WO2018149320A1 - 去抗凝肝素衍生物及其用于炎症性肠病的治疗 - Google Patents

去抗凝肝素衍生物及其用于炎症性肠病的治疗 Download PDF

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WO2018149320A1
WO2018149320A1 PCT/CN2018/075359 CN2018075359W WO2018149320A1 WO 2018149320 A1 WO2018149320 A1 WO 2018149320A1 CN 2018075359 W CN2018075359 W CN 2018075359W WO 2018149320 A1 WO2018149320 A1 WO 2018149320A1
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factor
heparin
iia
equal
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French (fr)
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邢新会
王怡
季洋
张翀
常智杰
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清华大学
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    • 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
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • 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
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the present invention relates to the preparation of an anti-coagulation heparin derivative, and its use for the prevention and/or treatment of inflammatory bowel disease.
  • IBD Inflammatory Bowel Disease
  • UC Ulcerative Colitis
  • CD Crohn's disease
  • Therapeutic drugs for UC have been studied in animal models and clinical trials for more than 40 years.
  • Traditional treatment of UC patients generally using thiopurine immunosuppressive agents, corticosteroid anti-inflammatory drugs, etc.
  • thiopurine immunosuppressive agents corticosteroid anti-inflammatory drugs, etc.
  • Cen, Y., et al. PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function. J Biol Chem, 2015.290 ( 33): p. 20580-9.
  • LMWHs Low Molecular Weight Heparins
  • Heparin is a type of sulfated, polydisperse, linear glycosaminoglycans (GAGs) and is one of the most important anticoagulant drugs. It is widely used clinically to prevent thromboembolic diseases. In addition, heparin and its derivatives have a wide range of biological activities, including coordination of cell adhesion, regulation of cell growth and proliferation, developmental processes, cell surface binding lipoprotein lipase and other proteins, neovascularization, viral invasion and tumor metastasis, etc. .
  • LMWHs The anti-inflammatory and other biological activities of LMWHs also play a role in the treatment of UC. Many clinical UC patients are relieved of symptoms after using LMWHs (Lean, QY, et al., Heparins in ulcerative colitis: proposed mechanisms of action and potential reasons for inconsistent clinical outcomes .Expert Rev Clin Pharmacol, 2015. 8(6): p. 795-811.). Heparanase is an important target for heparin drugs. Inhibiting heparanase activity and preventing further damage of intestinal mucosa is one of the therapeutic mechanisms (Waterman, M., et al., Heparanase upregulation by colonic epithelium). In inflammatory bowel disease. Mod Pathol, 2007.20(1): p.8-14.). However, at present, LMWHs have problems in the treatment of UC, such as the structure-activity relationship is not clear.
  • Heparin drugs are often used clinically in patients with inflammatory bowel disease with hypercoagulable state.
  • heparin itself has anticoagulant function, and heparin also has anti-inflammatory effect, but the retention of anticoagulant activity always induces bleeding. The risk of these side effects limits the use of heparin in inflammatory diseases that are not associated with coagulation.
  • anticoagulant heparin derivatives for the treatment of inflammatory bowel disease has not been reported.
  • the present inventors attempted to perform anticoagulant treatment of heparin by using commercially available heparin, commercially available low molecular weight heparin enoxaparin or the like by several different methods described later. Modification is critical to improve the therapeutic effect of inflammatory bowel disease.
  • Each of the treated anti-coagulated heparin derivatives showed good IBD therapeutic effects relative to various heparin or (ultra)low molecular weight heparin having anticoagulant activity.
  • the inventors of the present invention are committed to innovative research on heparin industrial technology, using a Maltose Binding Protein (MBP) fusion expression technology to achieve a series of heparinase (MBP-HepI, MBP-HepII, MBP-, respectively).
  • MBP Maltose Binding Protein
  • HepIII can be found in the high activity, soluble expression and industrial production of Chinese patents ZL200410038098.6, ZL201010259905.2, and ZL201010259913.7, respectively.
  • the series heparinase is listed as the Chinese Pharmacopoeia standard enzyme.
  • heparin is first modified to obtain an anticoagulant heparin derivative, and then heparinase is used to degrade the anticoagulant heparin derivative to obtain an enzymatically degraded low molecular weight and/or ultra low molecular weight anti-coagulation heparin derivative.
  • the present inventors attempted to de-coagulation treatment of commercially available heparin by several different methods described later, and then subjected to controlled degradation by heparinase I.
