WO2014183466A1 - 海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用 - Google Patents

海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用 Download PDF

Info

Publication number
WO2014183466A1
WO2014183466A1 PCT/CN2014/000470 CN2014000470W WO2014183466A1 WO 2014183466 A1 WO2014183466 A1 WO 2014183466A1 CN 2014000470 W CN2014000470 W CN 2014000470W WO 2014183466 A1 WO2014183466 A1 WO 2014183466A1
Authority
WO
WIPO (PCT)
Prior art keywords
sea cucumber
depolymerized
molecular weight
glycosaminoglycan
cucumber glycosaminoglycan
Prior art date
Application number
PCT/CN2014/000470
Other languages
English (en)
French (fr)
Inventor
王志国
刘全海
董玉琼
Original Assignee
上海开润生物医药有限公司
哈尔滨红豆杉生物制药有限公司
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 上海开润生物医药有限公司, 哈尔滨红豆杉生物制药有限公司 filed Critical 上海开润生物医药有限公司
Priority to US14/890,854 priority Critical patent/US20160082051A1/en
Publication of WO2014183466A1 publication Critical patent/WO2014183466A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/616—Echinodermata, e.g. starfish, sea cucumbers or sea urchins
    • 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
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • sea cucumber glycosaminoglycan in preparing medicine for preventing and treating thromboembolic diseases
  • the invention relates to the medical use of the sea cucumber glycosaminoglycan, in particular to the application of the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 54,500 Da or the sea cucumber glycosaminoglycan of the natural molecular segment in preparing a medicament for preventing and treating thromboembolic diseases
  • thromboembolic diseases include atherothrombotic thrombosis, venous thromboembolic disease, hypercoagulable state, and post-operative thrombosis or thrombosis after surgery.
  • thrombosis that is, local blood clot formation
  • thromboembolic disease including deep vein thrombosis and pulmonary embolism
  • Drugs for preventing and treating embolism can be divided into anticoagulant drugs, antiplatelet drugs, direct thrombolytic drugs, etc. according to the mechanism of action, and can be used for preventing and treating thrombotic diseases in clinical practice.
  • anticoagulant drugs prevent thrombosis or recurrence by affecting blood coagulation factors.
  • Anticoagulant drugs have no dissolution effect on the formed thrombus, but can prevent thrombus expansion and new thrombus formation, and promote the early autolysis of thrombus.
  • anticoagulant drugs have obvious preventive effects on venous thrombosis, and anticoagulant drugs are also It can be used in combination with prevention and treatment of thrombus in extracorporeal circulation and hemodialysis, aiming to prevent blood coagulation during therapeutic manipulation.
  • Hypercoagulable state or thrombosis in patients including factors such as increased blood coagulation protein, coagulation protein activation, and increased blood coagulation.
  • the principle of prevention and treatment of hypercoagulable state in addition to the removal of the cause of hypercoagulable state, must also use drugs that reduce or have inactivated coagulation protein, so that the hypercoagulable state turns to the normal direction, avoiding the tendency of thrombosis, use
  • the main drugs are heparin and low molecular weight heparin.
  • Oral drugs mainly include warfarin, dicoumarin, and new dicoumarin.
  • the drugs that reduce blood viscosity and prevent thromboembolism mainly include low molecular weight dextran.
  • warfarin can inhibit the vitamin K-dependent activation of certain coagulation factors, and is the most prescribed anticoagulant drug, and it is still the only oral effective vitamin K antagonist and the only one approved for long-term application.
  • Anticoagulation Blood drugs Clinical studies have confirmed that warfarin can reduce the incidence of stroke in 64% of patients with atrial fibrillation. However, despite its high effectiveness, warfarin also carries a serious and even fatal bleeding risk.
  • warfarin due to the large individual differences in pharmacokinetics and the vulnerability to diet, drug interactions are complex, so warfarin is difficult to optimally dose in clinical practice, and heparin and low molecular weight heparin drugs are used clinically. It is easy to cause bleeding, and people with different physiques are highly monitorable.
  • the object of the present invention is to provide an application of sea cucumber glycosaminoglycan in the preparation of a medicament for preventing and treating thromboembolic diseases, so as to overcome the above defects existing in the prior art and meet the needs of clinical application.
  • the weight average molecular weight is greater than 54,500 Da of depolymerized sea cucumber glycosaminoglycan or one or more of the sea cucumber glycosaminoglycans of the natural molecular segment
  • the anticoagulant activity is dose-dependent, with heparin and low molecular weight
  • heparin it increases the coagulation with increasing dose
  • the weight-dependent molecular weight increases with the onset of time delay and the duration of drug effect increases.
  • the sea cucumber glycosaminoglycan of the natural molecular segment can last for up to 16 hours at a certain dose.
  • the one or more of the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 54,500 Da or the sea cucumber glycosaminoglycan of the natural molecular segment may be used for preparing a medicament for preventing and treating thromboembolic diseases, specifically, Thromboembolic diseases include atherothrombotic disease, venous thromboembolic disease, hypercoagulable state, and post-operative thrombosis or thrombosis after surgery.
  • the "depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 54,500 Da or the sea cucumber glycosaminoglycan of a natural molecular segment” means that the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 54,500 Da has an arbitrary weight average a sea cucumber glycosaminoglycan having a molecular weight or a natural molecular segment, or a multi-stage mixture of sea cucumber glycosaminoglycan having a weight average molecular weight of more than 54,500 Da;
  • the weight average molecular weight of the depolymerized sea cucumber glycosaminoglycan is:
  • the weight average molecular weight of the depolymerized sea cucumber glycosaminoglycan is:
  • the drug including a therapeutically effective amount of the solution a sea cucumber glycosaminoglycan and a pharmaceutically acceptable carrier selected from the group consisting of mannitol, lactose, dextran, glucose, glycine, hydrolyzed gelatin, povidone or sodium chloride, preferably Mannitol
  • the drug is an injection for intravenous or subcutaneous injection or a lyophilized powder injection
  • the depolymerized sea cucumber glycosaminoglycan having a molecular weight of more than 54,500 Da or the sea cucumber glycosaminoglycan of the natural molecular segment is administered in a subcutaneous injection amount of 1 mg/kg to 100 mg/kg, preferably 2 mg/kg to 80 mg/kg ; intravenous injection
  • the dosage is 0.1 mg/kg to 40 mg/kg, preferably 0.2 mg/kg to 3 Omg/kg;
  • the depolymerized sea cucumber glycosaminoglycan has a purity of 90 to 99.99%, preferably 92% or more, more preferably 94% or more for achieving a more desirable effect;
  • the sea cucumber glycosaminoglycan of the natural molecular segment The purity of the sugar is 90 to 99.99%, preferably 92% or more, and more preferably 95% or more in order to achieve a more desirable effect;
  • the purity is weight purity.
  • the polydispersity of the depolymerized sea cucumber glycosaminoglycan is 1 ⁇ 2, preferably 1 ⁇ 1.6, more preferably 11.4;
  • the polydispersity refers to an index commonly used in the art to measure the molecular weight distribution of a polymer for characterizing the width of the molecular weight distribution of the polymer.
  • Polydispersity in this document or other literature, is also referred to as polydispersity index, polydispersity or distribution width index, which is the ratio of weight average molecular weight (Mw) to number average molecular weight (Mn), ie, Mw / Mn. This ratio varies with the width of the molecular weight distribution. In the case of monodispersion, M W / M n is equal to 1, and as the molecular weight distribution becomes wider, the Mw / Mn value becomes larger.
  • the weight average molecular weight is defined as follows: Weight-average Molecular Weight: The molecular weight of all synthetic polymer compounds and the molecular weight of most natural polymer compounds are not uniform, they are homologues of different molecular weights. mixture. The statistical average weight average molecular weight of the molecules in the polymer with different molecular weights was tested by high performance liquid chromatography.
  • the depolymerized sea cucumber glycosaminoglycan can be prepared by using a commercial product such as sea cucumber glycosaminoglycan or depolymerized sea cucumber glycosaminoglycan produced by Harbin Sequoia Biopharmaceutical Co., Ltd., or by the following method: (1) adding the enzyme to the ground sea cucumber, performing enzymatic hydrolysis and precipitation, collecting the crude sea cucumber glycosaminoglycan, purifying and decolorizing the crude sea cucumber glycosaminoglycan, and collecting the sea cucumber glycosaminoglycan;
  • the sea cucumber is selected from the group consisting of jade foot sea cucumber, rough sea cucumber, plum ginseng, two-color table ginseng or white radish anal ginseng, preferably jade foot sea cucumber or two-color table ginseng;
  • the enzyme is a hydrolyzed protease and a composite trypsin.
  • the hydrolyzed protease can be a commercial product, such as Alcalase of Novozymes (Shenyang) Biotechnology Co., Ltd., and the compound pancreatin can be used as a commercial product, such as Wuxi Xuemei enzyme preparation.
