明优选将所得洗脱液进行干燥，得到硫酸软骨素多糖金属盐。在本发明中，所述干燥优选为冷冻干燥，本发明对于所述冷冻干燥的温度和时间没有特殊的限定，能够将物料充分干燥即可。
本发明技术方案的第三方面是提供了一种药物组合物，该组合物含有第一方面所述的硫酸软骨素多糖金属盐和药学上可接受的载体。该药物组合物可根据本领域公知的方法制备。可通过将本发明化合物与一种或多种药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。本发明化合物在其药物组合物中的重量含量通常为0.1-95％。
本发明化合物或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括o/w型、w/o型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明化合物可以制成普通制剂、也制成是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将本发明化合物制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；湿润剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、 聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分本发明化合物与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分本发明化合物先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明化合物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明化合物的胶囊剂。
为将本发明化合物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、pH调剂剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调剂剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其它添加剂。
为达到用药目的，增强治疗效果，本发明的药物或药物组合物可用任何公知的给药方法给药。
本发明化合物药物组合物的给药剂量依照所要预防或治疗疾病的性质和严重程度，患者或动物的个体情况，给药途径和剂型等可以有大范围的变化。一般来讲，本发明化合物的每天的合适剂量范围为0.001-150mg/Kg体重，优选为0.1-100mg/Kg体重，更优选为1-60mg/Kg体重，最优选为2-30mg/Kg体重。上述剂量可以一个剂量单位或分成几个剂量单位给药，这取决于医生的临床经验以及包括运用其它治疗手段的给药方案。
本发明的化合物或组合物可单独服用，或与其他治疗药物或对症药物合并使用。当本发明的化合物与其它治疗药物存在协同作用时，应根据实际情况调整它的剂量。
本发明技术方案的第四方面提供了本发明第一方面所述硫酸软骨素多糖金属制备抗炎症疾病药物中的应用。所述的炎症疾病包括骨关节炎、类风湿性关节炎。
本发明提供了一种硫酸软骨素多糖金属盐，本发明提供的硫酸软骨素多糖金属盐通过抑制NF-κB信号通路、同时抑制与炎症相关的因子和酶的表达及功能，发挥抗炎作用。实施例的结果显示，本发明提供的硫酸软骨素多糖金属盐具有抗炎效果，能够应用于制备抗炎症疾病药物。
本发明提供了一种硫酸软骨素多糖金属盐，本发明提供的硫酸软骨素多糖金属盐不但具有抗炎活性，还具有提高骨密度功效。实施例的结果显示，本发明提供的硫酸软骨素多糖金属盐具有抗炎、骨保护功效，能够应用于制备抗类风湿性关节炎疾病药物。
本发明提供了一种硫酸软骨素多糖金属盐，本发明提供的硫酸软骨素多糖金属盐可以提高软骨含水量，具有软骨保护功效。实施例的结果显示，本发明提供的硫酸软骨素多糖金属盐具有抗炎、软骨保护功效，能够应用于制备抗骨关节炎疾病药物。
有益技术效果：本发明中的化合物当硫酸化程度较高时(硫酸根比羧酸根比值在一个特定范围内)，具有突出的体内抗炎活性，超过天然来源CS-E。本发明中的化合物硫酸化程度与体内抗炎活性存在明显构效关系。在急性溃疡性直肠炎动物模型中，本发明的化合物也表现出突出的生物活性，有明显治疗作用。相比于天然来源CS-A和CS-E，本发明的化合物在抗I型胶原诱导小鼠足趾肿胀(RA)动物模型、木瓜蛋白酶诱导大鼠关节炎(OA)模型中，均表现出突出生物活性，具有明显治疗作用。