  • the results showed that the low molecular weight and ultra-low molecular weight de-anti-coagulation heparin derivatives obtained by heparinase degradation can significantly improve the therapeutic effect of inflammatory bowel disease compared with high molecular weight anti-coagulation heparin.
  • the invention relates to the following:
  • a de-anti-coagulated heparin derivative having an anti-Xa factor of 70 IU/mg or less, preferably an anti-Xa factor of 60 IU/mg or less, preferably an anti-Xa factor of 50 IU/mg or less, preferably an anti-Xa factor of 40 IU or less.
  • the anti-Xa factor is 30 IU/mg or less, preferably the anti-Xa factor is 20 IU/mg or less, preferably the anti-Xa factor is 10 IU/mg or less, and
  • the anti-IIa factor is less than or equal to 175 IU/mg, preferably the anti-IIa factor is less than or equal to 170 IU/mg, preferably the anti-IIa factor is less than or equal to 160 IU/mg, preferably the anti-IIa factor is less than or equal to 150 IU/mg, and preferably the anti-IIa factor is less than or equal to 140 IU/mg.
  • the anti-IIa factor is less than or equal to 130 IU/mg, preferably the anti-IIa factor is less than or equal to 120 IU/mg, preferably the anti-IIa factor is less than or equal to 110 IU/mg, preferably the anti-IIa factor is less than or equal to 100 IU/mg, and preferably the anti-IIa factor is less than or equal to 90 IU/mg.
  • the anti-IIa factor is less than or equal to 80 IU/mg, preferably the anti-IIa factor is less than or equal to 70 IU/mg, preferably the anti-IIa factor is less than or equal to 60 IU/mg, preferably the anti-IIa factor is less than or equal to 50 IU/mg, and preferably the anti-IIa factor is less than or equal to 40 IU/mg.
  • the anti-IIa factor is 30 IU/mg or less, preferably the anti-IIa factor is 20 IU/mg or less, and preferably the anti-IIa factor is 10 IU/mg or less.
  • an anticoagulant heparin derivative for the preparation of a medicament for treating an inflammatory bowel disease, and a inflammatory bowel disease-related complication and a disease having a similar pathogenesis, wherein the inflammatory bowel disease is associated with concurrent Diseases with similar pathogenesis and disease include, but are not limited to, irritable bowel syndrome, arthritis and other parenteral complications including ankylosing spondylitis, gangrenous pyoderma, nodular erythema, ulceris, uveitis, sclera Outer inflammation and primary sclerosing cholangitis.
  • the anti-Xa factor of the anti-coagulation heparin derivative is 70 IU/mg or less, preferably the anti-Xa factor is 60 IU/mg or less, preferably the anti-Xa factor is 50 IU or less.
  • /mg preferably an anti-Xa factor of 40 IU/mg or less, preferably an anti-Xa factor of 30 IU/mg or less, preferably an anti-Xa factor of 20 IU/mg or less, preferably an anti-Xa factor of 10 IU/mg or less, and
  • the anti-IIa factor is less than or equal to 175 IU/mg, preferably the anti-IIa factor is less than or equal to 170 IU/mg, preferably the anti-IIa factor is less than or equal to 160 IU/mg, preferably the anti-IIa factor is less than or equal to 150 IU/mg, and preferably the anti-IIa factor is less than or equal to 140 IU/mg.
  • the anti-IIa factor is less than or equal to 130 IU/mg, preferably the anti-IIa factor is less than or equal to 120 IU/mg, preferably the anti-IIa factor is less than or equal to 110 IU/mg, preferably the anti-IIa factor is less than or equal to 100 IU/mg, and preferably the anti-IIa factor is less than or equal to 90 IU/mg.
  • the anti-IIa factor is less than or equal to 80 IU/mg, preferably the anti-IIa factor is less than or equal to 70 IU/mg, preferably the anti-IIa factor is less than or equal to 60 IU/mg, preferably the anti-IIa factor is less than or equal to 50 IU/mg, and preferably the anti-IIa factor is less than or equal to 40 IU/mg.
  • the anti-IIa factor is 30 IU/mg or less, preferably the anti-IIa factor is 20 IU/mg or less, and preferably the anti-IIa factor is 10 IU/mg or less.
  • the anti-coagulation heparin derivative according to (1) which has a weight average molecular weight of 600 to 8,000, preferably a weight average molecular weight of 1,000 to 7,800, more preferably a weight average molecular weight of 1,500 to 7,500, and further preferably a weight average
  • the molecular weight is from 2,000 to 7,000, the number average molecular weight is from 600 to 6,000, the number average molecular weight is more preferably from 1,200 to 5,800, the number average molecular weight is more preferably from 1,500 to 5,500, and the number average molecular weight is more preferably from 1,800 to 5,000.