  • Wuxi Xuemei brand compound pancreatin of Science and Technology Co., Ltd. the amount of hydrolyzed protease is 2% of the weight of sea cucumber, and the amount of compound pancreatin is 2 ⁇ 3% of the weight of sea cucumber;
  • step (1) adding the weight concentration of 5% acetic acid and 3% hydrogen peroxide to the product of step (1) for degradation, and collecting the depolymerized sea cucumber glycosaminoglycan having a weight average molecular weight of more than 54,5000 Da;
  • the preparation method of the medicine is a conventional method in the field of preparation, such as the method described in the Handbook of Traditional Chinese Medicine Preparation, and the injection or lyophilized powder injection is obtained;
  • the gel adsorption column such as a Sephadex-GlOO gel adsorption column, a Sephadex-G50 gel adsorption column or Sephadex-G200, the Sephadex-G100 gel adsorption column can be a GE gel of the United States GE company. column.
  • the depolymerized sea cucumber glycosaminoglycan drug of the present invention can be applied to a patient in need of treatment by subcutaneous or intravenous injection, and the dosage is administered by the physician according to the patient's specific conditions (such as age, weight, sex, time of illness, body). Status, etc.) OK.
  • the dosage of subcutaneous administration is 0.1 to 50 mg/kg, preferably 0.2 to 45 mg/kg
  • the dosage for intravenous administration is 0.01 to 30 mg/kg, preferably 0.05 to the depolymerized sea cucumber glycosaminoglycan. 20 mg/kg.
  • Sea cucumber glycosaminoglycan is an acid mucopolysaccharide contained in the wall of sea cucumber. It is unique to sea cucumber.
  • the present invention has found that sea cucumber glycosaminoglycan has significant anticoagulant, anti-platelet aggregation, blood viscosity reduction, fibrinolysis, and regulation of blood lipids, and can be used for treating thrombotic diseases.
  • sea cucumber glycosaminoglycans are further depolymerized into depolymerized sea cucumber glycosaminoglycans of different molecular weight fractions, which exhibit different anticoagulant activities, and their anticoagulant activity is gradually increased with increasing dose, relative to heparin drugs and Vitamin K antagonists are safer. It is clinically used for the treatment of thromboembolic diseases with wide window, high safety and good research and development value.
  • Figure 2-15 Molecular weight test report of depolymerized sea cucumber glycosaminoglycan with a weight average molecular weight of 121,017 Da.
  • Figure 3 is a linear relationship between the anticoagulant dose and clotting time of depolymerized sea cucumber glycosaminoglycans.
  • the method for extracting the glycosaminoglycan from sea cucumber refers to the sea cucumber glycosaminoglycan extracted from sea cucumber, and after degrading and depolymerizing, a glycosaminoglycan is produced, and the depolymerized sea cucumber glycosaminoglycan having the desired molecular weight is collected.
  • Methods for extracting sea cucumber glycosaminoglycans from sea cucumber body walls are well known to those skilled in the art, such as Chinese patent ZL200910305363.5.
  • Centrifuge collect and weigh the precipitate, add 8 times the weight of distilled water, heat to 85 °C ⁇ 2 ° C, after complete dissolution, add 6mol / L sodium hydroxide to adjust the pH to 9.0 ⁇ 0.2, add calcium chloride to the solution
  • concentration of calcium chloride reached 3% (w/v)
  • the temperature was raised to 92 ° C for 15 minutes, cooled to room temperature, centrifuged at 4 ° C, the supernatant was collected, and the pH was adjusted to 11.0 ⁇ 0.1 with a saturated sodium carbonate solution, and centrifuged.
  • the supernatant was collected, adjusted to pH 6.0 ⁇ 0.1 with 6 mol / L hydrochloric acid, 1 volume of ethanol was added, and refrigerated at 4 ° C for 12 h;
  • the liquid was centrifuged, and the precipitate was collected and weighed. Two times of distilled water was added, and the mixture was fully dissolved by heating. Potassium acetate was added to make a final concentration of 2 mol/L, and allowed to stand at 4 ° C for 12 hours. After centrifugation, the precipitate was collected and weighed, and 2 volumes of distilled water was added thereto, and the mixture was fully dissolved by heating, and potassium acetate was added thereto to have a final concentration of 2 mol/L, and allowed to stand at 4 ° C for 12 hours.
  • the precipitate was washed three times with a cold 2 mol/L potassium acetate solution, and then washed successively with 80% ethanol, 95% ethanol, and absolute ethanol. After the ethanol was evaporated, it was dried at 80 ° C, and weighed to obtain a crude product A.
  • the crude product A was added with 0.05 mol/L and pH 6.0 in HAc-NaAc buffer to prepare a 2% solution.
  • the solution was passed through a cellulose chromatography column and buffered with 1.5 column volumes of 0.4 mol/L NaCl in HAc-NaAc.
  • wash liquid CpH6.0 ⁇ 0.1 and then use Imol/L NaCl in HAc-NaAc buffer CpH6.0 ⁇ 0.i;> elution, collect the eluent according to the change speed of the UV detector at 220nm, set 60 In a °C water bath, adjust the pH to 11 ⁇ 0.1 with NaOH, add 3% hydrogen peroxide by volume, keep for 4 hours, cool, centrifuge, collect the supernatant, adjust the pH to 7.2 ⁇ 0.1 with HC1, add 1 times of ethanol, Allow to stand at 4 ° C for 12 h.
  • the crude product B is dissolved in 5% solution with distilled water, and concentrated to 1/2 of the original volume with an ultrafiltration membrane with a molecular weight cut off of 10,000. The water is added to the original volume, and then ultrafiltered to 1/2 volume, and water is added again. The filtrate is freeze-dried to obtain sea cucumber glycosaminoglycan,
  • the sea cucumber glycosaminoglycan obtained in this example can obtain a pure product with a purity of 99.0% by a differential refractive index detector (RID-10A, Shimadzu) (see Figure 1-1), and the depolymerized sea cucumber sugar obtained by the example.
  • the amine glycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 128,024 Da, and the D value was 1.26 (see Figure 2-1).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 4 h 50 min.
  • the solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • the glycosaminoglycans have a molecular weight of 54,500 Da to 57,000 Da, a value of ⁇ 1.5, and a purity of 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-2), and the depolymerization obtained by the example was obtained.
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH), and the weight average molecular weight of the product was 54,876 Da, and the D value was 1.28 (see Figure 2-2).
  • Example 2-2 The pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 4 h 20 min.
  • the solution was neutralized to neutral with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 58,000 Da to 62,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-3), and the depolymerization obtained by the example was obtained.
  • the sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 60,915 Da, and the D value was 1.36 (see Figure 2-3).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 °C for 3 hours and 50 minutes.
  • the solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 63,000 Da to 67,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product having a purity of 99.0% (see Figure 1-4), and the depolymerization obtained by the example was obtained.
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH).
  • the weight average molecular weight of the product was 64,904 Da, and the D value was 1.34 (see Figure 2-4).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 3 h 20 min.
  • the solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, and treated with 0.5 mol/l of sodium chloride. Elution, demineralization and small molecular impurities, the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber glycosaminoglycan, which has a molecular weight of 68,000 Da to 72,000 Da, a value of ⁇ 1.5, and a purity of 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-5), and the depolymerization obtained by the example.
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 71,147 Da, and the D value was 1.38 (see Figure 2-5 for the map).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 2 h 55 min.
  • the solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • the glycosaminoglycan has a molecular weight of 73,000 Da to 77,000 Da, a value of ⁇ 1.5, and a purity of 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Figure 1-6), and the depolymerization obtained by the example.
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 74,844 Da, and the D value was 1.26 (see Figure 2-6).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 2 h 30 min.
  • the solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • the glycosaminoglycan has a molecular weight of 78,000 Da to 82,000 Da, a value of ⁇ 1.5, and a purity of 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Fig. 1-7), and the depolymerization obtained by the example was obtained.
  • Chromatographic analysis of sea cucumber glycosaminoglycan by gel column (TSK gel G4000PWXL, TOSOH) showed that the product has a weight average molecular weight of 80,336 Da and a D value of 1.33 (see map Figure 2-7)
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 2 min 5 min.
  • the solution was neutralized to neutrality with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 83,000 Da to 87,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example can obtain a pure product with a purity of 99.0% by a differential refractive index detector (RID-10A, Shimadzu) (see Fig. 1-8 for the map), and the depolymerization obtained by the example
  • the weight fraction of the sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH).