本发明提供了所述硫酸软骨素多糖金属盐的制备方法，本发明通过半合成的手段，能够获得不同硫酸化程度的硫酸软骨素多糖金属盐，方法操作简单、适宜规模化生产。
附图说明
图1为实施例1制备的多糖1的高效液相色谱图；
图2为实施例2制备的多糖2的高效液相色谱图；
图3为实施例3制备的多糖3的高效液相色谱图；
图4为实施例4制备的多糖4的高效液相色谱图；
图5为实验例3中不同组别中UC小鼠体重变化对比图，注： ##p<0.01vs.Con；*p<0.05vs.Mod；
图6为实验例3中不同组别中UC小鼠结肠长度对比图，注： ##p<0.01vs.Con；*p<0.05vs.Mod；
图7为实验例3中不同组别中小鼠DAI评分对比图，注： ##p<0.01vs.Con；**p<0.01，*p<0.05vs.Mod。
图8为试验例5中不同组别中大鼠膝关节核磁图像，注：图8中CON组可见关节间隙正常，关节软骨面完整无破损，软骨下骨正常，软骨厚度正常，适量髌下脂肪，关节滑膜且有少许关节滑液；MOD组可见关节间隙变窄，关节软骨面受损缺失，软骨外侧边缘不完整，软骨形态不清晰，软骨厚度降低；5H组可见关节形态清晰，关节间隙恢复正常，有少许关节滑液。
图9为试验例5中不同组别中大鼠膝关节CT图，注：图9为CON组正常大鼠膝关节形态；MOD模型组可见膝关节软骨下骨出现骨质破坏，侵蚀严重，关节处有明显空洞形成，骨关节可见变形；POS阳性药组和5L组，与模型组相比骨质破坏及侵蚀现象较轻；5M组和5H组，与模型组相比骨质破坏及侵蚀现象得到明显改善。
具体实施方式
实施例1
(1)将商业化的多糖硫酸软骨素A(烟台东诚药业集团股份有限公司，NO.CSJ1170701)26.8g溶解在二甲基亚砜360mL中，加入三氧化硫三甲胺络合物24g(3eq.)，在搅拌、50℃条件下反应6小时；
(2)将所得反应体系冷却至室温，加入体积分数为95％的乙醇水溶液400mL，搅拌30min后，过滤，固体物料加水溶解(水与固体物料的用量比为1mL：1g)，用2mol/L的NaOH水溶液调节pH值为7.0，所得溶液装入透析袋(截留分子量为12000Da)，透析2天后旋蒸得到固体；
(3)将所得固体溶解于质量浓度2％的NaOAc水溶液中，然后加入无水乙醇，充分沉淀后离心，得到粗品；其中，所述固体、NaOAc水溶液和无水乙醇的用量比为1g：10mL：40mL；
(4)将所得粗品再次重复步骤(2)和步骤(3)，然后将所得物料在G25凝胶柱上纯化(采用的洗脱剂为H 2O)，最后将所得洗脱液冻干，得到21.3g硫酸软骨素多糖钠盐(记为多糖1)。
实施例2
按照实施例1的步骤制备糖硫酸软骨素多糖，不同之处在于步骤(1)中，将商业化的多糖硫酸软骨素A 6.7g溶解在二甲基亚砜90mL中，加入三氧化硫三甲胺络合物8g(4eq.)；在搅拌、60℃条件下反应24小时。最终得到4.4g硫酸软骨素多糖钠盐(记为多糖2)。
实施例3
按照实施例1的步骤制备糖硫酸软骨素多糖，不同之处在于步骤(1)中，将商业化的多糖硫酸软骨素A 6.7g溶解在二甲基亚砜90mL中，加入三氧化硫三甲胺络合物10g(5eq.)；在搅拌、60℃条件下反应24小时，透析袋的截留分子量为7000Da，最终得到4.1g硫酸软骨素多糖钠盐(记为多糖3)。
实施例4
按照实施例1的步骤制备糖硫酸软骨素多糖，不同之处在于步骤(1)中，将商业化的多糖硫酸软骨素A 6.7g溶解在二甲基亚砜90mL中，加入三氧化硫三甲胺络合物12g(6eq.)；在搅拌、70℃条件下反应20小时，透析袋的截留分子量为5000Da，最终得到3.9g硫酸软骨素多糖钠盐(记为多糖4)。
实施例5
按照实施例1的步骤制备糖硫酸软骨素多糖，不同之处在于步骤(1)中，将商业化的多糖硫酸软骨素A 1.0g溶解在二甲基亚砜13mL中，加入三氧化硫三甲胺络合物2.4g(8eq.)；在搅拌、100℃条件 下反应36小时，透析袋的截留分子量为3500Da，最终得到1.0g硫酸软骨素多糖钠盐(记为多糖5)。
药理实验
实验例1
对实施例1～5制备的多糖1～5的组成进行分析，包括平均分子量(Da)、-SO 3 -/-COO -(摩尔比值)、-SO 3 -含量、糖醛酸含量、氨基己糖含量、抗凝血效价(IU)、可降解多糖含量和各亚型成分的比例(用硫酸软骨素ABC酶降解实施例1～5制备的多糖1～5，并用高效液相色谱测试其所包含的各亚型成分，具体指多糖硫酸软骨素A、多糖硫酸软骨素C和多糖硫酸软骨素E的比例，其中，所述多糖1～5中还含有余量其它亚型的硫酸软骨素多糖，在此不做进一步限定；多糖1～4的高效液相色谱图如图1～4所示)，具体结果见表1。