  • a method of preparing an anti-coagulation heparin derivative comprising:
  • the de-agglomerated product is then enzymatically digested with an enzyme to obtain an anti-coagulated heparin derivative.
  • a method for treating inflammatory bowel disease, and inflammatory bowel disease-related complications and diseases having similar pathogenesis comprising:
  • the anticoagulated heparin derivative is administered to a subject in need thereof, wherein the de-anti-coagulated heparin derivative is a de-anti-coagulation heparin derivative of the invention.
  • Fig. 1 HE staining of pathological sections of colorectal tissue in Experimental Example 1-3.
  • Figure 2 Results of the full length of the colon in Experimental Examples 1-3, wherein (a) is a photograph of the colorectal and (b) is a histogram showing the length of the colorectal of each group.
  • Figure 4 shows the expression level of tight junction protein ZO-1 in intestinal epithelial cells in Experimental Examples 1-3.
  • Figure 5 shows the level of apoptosis of intestinal epithelial cells in Experimental Example 1-3, wherein (a) is a flow four-quadrant map of Annexin V/PI double staining, and (b) is a graph of percentage of apoptotic cells.
  • Figure 6 is a graph showing the percentage change in body weight of mice in Experimental Examples 4-6.
  • Figure 8 Histological scores of HE staining results of colorectal histopathological sections in Experimental Examples 4-6.
  • Fig. 9 Results of the entire length of the colorectal in Experimental Examples 4-6, wherein (a) is a photograph of the colorectal, and (b) is a histogram showing statistical results of the length of the colorectal of each group.
  • Figure 10 shows the level of apoptosis of intestinal epithelial cells in Experimental Examples 4-6, wherein (a) is a flow four-quadrant map of Annexin V/PI double staining, and (b) is a graph of percentage of apoptotic cells.
  • Figure 11 shows the expression level of tight junction protein ZO-1 in intestinal epithelial cells in Experimental Examples 4-6.
  • Anti-coagulation heparin derivative of the present invention (hereinafter also referred to as anti-coagulation heparin)>
  • the de-anti-coagulation heparin derivative according to the present invention is obtained by subjecting heparin or (super) low molecular weight heparin to de-coagulation treatment.
  • the de-anti-coagulation heparin derivative according to the present invention may also be a substance obtained by controlling degradation by heparinase on the basis of de-anticoagulation modification.
  • Heparin, low molecular weight heparin, and pentose are generally anticoagulant by accelerating the rate at which antithrombin III inactivates clotting factors.
  • the main role of this class of drugs is anti-Xa and anti-IIa activity.
  • anti-Xa activity and anti-IIa activity of heparin drugs it was found that anti-Xa activity is not sensitive to molecular mass, and anti-IIa activity depends on the molecular mass. The greater the molecular mass, the stronger the anti-IIa activity.
  • the inactivation of factor IIa by heparin relies on the formation of a heparin-anti-thrombin-IIa factor triple complex.
  • heparin binds to both antithrombin and factor IIa.
  • heparin must contain at least 18 sugar units. Among them, 13 monosaccharides are required for the role of "bridge", and 5 monosaccharides are required as identification fragments.
  • the average molecular mass of each monosaccharide is 300 Da, so the molecular mass must be above 5400 Da to have anti-IIa activity.
  • the average molecular weight of unfractionated heparin is 15000-19000 Da, most of the molecules are above 5400 Da, and the ratio of anti-Xa to anti-IIa activity is about 1.
  • the average molecular mass of low molecular weight heparin is 4000-5000 Da, and the molecular fragment with molecular mass above 5400 Da is relatively small. Generally, its anti-Xa:anti-IIa activity is about 1.5:1 to 5:1.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, preferably the anti-Xa factor is 60 IU/mg or less, preferably the anti-Xa factor is 50 IU/mg or less, preferably the anti-Xa factor is 40 IU/mg or less, preferably anti-Xa factor.
  • the factor Xa is 30 IU/mg or less, preferably the factor Xa is 20 IU/mg or less, and preferably the factor Xa is 10 IU/mg or less.
  • the anti-IIa factor of the anti-coagulation heparin of the present invention is 175 IU/mg or less, preferably the anti-IIa factor is 170 IU/mg or less, preferably the anti-IIa factor is 160 IU/mg or less, preferably the anti-IIa factor is 150 IU/mg or less, preferably anti-Ia factor.