  • the weight average molecular weight of the product was 84,481 Da, and the D value was 1.29 (see Figure 2-8).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, and 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization at 40 ° C for 40 min.
  • the solution was neutralized to neutrality with 0.1 mol/1 of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 88,000 Da to 92,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example can obtain a pure product with a purity of 99.0% by a differential refractive index detector (RID-10A, Shimadzu) (see Fig. 1-9 for the map), and the depolymerization obtained by the example Chromatographic analysis of sea cucumber glycosaminoglycan by gel column (TSK gel G4000PWXL, TOSOH) showed that the product had a weight average molecular weight of 90,919 Da and a D value of 1.26 (see Figure 2-9 for the spectrum).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization at 40 ° C for 15 min.
  • the solution was neutralized to neutral with 0.1 mol/ ⁇ sodium hydroxide, and 3 times volume of ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain depolymerized sea cucumber sugar amine.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 93,000 Da to 97,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Fig. 1-10), and the depolymerization obtained by the example was obtained.
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 95,821 Da, and the D value was 1.27 (see Figure 2-10 for the map).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled deagglomeration was carried out at 40 °C for 55 minutes.
  • the solution was neutralized to neutral with 0.1 mol/1 of sodium hydroxide, and 3 times by volume of ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 98,000 Da to 102,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Fig. 1-11), and the depolymerization obtained by the example was obtained.
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 101,250 Da, and the D value was 1.24 (see Figure 2-11 for the map).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 40 min.
  • the solution was neutralized to neutral with 0.1 mol/min of sodium hydroxide, and 3 times by volume of ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • the glycosaminoglycan has a molecular weight of 103,000 Da to 107,000 Da, a value of ⁇ 1.5, and a purity of 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example can be obtained by the differential refractive index detector (RID-10A, Shimadzu). 99.0% pure product (see Figure 1-12), the depolymerized sea cucumber glycosaminoglycan obtained by this example was analyzed by gel column (TSK gel G4000PWXL, TOSOH) to find the weight average molecular weight of the product 103,998Da, D The value is 1.26 (see Figure 2-12 for the map)
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out for 30 min at 40 °C.
  • the solution was neutralized to neutral with 0.1 mol/1 of sodium hydroxide, and 3 times by volume of ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • the glycosaminoglycan has a molecular weight of 108,000 Da to 112,000 Da, a value of ⁇ 1.5, and a purity of 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product with a purity of 99.0% (see Fig. 1-13), and the depolymerization obtained by the example was obtained.
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH). The weight average molecular weight of the product was 109,161 Da, and the D value was 1.22 (see Figure 2-13).
  • the pure sea cucumber glycosaminoglycan in the above Example 1 was formulated into a 5% solution with 5% acetic acid, 30% hydrogen peroxide was added to make the concentration of hydrogen peroxide in the solution 3%, and controlled depolymerization was carried out at 40 ° C for 20 min.
  • the solution was neutralized to neutral with 0.1 mol/min of sodium hydroxide, and 3 times by volume of ethanol was added for alcohol precipitation, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 113,000 Da to 117,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example was obtained by a differential refractive index detector (RID-10A, Shimadzu) to obtain a pure product having a purity of 99.0% (see Fig. 1-14), and the depolymerization obtained by the example was obtained.
  • the sea cucumber glycosaminoglycan was analyzed by gel column (TSK gel G4000PWXL, TOSOH) to find that the weight average molecular weight of the product was 115,268 Da, and the D value was 1.38 (see Figure 2-14).
  • the sea cucumber glycosaminoglycan pure product in the above Example 1 was formulated into a 5% solution with 5% acetic acid, and 30% dioxygen was added. Water allowed the concentration of hydrogen peroxide in the solution to be 3%, and controlled depolymerization at 40 °C for 10 min. The solution was neutralized to neutral with 0.1 mol/l of sodium hydroxide, and ethanol was precipitated by adding 3 volumes of ethanol, allowed to stand, and centrifuged to obtain a crude product of depolymerized sea cucumber glycosaminoglycan.
  • the crude product was dried, dissolved in 5 times by weight of water, passed through a sephadex-G75 column, eluted with 0.5 mol/l of sodium chloride to remove salts and small molecular impurities, and the desalted sample was freeze-dried to obtain 55 g of depolymerized sea cucumber.
  • Glycosaminoglycans have molecular weights ranging from 118,000 Da to 122,000 Da. The value is ⁇ 1.5 and the purity is 98% or more.
  • the depolymerized sea cucumber glycosaminoglycan obtained in this example can obtain a pure product with a purity of 99.0% by a differential refractive index detector (RID-10A, Shimadzu) (see Fig. 1-15 for the map), and the depolymerization obtained by the example
  • the weight fraction of the sea cucumber glycosaminoglycan was determined by gel column (TSK gel G4000PWXL, TOSOH).
  • the weight average molecular weight of the product was 121,017 Da, and the D value was 1.36 (see Figure 2-15).
  • Platelet aggregation clotting factor analyzer (model LG-PABER Beijing Shidi Scientific Instrument Co.;).
  • the plasma coagulation time of each sample solution was determined by using ⁇ different concentrations of sample solution instead of 10 ⁇ 1 0.9% sodium chloride solution. Each concentration was measured in parallel 4 times and averaged.
  • the experimental results show that the final concentration of the sample is in the dose range of DHG-1 (40.0 g/ml ⁇ 200. ( ⁇ g/ml), DHG-2 (30.0 g/ml ⁇ 200.0 ⁇ ⁇ / ⁇ 1), and the clotting time is prolonged with the dose. The clotting time is prolonged and the increasing trend is moderated. Therefore, the depolymerized sea cucumber glycosaminoglycan composition is safer and controllable for anticoagulation.
  • Test sample Name: Natural sea cucumber glycosaminoglycan, low molecular weight sea cucumber glycosaminoglycan (54, 876 Da, 60, 915 Da, 74,844Da 90,919Da) ; Preparation: After precision pipetting, dilute to the desired concentration with physiological saline for injection.
  • Line SD rat; Source: Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.; Gender: Male; Weight: 180-200 g; Animal certificate number: SCXK (Shanghai) 2008-0016; Feeding: Animals are fed in positive pressure Purified and ventilated animal room, room temperature 23 ⁇ 1 °C, humidity 50 70%, artificial lighting simulation of day and night changes, free to eat and drink.
  • Test sample Name: Depolymerized sea cucumber glycosaminoglycan (DHG); Preparation: After precision pipetting, dilute to the desired concentration with saline for injection.
  • Control sample Name: Heparin; Source: Sinopharm Chemical Reagent Co., Ltd.; Batch number: F20091029; Content: 150U/mg; Preparation: After precise weighing, dissolve and dilute to the desired concentration with physiological saline for injection.
  • Test animals strain: SD rat; Source: Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.; Gender: Male; Weight: 180-220 g; Animal certificate number: SCXK (Shanghai) 2008-0016; Feeding: Animal breeding In the positive pressure purification and ventilation animal room, room temperature 23 ⁇ 1 °C, humidity 50 ⁇ 70%, artificial lighting simulation changes day and night, free to eat and drink.
  • BS 110 s electronic balance, produced by SARTORIUS, with a minimum weight of 0.1 mg.
  • SD rats in each group were divided into different drug-administered groups, the negative control group (physiological saline 1 ml/kg), and the positive control low molecular weight heparin group (2 mg/kg). All drugs were administered subcutaneously in a volume of 0.5 ml.
  • thrombus wet weight inhibition rate thrombus wet weight (dissolved fine-blood sputum (fine) x recommended
  • both positive and test drugs can significantly inhibit thrombus formation after administration.
  • the test drug has a significant inhibitory effect on thrombosis.
  • Line SD rat; Source: Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.; Gender: Male; Weight: 180-220 g; Animal certificate number: SCXK (Shanghai) 2008-0016; Feeding: Animals are fed in positive pressure Purify and ventilate the animal room, room temperature 23 ⁇ 1 °C, humidity 50 ⁇ 70%, artificial lighting simulates day and night changes, free to eat and drink.
  • SD rats were divided into 10 groups, and the negative control group (subcutaneous injection of 0.5 ml of normal saline) was used to depolymerize different molecular weight fractions of sea cucumber glycosaminoglycan (54, 876 Da, 74, 844 Da), natural sea cucumber
  • the glycan composition was administered by subcutaneous injection at a dose ratio of 1:1 (10 mg/kg), and the blank was injected with a volume of 0.5 ml of physiological saline.
  • the plasma activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time ( ⁇ ) values were determined by abdominal aortic blood sampling at different time points after subcutaneous injection. See Table 5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Dermatology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Urology & Nephrology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