表1实施例1～5制备的多糖1～5的组成分析结果
Figure PCTCN2020078075-appb-000003
实验例2
对实施例1～5制备的多糖1～5的抗炎效果进行评价，具体如下：
(1)试验方法：CS化合物在巴豆油致小鼠耳部急性肿胀炎症模型中的药效学活性评价实验
动物：Balb/c小鼠，雄性(20～22g)；每组8只。
分组：模型组、阳性药吲哚美辛组(5mg/kg)、天然提取CS-E组(200mg/kg)、多糖组(200mg/kg，100mg/kg，50mg/kg)。阳性药和CS系列化合物均以质量浓度0.5％羧甲基纤维素钠配制，4℃冰箱保存。
给药和测试：每天1次，给药3天。末次给药后，计算化合物致小鼠耳肿率(％)及其对耳肿胀程度的抑制率(％)。
统计分析：实验结果以“均值±标准差”表示。两组间统计学差异采用t检验方法计算分析。*，表示p<0.05，**，表示p<0.01。
(2)多糖的抗炎效果见表2～4。
表2多糖的抗炎效果
Figure PCTCN2020078075-appb-000004
表3多糖的抗炎效果
Figure PCTCN2020078075-appb-000005
表4多糖的抗炎效果
Figure PCTCN2020078075-appb-000006
Figure PCTCN2020078075-appb-000007
由表2～4可知，多糖1-5的硫酸化程度与抗炎活性存在构效关系。多糖2～5在小鼠耳肿模型上表现出了良好的抗炎效果，优于天然来源CS-E多糖。特别是多糖3，其在50～200mg/kg范围内均表现出明显的抗炎作用，存在明显量效关系。
实验例3
对实施例3制备的多糖3(用SEMI5表示)进行抗DSS模型药效学评价，具体如下：
一、材料和方法
1、实验动物
C57BL/6J小鼠，雄性(18～20g)，每组6只。购自北京华阜康生物科技股份有限公司；许可证号：SCXK(京)2014-0004。
2、实验分组
(1)正常对照组(control组)：质量浓度0.5％羧甲基纤维素钠灌胃。
(2)UC(Ulcerative Colitis，溃疡性结肠炎)模型组(model组)：质量浓度0.5％羧甲基纤维素钠灌胃。
(3)SASP(阳性药柳氮磺吡啶)组：上海信谊药厂有限公司(批号：036151102)；500mg/kg，质量浓度0.5％羧甲基纤维素钠配制，4℃保存，每日一次灌胃给药。
(4)SEMI5-50组：50mg/kg，蒸馏水配制。4℃保存，每日一次灌胃给药。
(5)SEMI5-150组：150mg/kg，蒸馏水配制。4℃保存，每日一次灌胃给药。
3、实验方法
C57BL/6J小鼠于SPF级动物房(实验动物使用许可证编号：SYXK(京)2014-0023)适应性饲养一周后，随机按以上方案分为5组。UC模型组及给药组小鼠每天以DSS(MP，CA9011-18-1，US)按实验室已建溃疡性结肠炎造模方法建模。正常对照组(control组)及UC模型组(model组)以质量浓度0.5％羧甲基纤维素钠灌胃，每日一次。SASP组、SEMI5-50组及SEMI5-150组按照“实验分组”部分中的实验方案灌胃给药，每日一次。造模7天后，UC模型组动物出现明显精神萎靡、活动减少、体重下降、稀便、便血等UC典型病变。终止实验，处死各组动物，并检测结肠炎各项相关评价指标(见后续实验结果部分)，综合评价各化合物的抗UC药效学活性。
二、实验结果
1、SEMI5各浓度对DSS诱导UC模型小鼠体重的影响(表5及图5)。
表5不同组别中UC小鼠体重变化数据
Figure PCTCN2020078075-appb-000008
Figure PCTCN2020078075-appb-000009
##p<0.01vs.Con；*p<0.05vs.Mod
由表5和图5可知，与正常对照组相比，UC模型组小鼠体重明显降低，统计学上差异显著，提示UC造模成功。SASP组和SEMI5-150组未能有效缓解DSS诱导的急性UC C57BL/6J小鼠的体重降低，而SEMI5-50组与UC模型组相比有效缓解了体重降低，统计学检测差异显著。
2、SEMI5各浓度对UC小鼠结肠挛缩的影响(表6及图6)。
表6不同组别中UC小鼠结肠挛缩对比数据
Figure PCTCN2020078075-appb-000010