  • the factor IIa is less than or equal to 140 IU/mg, preferably the anti-IIa factor is less than or equal to 130 IU/mg, preferably the anti-IIa factor is less than or equal to 120 IU/mg, preferably the anti-IIa factor is less than or equal to 110 IU/mg, preferably the anti-IIa factor is less than or equal to 100 IU/mg, preferably anti-IIa factor.
  • the factor IIa is less than or equal to 90 IU/mg, preferably the anti-IIa factor is less than or equal to 80 IU/mg, preferably the anti-IIa factor is less than or equal to 70 IU/mg, preferably the anti-IIa factor is less than or equal to 60 IU/mg, preferably the anti-IIa factor is less than or equal to 50 IU/mg, preferably anti-IIa factor.
  • the factor IIa is 40 IU/mg or less, preferably the anti-IIa factor is 30 IU/mg or less, preferably the anti-IIa factor is 20 IU/mg or less, and preferably the anti-IIa factor is 10 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 175 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 175 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 175 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 175 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 175 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 175 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 175 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 170 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 170 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 170 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 170 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 170 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 170 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 170 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 160 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 160 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 160 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 160 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 160 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 160 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 160 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 150 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 150 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is less than 50 IU/mg, and the anti-IIa factor is 150 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 150 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 150 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 150 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 150 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 140 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the invention is less than or equal to 60 IU/mg, and the anti-IIa factor is less than or equal to 140 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 140 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 140 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 140 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 140 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 140 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 130 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 130 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 130 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 130 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 130 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 130 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 130 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 120 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the invention is less than or equal to 60 IU/mg, and the anti-IIa factor is less than or equal to 120 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 120 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 120 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 120 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 120 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 120 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 110 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 110 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 110 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 110 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 110 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 110 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 110 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 100 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 100 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 100 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 100 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 100 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 100 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 100 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 90 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 90 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 90 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 90 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 90 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 90 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 90 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 80 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 80 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 80 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 80 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 80 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 80 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 80 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 70 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 70 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 70 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 70 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 70 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 70 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 70 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 60 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 60 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 60 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 60 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 60 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 60 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 60 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 50 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 50 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 50 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 50 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 50 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 50 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 50 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 40 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 40 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 40 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 40 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 40 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 40 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 40 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 30 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 30 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 30 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 30 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 30 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 30 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 30 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 20 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 20 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 20 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 20 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 20 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 20 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 20 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 70 IU/mg or less, and the anti-IIa factor is 10 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 60 IU/mg or less, and the anti-IIa factor is 10 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 50 IU/mg or less, and the anti-IIa factor is 10 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 40 IU/mg or less, and the anti-IIa factor is 10 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 30 IU/mg or less, and the anti-IIa factor is 10 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 20 IU/mg or less, and the anti-IIa factor is 10 IU/mg or less.
  • the anti-Xa factor of the anti-coagulation heparin of the present invention is 10 IU/mg or less, and the anti-IIa factor is 10 IU/mg or less.
  • an anti-coagulant derivative has an anti-Xa factor of 5.8 IU/mg, an anti-IIa factor of 5.8 IU/mg, and a weight average molecular weight of the anticoagulant heparin derivative. It is 15158Da, the number average molecular weight is 13224Da, and its weight average molecular weight distribution is that the heparin molecule larger than 24k Da accounts for 8.57% of the total anticoagulated heparin derivative, and the heparin molecule of 16k ⁇ 24k Da accounts for the entire anticoagulation.
  • the proportion of heparin derivatives was 28%, the proportion of heparin molecules from 8k to 16k to the total anticoagulation heparin derivatives was 53.8%, and the ratio of heparin molecules less than 8k Da to the total anticoagulant heparin derivatives was 9.63%.
  • the de-anti-coagulation heparin derivative is obtained by de-coagulation treatment of unfractionated heparin purchased on the market.
  • an anti-coagulant derivative has an anti-Xa factor of 3.5 IU/mg, an anti-IIa factor of 5.1 IU/mg, and a weight average molecular weight of the anticoagulant heparin derivative. It is 4326Da, the number average molecular weight is 3254Da, and its weight average molecular weight distribution is that the ratio of heparin molecules less than 3k Da to the total anticoagulant heparin derivative is 35.55%, and the heparin molecule of 3k ⁇ 5k Da accounts for the entire anticoagulation.
  • the ratio of heparin derivatives was 31.94%, the proportion of heparin molecules from 5k to 8k to the total anticoagulation heparin derivatives was 24.07%, and the ratio of heparin molecules greater than 8k Da to the total anticoagulation heparin derivatives was 8.44%.