本发明公开了海参糖胺聚糖在制药物中的应用，动物试验证明，重均分子量为大于54,500Da的解聚海参糖胺聚糖或天然分子段的海参糖胺聚糖中的一段或者一段以上，其抗凝活性呈剂量依赖性，与肝素及低分子肝素比较，其随剂量增加凝血作用递增缓和，并且在同一剂量下随着重均分子量增加起效时间延迟的同时药效持续时间增加。天然分子段的海参糖胺聚糖在一定剂量下持续药效时间可达16小时，可用于制备防治动脉血栓栓塞疾病药物。相对肝素类药物以及维生素K拮抗剂类药物更具安全性。临床上用于血栓栓塞疾病治疗视窗宽，安全性高，具有良好的开发研究价值。

Description

海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用 技术领域
本发明涉及海参糖胺聚糖的医药用途， 具体涉及重均分子量为大于 54,500Da的解 聚海参糖胺聚糖或天然分子段的海参糖胺聚糖在制备防治血栓栓塞性疾病药物中的应 用， 血栓栓塞疾病包括动脉粥样硬化血栓性疾病、 静脉血栓栓塞性疾病、 血液高凝状态 以及手术后的血栓形成或治疗手术后的血栓。
背景技术
随着社会的发展和人口老龄化的进程，老年人血管老化，血管壁受损， 易患高血压、 动脉硬化、 糖尿病。 血管内皮细胞受损后， 产生的凝血激酶增多， 促进凝血酶形成，凝 血黄素 A2也增多， 同时制造抗凝物质前列环素减少， 易诱发血栓形成。 如血糖增高时， 糖与红血球中的血红蛋白结合， 使全身组织缺氧， 这时血小板凝集性增强， 粘度增大， 容易促进血栓形成。 因此中老年人血栓栓塞疾病的发病率逐年增加， 据世界卫生组织统 计， 全球每年有 1500万人死于血栓性疾病， 血栓形成， 即局部血液凝块形成， 是导致心 肌梗死和中风等动脉疾病以及静脉血栓栓塞性疾病 (包括深部静脉血栓形成和肺栓塞） 发生和患者死亡的主要原因。
防治栓塞形成药物可按作用机制分为抗凝血药物、抗血小板药物、直接溶栓药物等， 临床上均能用于预防和治疗血栓性疾病。 其中抗凝药物是通过影响凝血因子， 从而防止 血栓形成或复发。 抗凝药物对已经形成的血栓无溶解作用， 但可以防止血栓扩展和新血 栓形成，有利于促进血栓早期自溶，同时抗凝血药物对静脉血栓形成具有明显预防作用， 同时抗凝血药物还可以用于配合体外循环和血液透析中防治血栓的形成使用， 旨在防止 治疗操控时的血液凝固。
患者出现的血液高凝状态或血栓形成， 均涉及到凝血蛋白增高、 凝血蛋白激活和血 液凝固性增加等诸多因素。临床上对高凝状态的防治原则，除要去除高凝状态的病因外， 还须选用减低或具有灭活凝血蛋白的药物， 使高凝状态转向正常的方向发展， 避免导致 血栓倾向进展， 使用药物注射的主要有肝素及低分子肝素， 口服的主要有华法林、 双香 豆素、 新双香豆素等， 降低血粘度防治血栓栓塞的药物主要有低分子右旋糖苷。 其中华 法林能够抑制某些凝血因子的维生素 K依赖性激活， 是现处方量最大的抗凝血药物，且 至今仍是临床上唯一一个口服有效的维生素 K拮抗剂和唯一一个获准长期应用的抗凝 血药物。临床研究证实，华法林能够减少心房纤维性颤动患者 64%的中风发生率。不过， 尽管高度有效， 但华法林也会带来严重甚至致死性的出血风险。 此外， 因药动学的个体 差异性大且易受到饮食的影响， 药物相互作用又很复杂， 故华法林在临床实践中难以最 优剂量用药， 另外肝素及低分子肝素类药物， 临床使用易导致出血， 不同体质的人使用 监控性强。 因此就目前临床使用的抗凝药物来说均具有一定的副作用， 鉴于人口的老龄 化、 血栓性疾病发病率的增加， 以及现有抗凝药物在临床用于栓塞性疾病预防和治疗的 广泛性以及其安全隐患的严重性。 从中药中筛选、 分离更具疗效性、 安全性防治血栓栓 塞性疾病， 是预防和治疗血栓栓塞疾病的一种必然趋势。
发明内容
本发明的目的是提供一种海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用， 以 克服现有技术存在的上述缺陷， 满足临床应用的需要。
动物试验证明， 重均分子量为大于 54,500Da的解聚海参糖胺聚糖或天然分子段的 海参糖胺聚糖中的一段或者一段以上， 其抗凝活性呈剂量依赖性， 与肝素及低分子肝素 比较， 其随剂量增加凝血作用递增缓和， 并且在同一剂量下随着重均分子量增加起效时 间延迟的同时药效持续时间增加。天然分子段的海参糖胺聚糖在一定剂量下持续药效时 间可达 16小时。
因此， 所述重均分子量为大于 54,500Da的解聚海参糖胺聚糖或天然分子段的海参 糖胺聚糖中的一段或者一段以上， 可用于制备防治血栓栓塞疾病药物， 具体的， 所述血 栓栓塞疾病包括动脉粥样硬化血栓性疾病、 静脉血栓栓塞性疾病、 血液高凝状态以及手 术后的血栓形成或治疗手术后的血栓。
所述的 "重均分子量大于 54,500Da的解聚海参糖胺聚糖或天然分子段的海参糖胺 聚糖"指的是， 重均分子量大于 54,500Da的解聚海参糖胺聚糖任意一重均分子量或天 然分子段的海参糖胺聚糖， 或者是重均分子量大于 54,500Da的解聚海参糖胺聚糖任意 一重均分子量或然分子段的海参糖胺聚糖的多段混合物；
优选的： 所述解聚海参糖胺聚糖的重均分子量为：
54,500Da〜57,000Da、 57,010Da〜62,990Da、 63,000Da〜67,000Da、 67,010Da〜 72,990Da、 73,000Da〜77,000Da、 77,010Da〜82,990Da、 83,000Da〜87,000Da、 87,010Da〜 92,990Da、 93,000Da〜97,000Da、 97,010Da〜102,900Da、 103,000Da〜107,000Da、 107,010Da〜 112,990Da、 113,000Da〜 117,900Da或 118,000Da〜 122,050Da中的任意一段； 特别优选的， 所述解聚海参糖胺聚糖的重均分子量为：
54,500Da〜57,000Da、 58,000Da〜62,000Da、 63,000Da〜 67,000Da、 68,000Da〜 72,000Da、 73,000Da〜77,000Da、 78,000Da〜82,000Da、 83,000Da〜87,000Da、 88,000Da〜 92,000Da、 93,000Da〜 97,000Da、 98,000Da〜 102,000Da、 103,000Da〜 107,000、 108,000Da〜112,000Da、 113,000Da〜117,000Da、 118,000Da〜122,000Da中的任意一段； 所述药物， 包括治疗有效量的所述的解聚海参糖胺聚糖和药学上可接受的载体，所 述药学上可接受的载体选自甘露醇、 乳糖、 右旋糖酐、 葡萄糖、 甘氨酸、 水解明胶、聚 维酮或氯化钠的一段以上， 优选甘露醇；
所述的药物为静脉或皮下注射给药的注射液或者是冻干粉针剂；
分子量大于 54,500Da的解聚海参糖胺聚糖或天然分子段的海参糖胺聚糖， 皮下注 射给药量为大鼠 lmg/kg〜100mg/kg， 优选 2mg/kg〜80mg/kg; 静脉注射给药量为大鼠 0.1mg/kg〜40mg/kg， 优选 0.2mg/kg~ 3 Omg/kg；
所述药物中， 所述解聚海参糖胺聚糖的纯度为 90〜99.99%， 优选 92%以上， 为了 达到更理想的效果更优选 94%以上； 所述的天然分子段的海参糖胺聚糖的纯度为 90〜 99.99%， 优选 92%以上， 为了达到更理想的效果， 更优选 95%以上；
所述纯度为重量纯度。
所述的解聚海参糖胺聚糖的多分散度为 1~2， 优选 1~1.6， 更优选 1 1.4;
所述多分散度指的是本领域常用的衡量聚合物分子量分布的指数，用于表征聚合物 分子量分布的宽度。 多分散度在本文或其他文献中又被称多分散指数、 多分散性或分布 宽度指数， 是重均分子量 (Mw)与数均分子量 (Mn)之比， 即 Mw / Mn。 这个比值随分子 量分布宽度而变化。 在单分散时， MW / Mn等于 l， 随着分子量分布变宽， Mw / Mn 值逐渐变大。
所述重均分子量的定义如下： 重均分子量（Weight-average Molecular Weight): 所有 合成高分子化合物的分子量以及大多数天然高分子化合物的分子量都是不均一的， 它们 是分子量不同的同系物的混合物。聚合物中用不同分子量的分子重量平均的统计平均分 重均分子量是采用高效液相凝胶色谱法测试的。
所述解聚海参糖胺聚糖可采用商业化产品，如哈尔滨红杉生物制药有限公司生产的 海参糖胺聚糖或解聚海参糖胺聚糖， 或采用如下的方法制备： ( 1 ) 将酶加入绞碎的海参， 在进行酶解和沉淀， 收集海参糖胺聚糖粗品， 对海参 糖胺聚糖粗品进行纯化和脱色， 收集海参糖胺聚糖；
所述海参选自玉足海参、 糙海参、 梅花参、 二色桌片参或白底辐肛参中的一种以 上， 优选玉足海参或二色桌片参；
所述的酶为水解蛋白酶和复合胰酶， 水解蛋白酶可采用商业化产品， 如诺维信 （沈 阳） 生物技术有限公司的 Alcalase, 复合胰酶可采用商业化产品， 如无锡市雪梅酶制剂 科技有限公司的雪梅牌复合胰酶， 水解蛋白酶的用量为海参重量的 2%， 复合胰酶的用 量为海参重量的 2〜3%；
(2)将重量浓度为 5%醋酸和 3%的双氧水加入步骤（1 ) 的产物降解， 收集重均分 子量为大于 54,5000Da的解聚海参糖胺聚糖；
所述药物的制备方法， 为制剂领域常规的方法， 如 《中药制剂手册》 记载的方法， 获得所述的注射液或冻干粉针剂；
(3 ) 将所需分子量段的海参糖胺聚糖以及解聚海参糖胺聚糖采用凝胶柱收集所需 分子量段；
所述的凝胶吸附柱， 如 Sephadex-GlOO凝胶吸附柱、 Sephadex-G50凝胶吸附柱或 Sephadex-G200,所述 Sephadex-GlOO凝胶吸附柱，可以采用美国 GE公司的葡聚糖凝胶 柱。
本发明的含有解聚海参糖胺聚糖药物可通过皮下或者静脉注射的方法施加于需要 治疗的患者， 给药剂量由医师根据患者的具体情况 （如年龄、 体重、 性别、 患病时间、 身体状况等）确定。一般而言， 以解聚海参糖胺聚糖计， 皮下给药剂量为 0.1〜50mg/kg， 优选 0.2〜45mg/kg， 静脉给药的给药剂量为 0.01〜30 mg/kg， 优选 0.05〜20mg/kg。
海参糖胺聚糖是海参体壁中含有的一种酸性粘多糖是海参所特有的。 本发明发现， 海参糖胺聚糖具有显著抗凝血、 抗血小板聚集、 降低血粘度、 纤溶、 调节血脂等生物活 性， 可用于治疗血栓性疾病。
海参糖胺聚糖通过进一步解聚成为不同分子量段的解聚海参糖胺聚糖， 其呈现不 同的抗凝血活性， 且随着剂量增加其抗凝血活性递增趋势缓和， 相对肝素类药物以及维 生素 K拮抗剂类药物更具安全性。 临床上用于血栓栓塞疾病治疗视窗宽， 安全性高，具 有良好的开发研究价值。
附图说明 海参糖胺聚糖和解聚海参糖胺聚糖的纯度图：
-1天然分子段的海参糖胺聚糖的纯度图；
-2重均分子量为 54,876Da的解聚海参糖胺聚糖纯度图；
-3重均分子量为 60,915Da的解聚海参糖胺聚糖纯度图；
-4； 重均分子量为 64,904Da的解聚海参糖胺聚糖纯度图；