##p<0.01vs.Con；*p<0.05vs.Mod
由表6和图6可知，在DSS诱导C57BL/6J小鼠急性UC动物模型上，与正常对照组相比，UC模型组小鼠结肠明显挛缩变短，统计学上差异非常显著。SASP组未能有效改善模型动物的结肠挛缩。而SEMI5-50组、SEMI5-150组与UC模型组相比能有效改善其结肠挛缩，统计学上有显著性差异。
3、SEMI5各浓度对UC小鼠疾病综合指数DAI评分的影响(表7及图7)。
表7不同组别中疾病活动指数DAI及综合指数抑制率的对比数据
Figure PCTCN2020078075-appb-000011
Figure PCTCN2020078075-appb-000012
##p<0.01vs.Con；**p<0.01，*p<0.05vs.Mod
由表7与图7可知，与正常对照组相比，UC模型组动物疾病综合指数DAI明显增加，统计学上差异非常显著，提示造模成功。与UC模型组相比，SEMI5-50组和SEMI5-150组能明显降低实验动物疾病综合指数DAI的评分，统计学上差异非常显著。SASP组一定程度上改善,统计学检测有差异性。其中，DAI评分从动物体重下降程度、大便性状、便血等指标考核，DAI评分越低，提示越接近动物正常生理状态。DAI评分标准见表8。
表8 DAI评分标准
Figure PCTCN2020078075-appb-000013
注：①正常大便：成形大便；②松散大便：不粘附于肛门的糊状、半成形大便；③稀便：稀水样便。
实验例4
对实施例3制备的多糖3(用SEMI5表示)进行抗I型胶原诱导小鼠足趾肿胀模型评价，具体如下：
一、材料和方法
1动物：DBAI小鼠，雄性(20-22g)；每组7只。
2分组：空白对照组、模型组、阳性药吲哚美辛组(5mg/kg)、CSA组(200mg/kg)、CSE组(200mg/kg)、CS-E-semi3组(200mg/kg)和CS-Semi-5组(200mg/kg)。阳性药和CS系列化合物均以0.5％羧甲基纤维素钠配制，4℃冰箱保存。
3给药次数:每天1次，给药3天。
4实验方法：按照实验室已建方法，初次免疫后，每周测量各组小鼠的体重。实验21天二次冲击免疫后，每周观察各组小鼠的关节肿胀程度并进行关节肿胀度指数评分。
5统计分析：实验结果以“均值±标准差”表示。两两组间统计学差异采用t检验方法计算分析。*，表示p〈0.05，**，表示p<0.01。
二实验结果
表9、不同药物对小鼠体重的影响
Figure PCTCN2020078075-appb-000014
##，表示p<0.01vs.正常对照组。*，表示p<0.05，**，表示p<0.01vs.CIA模型组。
表10、不同药物对小鼠关节指数评分影响
Figure PCTCN2020078075-appb-000015
Figure PCTCN2020078075-appb-000016
##，表示p<0.01vs.正常对照组。*，表示p<0.05，**，表示p<0.01vs.CIA模型组。数值以“mean±sem”表示。
表11、小鼠足趾部骨密度检测结果
Figure PCTCN2020078075-appb-000017
测量范围为关节处1.00mm区域内的骨密度。
##，表示p<0.01vs.正常对照组。*，表示p<0.05，**，表示p<0.01vs.CIA模型组。
根据表9的小鼠结束体重结果对比可以看出，SEMI5药物对小鼠的体重降低有明显的恢复作用。表10可以得出小鼠关节通常在4w时出现肿胀，从足趾向足掌乃至踝关节发展，5w-6w时达到病变高峰，成模率可达100％。SEMI5药物对关节肿胀有明显的改善作用。表11提示SEMI5药物对骨密度有明显改善作用，且较阳性药的改善作用好。以上结果提示半合成SEMI5药物具有抗RA活性。
实验例5
对实施例3制备的多糖3(用SEMI5表示)进行抗木瓜蛋白酶诱导的OA模型评价，具体如下：
一材料和方法：
1动物：
SD大鼠，雄性(180-220g)；每组6只；购自北京维通利华实验动物技术有限公司；许可证号：SCXK(京)2012-0001。
2分组：
(1)正常对照组(Con组)：DDW灌胃。
(2)模型组(Mod组)：DDW灌胃。
(3)阳性药塞来昔布组(Pos组):购自辉瑞(W47055)；DDW配制，4℃保存，每日一次灌胃给药。
(4)化合物semi5低剂量组(5L组):50mg/kg，DDW配制，4℃保存，每日一次灌胃给药。
(5)化合物semi5中剂量组(5M组):100mg/kg，DDW配制，4℃保存,每日一次灌胃给药。