  • the anticoagulant heparin derivative is obtained by de-coagulation treatment of commercially available low molecular weight heparin, ie, enoxaparin.
  • an anti-coagulant derivative has an anti-Xa factor of 20.3 IU/mg, an anti-IIa factor of 33.1 IU/mg, and a weight average molecular weight of the anti-coagulated heparin derivative. It is 16427Da, the number average molecular weight is 13117Da, and its weight average molecular weight distribution is that the ratio of heparin molecules larger than 24k Da to the total anticoagulant heparin derivative is 20.62%, and the heparin molecule of 16k ⁇ 24k Da accounts for the entire anticoagulation.
  • the ratio of heparin derivatives is 20.00%, the proportion of heparin molecules from 8k to 16k to the total anticoagulation heparin derivatives is 36.71%, and the ratio of heparin molecules less than 8k Da to the total anticoagulation heparin derivatives is 22.67%.
  • the de-anti-coagulation heparin derivative is obtained by de-coagulation treatment of unfractionated heparin purchased on the market.
  • an anti-coagulant derivative has an anti-Xa factor of 60.6 IU/mg, an anti-IIa factor of 170.8 IU/mg, and a weight average molecular weight of the anticoagulant heparin derivative. It is 15793Da, the number average molecular weight is 13223Da, and its weight average molecular weight distribution is that the heparin molecule larger than 24k Da accounts for 13.44% of the total anticoagulated heparin derivative, and the heparin molecule of 16k ⁇ 24k Da accounts for the entire anticoagulation.
  • the proportion of heparin derivatives was 26.04%, the ratio of heparin molecules from 8k to 16k to the total anticoagulant heparin derivatives was 44.20%, and the ratio of heparin molecules less than 8k Da to the total anticoagulant heparin derivatives was 16.32%.
  • the de-anti-coagulation heparin derivative is obtained by de-coagulation treatment of unfractionated heparin purchased on the market.
  • an anti-coagulant derivative has an anti-Xa factor of 39 IU/mg, an anti-IIa factor of 124.9 IU/mg, and the weight average molecular weight of the de-anti-coagulated heparin derivative is 15212Da, the number average molecular weight is 12791Da, and its weight average molecular weight distribution is that the heparin molecule larger than 24k Da accounts for 10.91% of the total anticoagulated heparin derivative, and the heparin molecule of 16k ⁇ 24k Da accounts for the entire anticoagulated heparin.
  • the ratio of derivatives was 24.46%, the proportion of heparin molecules from 8k to 16k to the total anticoagulated heparin derivatives was 46.95%, and the ratio of heparin molecules less than 8k Da to the total anticoagulated heparin derivatives was 17.68%.
  • the de-anti-coagulation heparin derivative is obtained by de-coagulation treatment of unfractionated heparin purchased on the market.
  • an anti-coagulant derivative has an anti-Xa factor of 1.2 IU/mg, an anti-IIa factor of 7.5 IU/mg, and a weight average molecular weight of the anti-coagulated heparin derivative. It is 16706Da, the number average molecular weight is 13915Da, and its weight average molecular weight distribution is that the heparin molecule larger than 24k Da accounts for 17.10% of the total anticoagulated heparin derivative, and the heparin molecule of 16k ⁇ 24k Da accounts for the entire anticoagulation.
  • the ratio of heparin derivatives is 28.55%, the proportion of heparin molecules from 8k to 16k to the total anticoagulation heparin derivatives is 40.67%, and the ratio of heparin molecules less than 8k Da to the total anticoagulation heparin derivatives is 13.68%.
  • the de-anti-coagulation heparin derivative is obtained by de-coagulation treatment of unfractionated heparin purchased on the market.
  • the raw material heparin used for producing the anti-coagulation heparin derivative of the present invention may be unfractionated heparin or (super) low molecular weight heparin, and may be heparin having a weight average molecular weight of more than 8000 Da or a weight average molecular weight of 8000 Da or less. Heparin.
  • the activity of the anti-Xa factor and the anti-IIa factor of the anti-coagulation heparin derivative of the present invention simultaneously satisfies the above requirements.