-5重均分子量为 71, 147Da的解聚海参糖胺聚糖纯度图；
-6重均分子量为 74,844Da的解聚海参糖胺聚糖纯度图；
-7重均分子量为 80,336Da的解聚海参糖胺聚糖纯度图；
-8重均分子量为 84,481Da的解聚海参糖胺聚糖纯度图；
-9重均分子量为 90,919Da的解聚海参糖胺聚糖纯度图；
-10重均分子量为 95,821Da的解聚海参糖胺聚糖纯度图；
-11重均分子量为 10,1250Da的解聚海参糖胺聚糖纯度图
-12重均分子量为 10,3998Da的解聚海参糖胺聚糖纯度图
-13重均分子量为 10,9161Da的解聚海参糖胺聚糖纯度图
-14重均分子量为 115,268Da的解聚海参糖胺聚糖纯度图
- 15重均分子量为 121,017Da的解聚海参糖胺聚糖纯度图。
为海参糖胺聚糖和解聚海参糖胺聚糖的分子量测试报告。
- 1天然分子段的海参糖胺聚糖的分子量测试报告；
-2 重均分子量为 54,876Da的解聚海参糖胺聚糖分子量测试报告；-3重均分子量为 60,915Da的解聚海参糖胺聚糖分子量测试报告；-4重均分子量为 64,904Da的解聚海参糖胺聚糖分子量测试报告；-5重均分子量为 71, 147Da的解聚海参糖胺聚糖分子量测试报告；-6重均分子量为 74,844Da的解聚海参糖胺聚糖分子量测试报告；-7重均分子量为 80,336Da的解聚海参糖胺聚糖分子量测试报告；-8重均分子量为 84,481Da的解聚海参糖胺聚糖分子量测试报告；-9重均分子量为 90,919Da的解聚海参糖胺聚糖分子量测试报告；-10重均分子量为 95,821Da的解聚海参糖胺聚糖分子量测试报告；-11重均分子量为 10,1250Da的解聚海参糖胺聚糖分子量测试报告;-12重均分子量为 10,3998Da的解聚海参糖胺聚糖分子量测试报告; 图 2-13重均分子量为 10,9161Da的解聚海参糖胺聚糖分子量测试报告；
图 2-14重均分子量为 115,268Da的解聚海参糖胺聚糖分子量测试报告；
图 2-15重均分子量为 121,017Da的解聚海参糖胺聚糖分子量测试报告。
图 3为解聚海参糖胺聚糖体外抗凝血剂量与凝血时间的线性关系图。
图 3-1 DHG-1体外抗凝血实验结果。
图 3-2 DHG-2体外抗凝血实验结果。
具体实施方式
解聚海参糖胺聚糖的提取方法是指提取自海参的海参糖胺聚糖，经降解和解聚后产 生解聚糖胺聚糖， 收集所需分子量的解聚海参糖胺聚糖。 从海参体壁中提取海参糖胺聚 糖的方法是本领域技术人员所熟悉的， 如中国专利 ZL200910305363.5。
实施例 1
海参糖胺聚糖的提取：
称取玉足海参药材 4.5kg， 水浸 16小时。 将海参体壁淋干水分， 绞碎， 称重并补水 至 36kg， 置 60°C水浴中， 加入 6mol/L氢氧化钠调 pH至 8.2±0.2， 加入 90ml水解蛋白 酶 Alcalase (诺维信 （沈阳） 生物技术有限公司） 搅拌， 酶解 4小时， 85 °C以上灭活 10 分钟， 降温至 49°C±2°C， 加入 6mol/L氢氧化钠调 pH至 8.2±0.2， 再加 9g复合胰酶（无 锡市雪梅酶制剂科技有限公司， 雪梅牌）搅拌酶解 4小时， 煮沸 15分钟， 冷却。 5 离 心， 收集上清液， 加入 6mol/L盐酸调 pH至 2.5±0.2， 4°C冷藏 2小时， 离心， 收集上清 液， 加入 6mol/L氢氧化钠调 pH至 7.5±0.2， 加入 0.8倍体积的乙醇， 4°C静置 12h。
离心， 收集沉淀称重， 加 8倍重量的蒸馏水， 加热至 85 °C±2°C， 待完全溶解后， 加入 6mol/L氢氧化钠调 pH至 9.0±0.2，加入氯化钙至溶液中氯化钙浓度达到 3% (w/v)， 升温至 92°C保持 15分钟， 冷却至室温， 4°C离心， 收集上清液， 用饱和碳酸钠溶液调 pH至 11.0±0.1， 离心， 收集上清液， 用 6mol/L盐酸调 pH至 6.0±0.1， 加 1倍体积乙醇， 4°C冷藏 12h;
冷藏液离心， 收集沉淀称重， 加 2倍体积蒸馏水， 加热使其充分溶解， 加乙酸钾使 其最终浓度为 2mol/L， 4°C静置 12h。 离心， 收集沉淀称重， 加 2倍体积蒸馏水， 加热 使其充分溶解，加乙酸钾使其最终浓度为 2mol/L， 4°C静置 12h。离心，沉淀用冷 2mol/L 乙酸钾溶液洗涤三次， 然后依次用 80%乙醇、 95%乙醇、 无水乙醇洗涤， 待乙醇挥发尽 后 80°C干燥， 称重， 得粗品 A。 粗品 A加 0.05mol/L、 pH6.0的 HAc-NaAc缓冲液溶解制成 2%溶液上柱，溶液通过 纤维素层析柱后，用 1.5倍柱体积的 0.4mol/LNaCl的 HAc-NaAc缓冲液 CpH6.0±0.1)洗涤， 再用 Imol/LNaCl的 HAc-NaAc缓冲液 CpH6.0±0.i;>洗脱， 根据紫外检测仪在 220nm处数 值的变化速度收集洗脱液， 置 60°C水浴中， 用 NaOH调 pH至 11±0.1， 按体积量加入 3%双氧水， 保持 4小时， 冷却， 离心， 收集上清液， 用 HC1调 pH至 7.2±0.1， 加 1倍 量乙醇， 4°C静置 12h。
离心， 收集沉淀， 依次用 80%乙醇、 95%乙醇、 无水乙醇洗涤， 得粗品 B。
粗品 B用蒸馏水溶解成 5%的溶液，用截留分子量 1万的超滤膜浓缩至原体积的 1/2， 补加水至原体积， 再超滤至 1/2体积， 再加水重复一次， 超滤液冷冻干燥， 得海参糖胺 聚糖，
该实例得到的海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-1 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 128,024Da， D值为 1.26 (图谱见 图 2-1 )
实施例 2
解聚海参糖胺聚糖的制备
实施例 2-1
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 4h 50min。 将该溶液用 0.1mol/l的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 54,500Da〜57,000Da， 0值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-2 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 54,876Da， D值为 1.28 (图谱见 图 2-2)
实施例 2-2 将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 4h 20min。 将该溶液用 0.1mol/l的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 58,000Da〜62,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-3 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 60,915Da， D值为 1.36 (图谱见 图 2-3 )
实施例 2-3
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40 °C进行控制解聚 3h 50min。 将该溶液用 0.1mol/l的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 63,000Da〜67,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-4 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 64,904Da， D值为 1.34 (图谱见 图 2-4)
实施例 2-4
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 3h 20min。 将该溶液用 0.1mol/l的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 68,000Da〜72,000Da， 0值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-5 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 71,147Da， D值为 1.38 (图谱见 图 2-5 )
实施例 2-5
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 2h 55min。 将该溶液用 0.1mol/l的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 73,000Da〜77,000Da， 0值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-6 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 74,844Da， D值为 1.26 (图谱见 图 2-6)
实施例 2-6
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 2h 30min。 将该溶液用 0.1mol/l的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 78,000Da〜82,000Da， 0值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-7 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 80,336Da， D值为 1.33 (图谱见 图 2-7)
实施例 2-7
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 2h 5min。 将该溶液用 0.1mol/l的氢 氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺聚 糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 83,000Da〜87,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-8 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 84,481Da， D值为 1.29 (图谱见 图 2-8)
实施例 2-8