(6)化合物semi5高剂量组(5H组):200mg/kg，DDW配制，4℃保存,每日一次灌胃给药。
二实验结果
1膝关节净宽度
使用游标卡尺测量去皮去脂肪的膝关节宽度，可以直观地检测出关节的肿胀程度。
表12、各组膝关节净宽度
Figure PCTCN2020078075-appb-000018
注：模型组(Mod)动物的膝关节净宽度相比于正常对照组(Con)明显增加，统计学上差异非常显著，提示模型组动物关节出现明显肿胀；阳性药组(Pos)相比于模型组无统计学差异； 各给药组动物的膝关节净宽度相比于模型组(Mod)具有统计学差异，提示化合物具有较好的抗炎活性。**p<0.01vs Con； ##p<0.01vs Mod。
2核磁结果
通过核磁检测大鼠膝关节软骨的损伤程度(见图8)，并计算相应条件下所测得的软骨灰度值的变化数据，反应出各组关节软骨的含水量，含水量越高提示关节软骨越接近正常生理状态(见表13)。
表13、膝关节灰度值(n＝6)
Figure PCTCN2020078075-appb-000019
表13中可见模型组(Mod)动物的灰度值相比于正常对照组(Con)统计学差异非常显著，提示软骨破坏明显造模成功；给药高剂量组动物的灰度值相比于模型组(Mod)统计学上差异非常显著。以上结果提示模型组软骨破坏严重，而给药组具有明显的改善效应。**p<0.01vs Con；##p<0.01vs Mod。
3CT检测结果
CT可检测大鼠膝关节的软骨下骨，判断软骨下骨重塑情况。
三、实验结论
1、抗DSS诱导急性溃疡性结肠炎模型药效学评价
1)、UC模型组动物出现明显的体重降低、便血、稀便、结肠挛缩、DAI综合指数评分和结肠组织病理评分升高，提示DSS诱导小鼠溃疡性结肠炎模型造模成功，体系可靠，适于进行抗UC化合物的活性评价。
2)、SASP组，本次实验未能有效减少UC模型动物的体重降低，但是能够改善UC模型动物稀便、便血、结肠挛缩等现象，提示SASP具有一定抗UC活性。
3)、SEMI5-50组能有效减轻模型动物的体重降低，且在UC模型动物稀便、便血以及降低DAI评分等方面显示出一定的抗UC活性，统计学上有显著性差异。以上结果说明，在本测试体系下SEMI5-50对DSS诱导的UC动物模型有明显治疗作用。
4)、SEMI5-150组未能减轻UC模型动物的体重降低，统计学上无显著性差异，但能改善结肠挛缩，统计学上有显著性差异，且在UC模型动物稀便、便血以及降低DAI和组织评分等方面均显示出抗UC活性，统计学上有显著性差异。以上结果说明，在本测试体系下SEMI5-150对DSS诱导的UC动物模型具有一定的治疗作用。
2、抗I型蛋白诱导小鼠足趾肿胀模型药效学评价
1)、小鼠结束体重结果对比可以看出，SEMI5对关节炎小鼠的体重降低有明显的恢复作用。
2)、SEMI5对关节肿胀有明显的改善作用。
3)、SEMI5对关节骨密度有明显改善作用，且较阳性药的改善作用好。以上结果提示半合成SEMI5具有抗RA活性。
3、抗木瓜蛋白酶诱导大鼠骨关节炎模型药效学评价
1)、模型组(Mod)动物的膝关节净宽度相比于正常对照组(Con)明显增加，统计学上差异非常显著，提示模型组动物关节出现明显肿胀；阳性药组(Pos)相比于模型组无统计学差异；各给药组动物的膝关节净宽度相比于模型组(Mod)具有统计学差异，提示SEMI5具有较好的抗炎活性。
2)、CON组可见关节间隙正常，关节软骨面完整无破损，软骨下骨正常，软骨厚度正常，适量髌下脂肪，关节滑膜且有少许关节滑液；MOD组可见关节间隙变窄，关节软骨面受损缺失，软骨外侧边缘不完整，软骨形态不清晰，软骨厚度降低；SEMI5H组可见关节形态清晰，关节间隙恢复正常，有少许关节滑液。
3)、模型组(Mod)动物的灰度值相比于正常对照组(Con)统计学差异非常显著，提示软骨破坏明显造模成功；给药SEMI5高剂量组动物的灰度值相比于模型组(Mod)统计学上差异非常显著。以上结果提示模型组软骨破坏严重，而给药组具有明显的改善效应。
4)、对比于CON组正常大鼠膝关节形态，MOD模型组可见膝关节软骨下骨出现骨质破坏，侵蚀严重，关节处有明显空洞形成，骨关节可见变 形；POS阳性药组和SEMI5L组，与模型组相比骨质破坏及侵蚀现象较轻；SEMI5M组和SEMI5H组，与模型组相比骨质破坏及侵蚀现象得到明显改善。
以上结果提示半合成SEMI5具有抗OA活性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (12)