  • the anti-Xa factor of the anti-coagulation heparin derivative of the present invention is in the range of 0 to 70 IU/mg, and the anti-IIa factor is in the range of 0 to 175 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin derivative of the present invention is in the range of 0 to 60 IU/mg, and the anti-IIa factor is in the range of 0 to 140 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin derivative of the present invention is in the range of 0 to 50 IU/mg, and the anti-IIa factor is in the range of 0 to 110 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin derivative of the present invention is in the range of 0 to 40 IU/mg, and the anti-IIa factor is in the range of 0 to 80 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin derivative of the present invention is in the range of 0 to 30 IU/mg, and the anti-IIa factor is in the range of 0 to 50 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin derivative of the present invention is in the range of 0 to 20 IU/mg, and the anti-IIa factor is in the range of 0 to 30 IU/mg.
  • the anti-Xa factor of the anti-coagulation heparin derivative of the present invention is in the range of 0 to 10 IU/mg, and the anti-IIa factor is in the range of 0 to 10 IU/mg.
  • the anti-coagulation heparin derivative of the present invention has a weight average molecular weight of 1,000 to 8,000, a weight average molecular weight of 600 to 8000, preferably a weight average molecular weight of 1000 to 7800, and further preferably a weight average molecular weight of 1500 to 7500, more preferably a weight average molecular weight of 2,000 to 7,000, a number average molecular weight of 600 to 6,000, still more preferably a number average molecular weight of 1200 to 5800, still more preferably a number average molecular weight of 1,500 to 5,500, still more preferably a number average molecular weight of 1800. ⁇ 5000.
  • the weight average molecular weight is preferably 2,000 to 7,500, more preferably 2,500 to 7,300, still more preferably 2,800 to 7,100, and the number average molecular weight is 600 to 6,000, preferably the number average molecular weight is 1,500 to 5,000, and more preferably 1800 to 4,800. More preferably, it is 1900 - 4600.
  • the ratio of the weight average molecular weight to the number average molecular weight (weight average molecular weight/number average molecular weight) of the anti-coaglysin derivative of the present invention is 1.40 or more, preferably 1.41 or more, and more preferably 1.42 or more. More preferably, it is 1.43 or more, further preferably 1.44 or more, further preferably 1.45 or more, further preferably 1.46 or more, further preferably 1.47 or more, further preferably 1.48 or more, further preferably 1.49 or more, and further preferably 1.50 or more.
  • the 2-saccharide unit of the anti-coagulation heparin derivative of the present invention accounts for 40% by weight or more, more preferably 45% by weight or more, and still more preferably 50% by weight or more, further. It is preferably at least 55% by weight.
  • the 4-saccharide unit of the anti-coagulation heparin derivative of the present invention accounts for 10 to 30% by weight, and more preferably 15 to 25% by weight, based on the total sugar component.
  • the 6-saccharide unit of the anti-coaglysin derivative of the present invention accounts for 15% by weight or less, preferably 12% by weight or less, more preferably 10% by weight or less, further preferably 10% by weight or less, more preferably It is 8 wt% or less.
  • the 8-saccharide unit of the anti-coagulation heparin derivative of the present invention accounts for 10% by weight or less, preferably 7% by weight or less, more preferably 6% by weight or less, further preferably 6% by weight or less, more preferably It is 5% by weight or less, more preferably 4.5% by weight or less, still more preferably 4% by weight or less.
  • the total sugar component in the present invention means that all the sugar components are detected by the method described in the following examples, and the content of the two sugar units in the total sugar component means that the total of all the 2 sugar units that can be detected is all
  • the proportion of the sugar component, the content of the four sugar units in the total sugar component is the ratio of the total amount of all the four sugar units that can be detected to all the sugar components
  • the content of the six sugar units in the total sugar component is The total amount of all the six saccharide units that can be detected is the ratio of all the sugar components
  • the content of the eight saccharide units to the total of the saccharide components is the ratio of the total amount of all the saccharide units that can be detected to the total amount of all the saccharide components.
  • the heparin molecule from which anticoagulant activity is removed can be obtained by periodic acid oxidation, and other biological activities can be largely retained, and the degree and form of sulfation remain substantially unchanged.
  • Periodic acid can selectively oxidize the ortho carbon atom containing an unsubstituted hydroxy group or an amino group, so that the unsulfated uronic acid C(2)-C(3) bond is cleaved, and the antithrombin in the heparin molecule binds five.
  • heparin pyridinium salt Reaction with heparin pyridinium salt using a silylating agent such as N,O-bis(trimethylsilyl)acetamide (BTSA) or N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MTSTFA)
  • BTSA N,O-bis(trimethylsilyl)acetamide
  • MTSTFA N-methyl-N-(trimethylsilyl)-trifluoroacetamide
  • MTSTFA can completely remove the 6-O-sulfate group of heparin and has few side reactions, and only a small amount of 2-O-sulfate group will be affected (Kariya, Y., et al., Preparation of completely 6-O-desulfated heparin). And its ability to enhance activity of basic fibroblast growth factor. Journal of Biological Chemistry, 2000. 275(34): p. 25949-25958.).