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 lh 40min。 将该溶液用 O.lmol/1的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 88,000Da〜92,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-9 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 90,919Da， D值为 1.26 (图谱见 图 2-9)
实施例 2-9
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 lh 15min。 将该溶液用 O.lmol/Ι的 氢氧化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺 聚糖的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 93,000Da〜97,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-10)， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 95,821Da， D值为 1.27 (图谱见 图 2-10)
实施例 2-10
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40 °C进行控制解聚 55min。将该溶液用 O.lmol/1的氢氧 化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺聚糖 的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 98,000Da〜102,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-11 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 101,250Da， D值为 1.24 (图谱见 图 2-11 )
实施例 2-11
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 40min。将该溶液用 O.lmol/Ι的氢氧 化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺聚糖 的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 103,000Da〜107,000Da， 0值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-12)， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 103,998Da， D值为 1.26 (图谱见 图 2-12)
实施例 2-12
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40 °C进行控制解聚 30min。将该溶液用 O.lmol/1的氢氧 化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺聚糖 的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 108,000Da〜112,000Da， 0值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-13 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 109,161Da， D值为 1.22 (图谱见 图 2-13 )
实施例 2-13
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40°C进行控制解聚 20min。将该溶液用 O.lmol/Ι的氢氧 化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺聚糖 的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 113,000Da〜117,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-14)， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 115,268Da， D值为 1.38 (图谱见 图 2-14)
实施例 2-14
将上述实施例 1中的海参糖胺聚糖纯品用 5%醋酸配成 5%的溶液，加入 30%的双氧 水使溶液中双氧水的浓度为 3%， 40 °C进行控制解聚 10min。将该溶液用 0.1mol/l的氢氧 化钠中和至中性， 加入 3倍体积的乙醇进行醇沉， 静置， 离心， 得到解聚海参糖胺聚糖 的粗品。
该粗品干燥， 溶于 5倍重量的水中， 过 sephadex-G75柱， 用 0.5mol/l的氯化钠进行 洗脱， 脱去盐及小分子杂质， 脱盐后的样品冷冻干燥既得 55g解聚海参糖胺聚糖， 其分 子量都在 118,000Da〜122,000Da， 。值< 1.5， 纯度为 98%以上。
该实例得到的解聚海参糖胺聚糖， 经示差折光检测器 (RID-10A,岛津)可得到纯度为 99.0%的纯品 （图谱见图 1-15 )， 经该实例得到的解聚海参糖胺聚糖经凝胶柱 (TSK gel G4000PWXL, TOSOH)色谱分析知该产品的重均分子量 121,017Da， D值为 1.36 (图谱见 图 2-15 )
实施例 3
将以上获得的 40.0g海参糖胺聚糖或者解聚海参糖胺聚糖加入 80g甘露醇、加入注 射用水 1000ml溶解， 经过超滤、 灌装、 冻干， 得到 1000瓶注射用海参糖胺聚糖或者解 聚海参糖胺聚糖的冻干粉针剂。
实施例 4
海参糖胺聚糖和解聚海参糖胺聚糖的药效学实验
4.1体外抗凝血实验
4.1.1 试验材料
供试样品：
名称： 解聚海参糖胺聚糖， 以下缩写： DHG; DHG-1 (实施例 2-1 )、 DHG-2 (实 施例 2-6); 配制： 精密吸取后以注射用生理盐水稀释至所需浓度。
试验动物
品系： 兔； 来源： 上海陈行实验用兔有限公司； 性别： 雄性； 体重： 1850克； 动物合格证号： SCXK (沪） 2008-0010。
4.1.2 试验仪器
血小板聚集凝血因子分析仪 （ 型号 LG-PABER北京世帝科学仪器公司；)。
4.1.3实验方法
实验当日， 于样品池中分别加入兔血浆 80μ1、 0.9%氯化钠溶液 10μ1， 预热 180s后， 加入 1 %氯化钙溶液 10μ1， 立即混匀， 避免产生气泡， 用血小板聚集凝血因子分析仪开 始计算时间， 记录各样品池凝结时间， 即为空白。
精密量取对照品溶液， 用 0.9 %氯化钠溶液稀释成不同浓度的溶液， 即为不同浓度 的样品溶液 DHG-1 (40.0 g/ml〜200.(^g/ml)、 DHG-2 (30.0 g/ml〜200.(^g/ml)。
用 ΙΟμΙ不同浓度的样品溶液代替 10μ1 0.9%氯化钠溶液， 分别测定各浓度的样品溶 液的血浆凝结时间。 每个浓度平行测定 4次， 求平均值。
4.1.4 实验结果
实验结果显示样品终浓度在 DHG-1 (40.0 g/ml〜200.(^g/ml)、 DHG-2 (30.0 g/ml〜 200.0μ§/ιη1 ) 剂量范围内， 随剂量增加凝血时间延长， 凝血时间延长递增趋势缓和。 因 此， 解聚海参糖胺聚糖组合物用于抗凝血更具安全性， 可控性。
DHG-1体外抗凝血实验结果
Figure imgf000015_0001
4.2 皮下注射解聚海参糖胺聚糖和天然海参糖胺聚糖对大鼠凝血系统的影响
4.2.1 试验材料
供试样品： 名称： 天然海参糖胺聚糖、低分子海参糖胺聚糖（54,876Da、 60,915Da、 74,844Da 90,919Da); 配制： 精密吸取后以注射用生理盐水稀释至所需浓度。
4.2.2 试验动物
品系： SD大鼠； 来源： 上海西普尔 -必凯实验动物有限公司； 性别： 雄性； 体 重： 180-200克； 动物合格证号： SCXK (沪） 2008-0016; 饲养： 动物饲养于正压净化 通风动物房内， 室温 23±1 °C， 湿度 50 70 %， 人工照明模拟昼夜变化， 自由进食与饮 水。
4.2.3 试验仪器
自动凝血分析仪 Sysmex CA-1500
4.2.4 实验方法
将 SD大鼠每组 10只， 分为给药组， 阴性对照组 （皮下注射生理盐水 0.5ml)， 两 个剂量组（10 20mg/kg)皮下注射给药，体积 0.5ml。皮下注射给药后不同时间段（0.5h 1.0h 2.0h 4h 6h 8h 12h) 腹主动脉采血测定凝血酶原时间 （PT)、 活化部分凝 血活酶时间 （ΑΡΤΤ) 和凝血酶时间 （ΤΤ) 数值， 参见表 2 4
各组动物在手术前 lOmin用 3%速可眠腹腔注射麻醉（O. lml/lOOg体重）， 仰卧固 定后腹腔手术， 用一次性 3.2%柠檬酸钠抗凝真空采血管采血。
4.2.5 试验结果
解聚海参糖胺聚糖和天然海参糖胺聚糖 10mg/kg和 20mg/kg对 APTT TT ΡΤ 产生明显影响， 不同重均分子量的解聚海参糖胺聚糖随时间递增抗凝血活性递增，抗 凝血延长率到达峰值的时间 2-8h之间， 重均分子量较小的达到峰值时间比重均分子 量大的达到峰值时间早。 天然分子段的海参糖胺聚糖皮下注射后产生的抗凝血作用 10mg/kg 剂量下， 6h时达到峰值； 20mg/kg 剂量下， 6h时达到峰值。 解聚海参糖氨 基糖 10mg/kg 20mg/kg剂量下不同分子段起效时间以及到达作用峰值的时间不同， 皮下注射解聚海参糖胺聚糖对 APTT产生的极显著影响使得 APTT延长超过了 150%— 250%的范围， 参见表 2-1 2-2、 2-3 2-4
DHG大鼠皮下注射抗凝血实验结果
好段 凝血时间 (s)
( 10mg kg) 0.5h l.Oh 2h 4h 6h 8h 12h
54,876Da 22.3±1.1 28.1 1.4 35.3±0.9 34.5±0.6 21.9 0.7 20.1 0.9 17.4 0.3
APTT
60,915Da 21.5±0.9 22.6 0.5 29.0 0.7 31.9 0.8 25.1 0.4 22.4±0.6 17.7 0.4
74,844Da 18.7 0.5 21.5±1.0 26.3±0.6 27.6 0.7 21.6 0.5 20.2 0.6 17.4 0.5 %£ΖΌΙ %26'ξΖ %OVZV %ξΥ£Ζ %ΖΟ'ξ\ %U'U %ZO'L