  1. 一类如通式(I)所示的硫酸软骨素多糖金属盐，具有以下的结构通式，
    Figure PCTCN2020078075-appb-100001
    整体呈电中性，包括硫酸软骨素多糖阴离子和金属阳离子；
    所述硫酸软骨素多糖阴离子的平均分子量为1000～15000Da；
    所述硫酸软骨素多糖阴离子中-SO 3 -与-COO -的摩尔比值为1.46～2.73。
    所述硫酸软骨素多糖阴离子n的范围为2≤n≤45。
  2. 根据权利要求1所述的硫酸软骨素多糖金属盐，其特征在于，所述硫酸软骨素多糖阴离子的平均分子量为4000～15000Da；所述硫酸软骨素多糖阴离子中-SO 3 -与-COO -的摩尔比值为1.9～2.5。
  3. 根据权利要求1或2任一项所述的硫酸软骨素多糖金属盐，其特征在于，所述金属阳离子包括钠离子和/或钙离子和/或钾离子。
  4. 权利要求1～3任一项所述硫酸软骨素多糖金属盐的制备方法，包括以下步骤：
    将多糖硫酸软骨素原料、硫酸化试剂和有机溶剂混合，进行硫酸化反应，得到硫酸化产物体系；
    将所述硫酸化产物体系依次进行第一沉降处理、成盐处理、透析、第二沉降处理和凝胶柱纯化，得到硫酸软骨素多糖金属盐；所述成盐处理采用的成盐试剂为金属氢氧化物水溶液。
  5. 根据权利要求4所述的制备方法，其特征在于，所述多糖硫酸软骨素原料中包括硫酸软骨素多糖A和硫酸软骨素多糖C，其中，所述硫酸软骨素多糖A的重量含量为70～90％，硫酸软骨素多糖C的重量含量为10～30％；
    所述多糖硫酸软骨素原料的平均分子量为20000～23000Da；
    所述多糖硫酸软骨素原料中-SO 3 -与-COO -的摩尔比值为0.9～1.1。
  6. 根据权利要求4或5任一项所述的制备方法，其特征在于，
    所述硫酸化试剂包括三氧化硫三甲胺络合物、三氧化硫吡啶络合物和三氧化硫三乙胺络合物中的一种或几种；
    所述硫酸化试剂与多糖硫酸软骨素原料中重复二糖片段的当量比为(1～10)：1。
  7. 根据权利要求6所述的制备方法，其特征在于，所述硫酸化反应的温度为40～120℃，时间为2～36小时；所述硫酸化试剂与多糖硫酸软骨素原料中重复二糖片段的当量比为(3-8)：1。
  8. 根据权利要求4所述的制备方法，其特征在于，所述成盐试剂为氢氧化钠水溶液和/或氢氧化钾水溶液；所述成盐试剂的浓度为1～4mol/L。
  9. 根据权利要求4所述的制备方法，其特征在于，所述第一沉降处理所采用的第一沉降试剂为体积分数为90～95％的乙醇水溶液；
    所述第二沉降处理所采用的第二沉降试剂为乙醇；
    所述透析所采用的透析袋的截留分子量为3000～12000Da。
  10. 一种药物组合物，其特征在于，所述的药物组合物包含权利要求1-3任一项所述的硫酸软骨素多糖金属盐以及药学上可接受的载体或赋形剂。
  11. 权利要求1～3任一项所述硫酸软骨素多糖金属盐在制备抗炎症疾病药物中的应用。
  12. 根据权利要求11的应用，其特征在于，所述的炎症疾病包括溃疡性结肠炎、骨关节炎和类风湿性关节炎。
PCT/CN2020/078075 2019-03-05 2020-03-05 硫酸软骨素多糖，其半合成制备方法和应用 WO2020177749A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US17/436,675 US20220143074A1 (en) 2019-03-05 2020-03-05 Chondroitin sulfate polysaccharide, and semi-synthetic preparation method therefor and use thereof
JP2021552974A JP2022531825A (ja) 2019-03-05 2020-03-05 硫酸コンドロイチン多糖類、その半合成調製方法および使用
CN202080023949.7A CN113631584B (zh) 2019-03-05 2020-03-05 硫酸软骨素多糖,其半合成制备方法和应用
KR1020217031157A KR20210151798A (ko) 2019-03-05 2020-03-05 콘드로이틴 설페이트 다당류, 이의 반합성 제조방법 및 이의 용도
EP20765896.4A EP3936527A4 (en) 2019-03-05 2020-03-05 CHONDROITIN SULPHATE POLYSACCHARIDE AND SEMI-SYNTHETIC PROCESS FOR ITS PRODUCTION AND ITS USE
JP2023196288A JP2024020460A (ja) 2019-03-05 2023-11-17 硫酸コンドロイチン多糖類、その半合成調製方法および使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910163208.8 2019-03-05
CN201910163208.8A CN111662394B (zh) 2019-03-05 2019-03-05 硫酸软骨素多糖半合成制备方法和应用