  • the base catalysis method is a common modification means for removing the heparin partial O-sulfate group.
  • heparin When heparin is lyophilized under alkaline conditions (pH 11-14), the 2-O-sulfate- ⁇ -L-iduronic acid residue is desulfated to form a 2,3-epoxy compound intermediate, further Hydrolysis forms desulfated alpha-L-iduronic acid to give 2-O desulfated heparin.
  • the 3-O sulfate group which is rare on the D-glucosamine residue in the heparin chain, is also partially affected by exfoliation, while the remaining sulfate groups remain intact.
  • Removal of the N-sulfate group on the heparin glucosamine residue is often carried out by solvent solution. Hydrolysis of the N-site sulfate group may be accompanied by cleavage of the glycosidic bond and removal of the O-sulfate group, while the heparin pyridinium salt can remove the N-sulfate group in the DMSO containing a small amount of water and is free of glycosidic bond breaks (Inoue, Y., &Nagasawa, K. Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. Carbohydrate research, 1976. 46(1): p. 87-95.).
  • N-reacetylation can be achieved using an acetylating reagent such as acetic anhydride (Purkerson M L, Tollefsen D M, Klahr SN-desulfated/acetylated heparin ameliorates the progression of renal disease in rats with subtotal renal ablation. Journal of Clinical Investigation, 1988 , 81(1): p.69.).
  • an acetylating reagent such as acetic anhydride
  • the substrate heparin for example, sodium heparin
  • a sodium periodate solution is added to carry out the reaction.
  • ethylene glycol is added to neutralize excess sodium periodate, and sodium borohydride is added to the reaction.
  • the filtered sample was collected by filtration.
  • the dialysis bag or the like is used for concentration and desalting, and finally an anticoagulant heparin derivative having anticoagulant activity is obtained.
  • the concentration of the substrate heparin after de-coagulation treatment used in the present invention can be determined by those skilled in the art, and is not particularly limited, and is preferably from 1 to 100 g/L.
  • the substrate heparin used in the present invention is a large molecular weight unfractionated heparin having a molecular weight of, for example, 5,000 to 30,000 and an average molecular weight of 20,000.
  • a heparinase such as heparinase I
  • heparinase I is used in the production of the anti-coagulation heparin derivative of the invention.
  • the E.C. number of heparinase I is E.C. 4.2.2.7. It is also possible to use purchased heparinase I, such as heparinase I available from Sigma or IBEX.
  • the heparanase may also be a fusion protein formed by recombinant heparinase I or heparinase I constructed by molecular biological methods with any fusion partner.
  • heparinase I is a fusion protein of heparinase I, in particular a fusion protein of heparinase I comprising MBP.
  • Heparinase I may also be a fusion protein formed with any fusion partner as long as it has the activity of heparinase I.
  • heparinase I is a fusion protein formed by heparinase I and a fusion partner, in particular a fusion protein of maltose binding protein (MBP) and heparinase I.
  • MBP maltose binding protein
  • the fusion protein of heparinase I and MBP is sometimes referred to as MBP-HepA hereinafter (see Chinese Patent ZL 200410038098.6, Grant No. CN1312183C).
  • the manner in which heparinase I reacts with the substrate heparin may be batchwise, continuous or semi-continuous,
  • One of ordinary skill in the art can appropriately select according to the needs of production.
  • the time of the reaction and the reaction apparatus can be appropriately determined by those skilled in the art as long as the target low molecular weight heparin can be obtained.
  • the anti-coagulation substrate heparin solution obtained by the method described above is added to the reactor, and then heparinase I is added. And react with the substrate heparin after de-coagulation treatment. As the reaction progresses, the anticoagulated heparin substrate is gradually degraded, and the reaction solution is monitored at intervals, and the reaction is terminated at an appropriate time.
  • the mixed solution in which the above reaction was terminated was subjected to preliminary filtration using a cellulose membrane vacuum priming apparatus, and ultrafiltration was carried out by an ultrafiltration apparatus to obtain a secondary filtrate.
  • the amount of heparinase I can be appropriately determined by those skilled in the art with reference to the activity of different enzymes.
  • the amount of each enzyme is heparinase I in the range of 50 IU to 500 IU per liter of the reaction solution, preferably Range of 100 IU to 250 IU.