%li'LZ %ξΖ'6ί %ΖίΊ9 % 9'9V %ΖΥξί %9V6l Βα6Ι6'06
%8£'^T %ζο'θί %6νςν %ςνο %οο'ςί %ζς'θί ttn'tL
丄丄 <IV
%ZO'Ll %Zi'VV %Ζξ' 9 %9^'80Τ %ξί'ί6 %0ξ'\9 %69'Οξ ^aSI6'09
%66 T %68'6Ζ %ZZ'LV %\ 9Zl % Ζ'ξί\ %69'ΟΟΤ %0V99 ^Q9Z,8'fS
mi ¾8 ¾9 qi m (§5[/§UI0T)
目 傘^ 教 OHa
YO tlf Ό ς·0 9ΌΤ8Ό17 ς'θ ί'Ι 9·0 i'O tOf ΓΙ Γ6£
9'0 'ZV VO L'VV 6ΌΤΓ917 ξ'θ ί'ςν 8'0 9'O i'ZV ΓΙ 6'6£
£Ί 6 t 9'θ ς'ίν ς-Q L'VV 8·0 6 t
Figure imgf000017_0001
Βα6Ι6'06
9Ό .St VO i'VV ς-O i'LV 6.0 Δ.Ο 9.617 t.I S.St ΓΙ tt 'tL XX
Figure imgf000017_0002
mi ¾8 ¾9 qi wi o (§>l/§uioi) Τ ε ο rsi τ οτε ςτ to osi ro rsi ε ο 6ti to rsi
ί'ο ς-ςι Γ9ΐ so 9'9i 6' '9 Υθ β'ςι ί'θ ς·ςι νο νςι
Ζ'0 9'ξΙ to 091 ς'ο ς·9ΐ ΔΟ 9·9Ι so ^ 91 ί'θ β'ξΐ Υθ ί'ξΙ ^α6ϊ6'06
S O Γ9Ι ξ'0 ί'91 9'0 VLl Δ0 ΓΔΙ Γ9Ι ξθ β ξΐ tt 'tL Id
90 90 ΓΔΙ O L'Ll SO 6ΔΙ SO ^ ΔΙ VO i'91 90 Γ9Ι ^aSI6'09
ξ'θ ί'ςΐ SO ε·9Ι Ε'ΟΤΟ'ΔΤ 8·0 ^ 8Ι 0 ε·6Ι 9Ό t l ^ 91 Ba9
mi ¾8 ¾9 qi mi o (§>l/§uioi)
νο γςι νο ς-ςι t.O 6.tl t'O fOTO'^T 8Ό 0 I 90 sti φ
9'0=F9'9T νθ ς'61 t.O 6.81 ε'ο ε'Δΐ 9' 9'^T
ΔΌ ε'ΔΙ ε'Ο Δ'6Ι O L'OZ ς'ο β'ΐζ t.o 0.61 90 ΓΔΙ ^Q6I6'06
OLtOOO/tlOZSD/lDd 99 8Ϊ/ 0Ζ OAV 项目 分子段 时间延长率(％)
( 10mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h
54,876Da 8.96% 16.52% 27.20% 23.31% 11.25% 7.04% 4.65%
60,915Da 7.31% 9.68% 15.43% 19.04% 15.83% 12.74% 5.69%
PT
74,844Da 5.37% 7.79% 12.31% 16.18% 9.24% 6.87% 4.21%
90,919Da 4.01% 6.86% 8.37% 10.89% 7.64% 5.34% 3.81% 天然 1.97% 3.87% 4.84% 6.69% 8.62% 6.48% 3.17% 项目 分子段 时间延长率(％)
( 10mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h
54,876Da 18.64% 26.09% 33.52% 31.52% 18.02% 10.21% 6.42% 60,915Da 16.55% 18.67% 25.48% 28.24% 19.50% 11.02% 7.61%
TT
74,844Da 10.64% 13.09% 16.77% 24.98% 15.85% 9.36% 6.01% 90,919Da 6.35% 8.21% 11.68% 14.83% 9.57% 7.32% 4.23% 天然 2.10% 5.32% 7.21% 9.52% 13.08% 10.27% 3.97% 表 2-3 DHG大鼠皮下注射抗凝血实验结果
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000019_0002
Figure imgf000019_0003
OLtOOO/nOZSLJ/∑Jd OAV 项目 分子段 时间延长率(％)
(20mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h
54,876Da 52.88% 57.35% 71.20% 81.34% 73.93% 65.37% 54.69%
60,915Da 48.36% 54.27% 61.04% 74.21% 87.55% 79.62% 58.78% ττ
74,844Da 44.31% 50.31% 58.39% 73.22% 82.55% 65.77% 51.25%
90,919Da 32.62% 41.41% 47.90% 55.02% 58.11% 53.28% 40.05% 天然 9.98% 18.66% 24.16% 34.20% 43.26% 38.68% 30.47%
4.3 解聚海参糖胺聚糖对大鼠动静脉导管血栓形成模型的影响
4.3.1 试验材料
供试样品- 名称： 解聚海参糖胺聚糖 （DHG); 配制： 精密吸取后以注射用生理盐水稀释至 所需浓度。对照样品：名称：肝素；来源：国药集团化学试剂有限公司；批号： F20091029; 含量： 150U/mg; 配制： 精密称取后以注射用生理盐水溶解并稀释至所需浓度。
试验动物： 品系： SD大鼠； 来源： 上海西普尔 -必凯实验动物有限公司； 性别： 雄性； 体重： 180-220克； 动物合格证号： SCXK (沪） 2008-0016; 饲养： 动物饲养于 正压净化通风动物房内， 室温 23±1 °C， 湿度 50〜70%， 人工照明模拟昼夜变化， 自由 进食与饮水。
4.3.2 试验仪器
BS 110 s型电子天平， SARTORIUS公司生产， 最小称量值 0.1mg。
4.3.3 试验方法
将 SD大鼠每组 10只分为不同的给药组， 阴性对照组 （生理盐水 lml/kg)， 阳 性对照低分子肝素组 （2mg/kg)。 所有药物均为皮下注射给药， 体积 0.5ml。
各组动物在手术前 lOmin用 12%的水合氯醛腹腔注射麻醉 （350〜400 mg/kg) 后， 仰卧固定， 切开颈部皮肤， 分离左侧颈动脉和右侧颈外静脉， 以一旁路管连接， 管中置一 7cm长 4号手术丝线。 分别在给药 2小时后开放血流 15分钟， 然后取出 丝线称重， 减去丝线重量， 即为血栓湿重。 计算出各试验组的血栓湿重平均值及标 准差， 用 t-检验同生理盐水组进行比较。并按下式计算各试验组的血栓湿重抑制率: 血栓抑制率 (%) =血栓湿重 （溶細 -血誦 （細) x薦
血栓湿重 （溶剂组） 4.3.4 试验结果
参见表 3， 阳性药和受试药物在给药后测试都可以显著抑制血栓的形成。受试药 物对血栓形成的抑制作用显著。
表 3 DHG对大鼠动静脉导管血栓形成模型的影响
Figure imgf000021_0001
与阴性组相比较： * P<0.05， ** P<0.01
4.4 不同分子量段解聚海参糖胺聚糖组合物皮下注射对大鼠凝血系统的影响
4.4.1 试验材料
供试样品：
名称： 解聚海参糖胺聚糖 54,876Da、 74,844Da; 天然海参糖胺聚糖； 来源： 上 海开润生物医药有限公司； 配制： 精密吸取后以注射用生理盐水稀释至所需浓度。 4.4.2 试验动物
品系： SD大鼠； 来源： 上海西普尔 -必凯实验动物有限公司； 性别： 雄性； 体 重： 180-220克； 动物合格证号： SCXK (沪） 2008-0016; 饲养： 动物饲养于正压净化 通风动物房内， 室温 23±1 °C， 湿度 50〜70 %， 人工照明模拟昼夜变化， 自由进食与饮 水。
4.4.3 试验仪器
自动凝血分析仪 Sysmex CA-1500
4.4.4 实验方法
将 SD大鼠每组 10只， 分成不同的给药组， 阴性对照组 （皮下注射生理盐水 0.5ml ), 解聚海参糖胺聚糖不同分子量段（54,876Da、 74,844Da)， 天然海参糖胺聚糖 组合物剂量比为 1 : 1 ( 10mg/kg) 皮下注射给药， 空白注射生理盐水体积 0.5ml。 皮下 注射给药后不同时间段腹主动脉采血测定血浆活化部分凝血活酶时间 （APTT)、 凝血 酶原时间 （PT)、 凝血酶时间 (ΤΤ)数值， 参见表 5。 各组动物在手术前 lOmin用 3%速可眠腹腔注射麻醉 （O. lml/lOOg体重）， 仰卧 固定后腹腔手术， 用一次性 3.2%柠檬酸钠抗凝真空采血管采血。
.5 试验结果
实验结果显示， 不同分子量段的解聚海参糖胺聚糖组合物皮下注射具有显著延 长 APTT TT活性， 并且可以克服单一分子量段起效时间缓慢或者持续时间短， 实 验数据参见表 4-1 4-2
表 4-1 不同分子段 DHG大鼠皮下注射抗凝血实验结果
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000023_0001
表 4-2 不同分子段 DHG大鼠凝血时间延长率 项目 组合物 时间延长率(％)
(20mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h
54,876Da
( 10mg kg) + 74,844Da 106.65% 159.85% 223.83% 290.36% 303.58% 260.48% 159.61%
APTT (10mg/kg)
60,915Da
(10mg/kg)+天然 64.56% 96.25% 143.82% 201.41% 278.11% 206.51% 128.37% (lOmg/kg) 项目 分子段 时间延长率(％)
(20mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h
54,876Da
( lOmg kg) + 74,844Da 16.95% 20.23% 27.64% 32.15% 33.47% 30.51% 19.94%
PT (lOmg/kg)
60,915Da
(10mg/kg)+天然 8.95% 13.52% 18.72% 25.95% 30.64% 26.85% 17.01% (lOmg/kg) 项目 分子段 时间延长率(％)
(20mg/kg) 0.5h l.Oh 2h 4h 6h 8h 12h
54,876Da
( lOmg kg) + 74,844Da 50.13% 55.26% 67.39% 76.26% 82.71% 74.15% 55.23%
TT (lOmg/kg)
60,915Da
(10mg/kg)+天然 38.52% 48.79% 52.95% 60.87% 74.52% 62.28% 49.35% (lOmg/kg)