Publications (1)

Publication Number Publication Date
WO2020177749A1 true WO2020177749A1 (zh) 2020-09-10

Family

ID=72337668

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/078075 WO2020177749A1 (zh) 2019-03-05 2020-03-05 硫酸软骨素多糖，其半合成制备方法和应用

Country Status (6)

Country Link
US (1) US20220143074A1 (zh)
EP (1) EP3936527A4 (zh)
JP (2) JP2022531825A (zh)
KR (1) KR20210151798A (zh)
CN (2) CN111662394B (zh)
WO (1) WO2020177749A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111662394B (zh) * 2019-03-05 2022-11-04 中国医学科学院药物研究所 硫酸软骨素多糖半合成制备方法和应用
WO2023250170A2 (en) * 2022-06-24 2023-12-28 The Board Of Regents Of The University Of Oklahoma Heparanase inhibitors and methods of use
CN115353574B (zh) * 2022-09-06 2023-06-13 福建省深蓝生物科技有限公司 高硫酸化度的硫酸软骨素cs-e及其提取方法
WO2024072942A2 (en) * 2022-09-29 2024-04-04 Adora Animal Health Corporation Skin penetrating formulations of sulfated glycosaminoglycans and fragments derived therefrom for the treatment of pain and other medical conditions

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0340628A2 (en) * 1988-05-02 1989-11-08 Crinos Industria Farmacobiologica S.p.A. Sulfoamino derivatives of chondroitin sulfates, of dermaten sulfate and of hyaluronic acid and their pharmacological properties
US5384398A (en) * 1991-11-28 1995-01-24 Elf Sanofi High molecular mass N,O-sulphated heparosans, process for their preparation and the pharmaceutical compositions which contain them
WO2016159296A1 (ja) * 2015-03-31 2016-10-06 生化学工業株式会社 グリコサミノグリカンの硫酸化方法

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000309537A (ja) * 1999-04-28 2000-11-07 Seikagaku Kogyo Co Ltd 腸疾患予防及び／または治療剤
WO2001051003A2 (en) * 2000-01-10 2001-07-19 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of disease
CN1470533A (zh) * 2003-05-07 2004-01-28 毅 汤 低分子量硫酸软骨素的制备方法
CN1486993A (zh) * 2003-08-19 2004-04-07 毅 汤 中分子量硫酸软骨素的制备方法
CN100389777C (zh) * 2003-08-19 2008-05-28 汤毅 中分子量硫酸软骨素注射剂及其制备方法
CN1273492C (zh) * 2003-12-04 2006-09-06 中国药科大学 一种多糖及其制法和用途
JP2006327955A (ja) * 2005-05-24 2006-12-07 Taisho Pharmaceut Co Ltd 低分子量コンドロイチン硫酸を含む医薬
CN1789287A (zh) * 2005-12-20 2006-06-21 山东大学 一种多硫酸化硫酸软骨素及其制备方法
WO2009044765A1 (ja) * 2007-10-01 2009-04-09 Seikagaku Corporation 新規な低分子化コンドロイチン硫酸およびその用途
JP6026717B2 (ja) * 2010-08-02 2016-11-16 クラシエホームプロダクツ株式会社 ヒアルロン酸産生促進剤
FR2969618B1 (fr) * 2010-12-28 2014-05-16 Pf Medicament Procede de preparation de chondroitine sulfate de sodium
DK2707396T3 (en) * 2011-05-12 2019-01-28 Gnosis Spa Biotechnological sulfated chondroitin sulfate at position 4 or 6 on the same polysaccharide chain and process for its preparation
CN103735572A (zh) * 2012-10-17 2014-04-23 山东明仁福瑞达制药股份有限公司 一种具有增加骨密度、抗炎镇痛作用的组合物及其制备方法
CN105399864A (zh) * 2015-12-23 2016-03-16 青岛九龙生物医药有限公司 高纯度低分子量硫酸软骨素钠的制备工艺
CN105924544B (zh) * 2016-05-16 2018-03-09 山东大学 一种高硫酸化硫酸软骨素及其制备方法与应用
CN106349407A (zh) * 2016-08-29 2017-01-25 中国海洋大学 一种低分子量岩藻糖化硫酸软骨素及其制备方法和在制备抗特鲁索综合征的药物中的应用
CN108484795B (zh) * 2018-04-28 2021-02-09 山东第一医科大学第二附属医院 硫酸软骨素锂及其制备方法和应用
CN111662394B (zh) * 2019-03-05 2022-11-04 中国医学科学院药物研究所 硫酸软骨素多糖半合成制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0340628A2 (en) * 1988-05-02 1989-11-08 Crinos Industria Farmacobiologica S.p.A. Sulfoamino derivatives of chondroitin sulfates, of dermaten sulfate and of hyaluronic acid and their pharmacological properties
US5384398A (en) * 1991-11-28 1995-01-24 Elf Sanofi High molecular mass N,O-sulphated heparosans, process for their preparation and the pharmaceutical compositions which contain them
WO2016159296A1 (ja) * 2015-03-31 2016-10-06 生化学工業株式会社 グリコサミノグリカンの硫酸化方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3936527A4 *

Also Published As

Publication number Publication date
CN113631584A (zh) 2021-11-09
EP3936527A1 (en) 2022-01-12
CN111662394B (zh) 2022-11-04
EP3936527A4 (en) 2022-11-23
JP2022531825A (ja) 2022-07-12
KR20210151798A (ko) 2021-12-14
US20220143074A1 (en) 2022-05-12
CN111662394A (zh) 2020-09-15
JP2024020460A (ja) 2024-02-14
CN113631584B (zh) 2023-09-29

Similar Documents

Publication Publication Date Title
WO2020177749A1 (zh) 硫酸软骨素多糖，其半合成制备方法和应用
AU2021383828A1 (en) Compositions of peptide inhibitors of interleukin-23 receptor
WO2018040989A1 (zh) 依达拉奉与(+)-2-莰醇的舌下用药物组合物
JP6158259B2 (ja) 高硫酸化二糖製剤
CA2449962A1 (en) Use of glycosaminoglycans for the treatment of hiv-associated nephropathy
EA009944B1 (ru) Пероральные препаративные формы кладрибина
JPH05504785A (ja) 新規なへパリン誘導体
KR20130040825A (ko) 과황산화 글루코피라노사이드
US6750249B1 (en) Controlled release oral preparations of esculetin and its derivatives
JP7406192B2 (ja) 線維症治療薬
CN110652510B (zh) 中乌宁在制备防治肾纤维化药物中的应用
EP2793853B1 (en) Pharmaceutical formulations of flurbiprofen and glucosamin
CN111991387B (zh) 去氢二异丁香酚在制备防治溃疡性结肠炎的药物中的应用
WO2023150500A1 (en) Peptide inhibitors of interleukin-23 receptor and pharmaceutical compositions thereof
CN106967073A (zh) 一种治疗类风湿性关节炎的药物
AU2002311591A1 (en) Use of glycosaminoglycans for the treatment of HIV-associated nephropathy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20765896

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021552974

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020765896

Country of ref document: EP

Effective date: 20211005