Claims

权 利 要 求 书
1. 重均分子量大于 54,500Da的解聚海参糖胺聚糖或天然分子段的海参糖胺聚糖中 的一段或者一段以上， 在制备防治动脉血栓栓塞疾病药物中的应用。
2. 根据权利要求 1所述的应用，其特征在于，所述血栓栓塞疾病包括动脉粥样硬化 血栓性疾病、 静脉血栓栓塞性疾病、 血液高凝状态、 手术后形成的血栓或者是预防手术 后血栓的形成。
3. 根据权利要求 1〜2任一项所述的应用， 其特征在于， 所述的重均分子量大于 54,500Da的解聚海参糖胺聚糖和天然分子段的海参糖胺聚糖包括重均分子量大于 54,500Da的解聚海参糖胺聚糖任意一重均分子量和天然分子段的海参糖胺聚糖，或者是 重均分子量大于 54,500Da的解聚海参糖胺聚糖任意一重均分子量和天然分子段的海参 糖胺聚糖的多段混合物。
4. 根据权利要求 1〜2任一项所述的应用， 其特征在于， 所述解聚海参糖胺聚糖的 重均分子量为：
54,500Da〜57,000Da、 57,010Da〜62,990Da、 63,000Da〜 67,000Da、 67,010Da〜 72,990Da、 73,000Da〜77,000Da、 77,010Da〜82,990Da、 83,000Da〜87,000Da、 87,010Da〜 92,990Da、 93,000Da〜 97,000Da、 97,0 lODa〜 102,900Da、 103,000Da〜 107,000Da、 107,010Da〜 112,990Da、 113,000Da〜 117,900Da或 118,000Da〜 122,050Da中的任意一段。
5. 根据权利要求 1〜2任一项所述的应用， 其特征在于， 所述解聚海参糖胺聚糖的 重均分子量为：
54,500Da〜57,000Da、 58,000Da〜62,000Da、 63,000Da〜 67,000Da、 68,000Da〜 72,000Da、 73,000Da〜77,000Da、 78,000Da〜82,000Da、 83,000Da〜87,000Da、 88,000Da〜 92,000Da、 93,000Da〜 97,000Da、 98,000Da〜 102,000Da、 103,000Da〜 107,000、 108,000Da〜112,000Da、 113,000Da〜117,000Da、 118,000Da〜122,000Da中的任意一段。
6. 根据权利要求 1〜2任一项所述的应用， 其特征在于， 所述药物包括治疗有效量 的所述的解聚海参糖胺聚糖和药学上可接受的载体，为静脉或皮下注射给药的注射液或 者是冻干粉针剂。
7. 根据权利要求 3所述的应用，其特征在于，所述药物包括治疗有效量的所述的解 聚海参糖胺聚糖和药学上可接受的载体，为静脉或皮下注射给药的注射液或者是冻干粉 针剂。
8. 根据权利要求 4所述的应用，其特征在于，所述药物包括治疗有效量的权利要求 所述的解聚海参糖胺聚糖和药学上可接受的载体，为静脉或皮下注射给药的注射液或者 是冻干粉针剂。
9. 根据权利要求 5所述的应用，其特征在于，所述药物包括治疗有效量的所述的解 聚海参糖胺聚糖和药学上可接受的载体，为静脉或皮下注射给药的注射液或者是冻干粉 针剂。
PCT/CN2014/000470 2013-05-13 2014-05-07 海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用 WO2014183466A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/890,854 US20160082051A1 (en) 2013-05-13 2014-05-07 Use of sea cucumber glycosaminoglycan in preparing medicine for prevention and treatment of thromboembolic disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310175750.8 2013-05-13
CN201310175750.8A CN104147040A (zh) 2013-05-13 2013-05-13 海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用

Publications (1)

Publication Number Publication Date
WO2014183466A1 true WO2014183466A1 (zh) 2014-11-20

Family

ID=51872805

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/000470 WO2014183466A1 (zh) 2013-05-13 2014-05-07 海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用

Country Status (3)

Country Link
US (1) US20160082051A1 (zh)
CN (1) CN104147040A (zh)
WO (1) WO2014183466A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112083083A (zh) * 2019-06-14 2020-12-15 苏州融析生物科技有限公司 一种海参糖胺聚糖含量的测定方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1579415A (zh) * 2004-05-20 2005-02-16 陈任重 玉足海参糖胺聚糖针剂及其制备方法
CN101624426A (zh) * 2009-08-07 2010-01-13 黑龙江红豆杉药业有限责任公司 一种玉足海参糖胺聚糖的提取方法
CN103417565A (zh) * 2012-05-17 2013-12-04 上海开润生物医药有限公司 解聚海参糖胺聚糖在制备防治血栓性疾病药物中的应用
CN103536621A (zh) * 2012-07-11 2014-01-29 上海开润生物医药有限公司 海参糖胺聚糖在制备治疗冠脉综合症药物中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101724086B (zh) * 2009-11-25 2012-09-26 深圳海王药业有限公司 低聚凤梨参糖胺聚糖及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1579415A (zh) * 2004-05-20 2005-02-16 陈任重 玉足海参糖胺聚糖针剂及其制备方法
CN101624426A (zh) * 2009-08-07 2010-01-13 黑龙江红豆杉药业有限责任公司 一种玉足海参糖胺聚糖的提取方法
CN103417565A (zh) * 2012-05-17 2013-12-04 上海开润生物医药有限公司 解聚海参糖胺聚糖在制备防治血栓性疾病药物中的应用
CN103536621A (zh) * 2012-07-11 2014-01-29 上海开润生物医药有限公司 海参糖胺聚糖在制备治疗冠脉综合症药物中的应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112083083A (zh) * 2019-06-14 2020-12-15 苏州融析生物科技有限公司 一种海参糖胺聚糖含量的测定方法

Also Published As

Publication number Publication date
US20160082051A1 (en) 2016-03-24
CN104147040A (zh) 2014-11-19

Similar Documents

Publication Publication Date Title
JP6755980B2 (ja) 高純度ヘパリンおよびその製造方法
CN103285031B (zh) 解聚海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用
JPH0323528B2 (zh)
CN100525777C (zh) 一种解聚海参糖胺聚糖组合物及其制备方法和用途
JPH03243601A (ja) 血液凝固の制御能をもつムコ多糖組成物およびその製造方法
CN111499770A (zh) 一种褐藻岩藻多糖及其制备方法和改善肠道菌群的应用
WO2014183466A1 (zh) 海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用
EP4360638A1 (en) Application of sulfate gluco-galacto-oligosaccharide in preparation of anticoagulant and/or antithrombotic drug
KR101780643B1 (ko) 효소분해를 이용한 헤파린의 정제 방법
CN104725532B (zh) 一种精确定量控制肝素/类肝素中硫酸软骨素及硫酸皮肤素含量的方法
CN103536621A (zh) 海参糖胺聚糖在制备治疗冠脉综合症药物中的应用
WO2014183467A1 (zh) 黑海参糖胺聚糖在制备防治血栓栓塞疾病药物中的应用
CN103417565A (zh) 解聚海参糖胺聚糖在制备防治血栓性疾病药物中的应用
CN103830263A (zh) 解聚海参糖胺聚糖在制药中的应用
CN103041176A (zh) 见血清提取物及其用途
AU2015203727B2 (en) High Purity Heparin and Production Method Therefor
CN103830264A (zh) 解聚海参糖胺聚糖在制备防治血栓性疾病药物中的应用

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: 14797534

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 14890854

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14797534

Country of ref document: EP

Kind code of ref document: A1