  • IU means the amount of enzyme which produces 1 ⁇ mol of 4,5 unsaturated end product per minute at a temperature of 30 ° C and pH 7.4.
  • the substrate heparin used to produce the anti-coagulation heparin derivative used in the present invention is commercially available or can be directly extracted from an animal, for example, can be extracted from the porcine small intestine mucosa.
  • the heparin disaccharide unit is mainly L-iduronic acid and N-sulfated glucosamine linked by an ⁇ (1 ⁇ 4) glycosidic bond.
  • the substrate for producing the anti-coagulation heparin derivative of the present invention can be purchased from, for example, heparin of Hebei Changshan Biochemical Pharmaceutical Co., Ltd., Yantai Dongcheng Biochemical Co., Ltd., Shenzhen Haipurui Pharmaceutical Co., Ltd., Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd., US Medicine Star (Nanjing) Pharmaceutical Co., Ltd., etc.
  • the concentration of the substrate heparin after de-coagulation treatment used in the present invention can be determined by those skilled in the art, and is not particularly limited, and is preferably from 1 to 100 g/L.
  • the substrate heparin after de-coagulation treatment can be added to a buffer to prepare a suitable concentration.
  • the buffer used should not impair the enzymatic activity of heparinase I.
  • 20 mM Tris, 20 mM CaCl 2 , 50 mM NaCl was used, and a pH of about 7, for example, a buffer of 7.4 to 7.6 was adjusted with 1 mM hydrochloric acid.
  • a deionized water solution of 5.0 mM CaCl 2 and 200 mM NaCl was used, and then a buffer solution having a pH of 7.0 was adjusted with a 1 M HCl solution.
  • the temperature at which the heparinase I reacts with the substrate heparin after the anticoagulation treatment is not particularly limited, and may be any temperature that does not inactivate heparinase I, and may be, for example, 10 to 45 ° C. Most preferably 30 °C.
  • the time for the reaction of heparinase I with the substrate heparin after de-coagulation treatment is not particularly limited, and those skilled in the art can appropriately according to the enzyme activity of the added heparinase, the concentration of the substrate, and the temperature of the reaction.
  • the reaction time of heparinase with the substrate may be from 5 minutes to 10 hours, or from 10 minutes to 4 hours.
  • the method of monitoring the reaction solution may be appropriately selected according to the reaction system, and in a specific method, using an ultraviolet spectrophotometer The change in absorbance at 231 nm was detected, and the absorbance A 231 at 231 nm was continuously increased as the reaction progressed, whereby the degree of progress of the reaction was determined by the increase in absorbance.
  • the reaction can be terminated for further separation to obtain ultra low molecular weight heparin or low molecular weight heparin.
  • methods for terminating the reaction those skilled in the art can select according to their knowledge, such as adding a reagent for terminating the reaction, or increasing the temperature to inactivate the enzyme.
  • the reaction is terminated with hydrochloric acid to adjust the pH to 2.0 for 3 minutes, and the pH is adjusted back to 7.0 with 2.0 M NaOH. From the viewpoint of not adding other impurities, it is preferred to increase the temperature of the reaction system to inactivate the enzyme to terminate the reaction.
  • the entire reaction system is placed in a 100 ° C water bath for 10 minutes to terminate the reaction of the enzyme degradation substrate.
  • the de-anti-coagulation heparin derivative according to the present invention can be used for the treatment of inflammatory bowel disease, for example, for the treatment of ulcerative colitis and Crohn's disease.
  • the anti-anticoagulin derivative of the present invention After the administration of the anti-anticoagulin derivative of the present invention to treat a mouse model of colitis induced, it can effectively alleviate the intestinal shortening caused by intestinal fistula during the onset of inflammatory bowel disease, and the colonic epithelial mucosal tissue of the mouse Compared with the disease group, the gland structure was clear and the inflammatory cell infiltration was reduced, which significantly reduced the intestinal inflammation in the drug-treated group and effectively reduced the spleen enlargement caused by inflammation.
  • the de-anti-coagulation heparin derivative of the invention can effectively repair the abnormal expression of tight junction protein ZO-1 in colonic epithelial cells when inflammatory bowel disease occurs, thereby alleviating the DSS-induced increase in cell membrane permeability, thereby The effect of protecting the intestinal integrity of the intestinal epithelial cells and improving the intestinal epithelial barrier function is achieved.
  • the anti-coagulation heparin derivative of the present invention can effectively reduce inflammatory cell infiltration, and can effectively attenuate spleen enlargement caused by inflammation, the colon is shortened, and intestinal inflammation is remarkably reduced.
  • the anti-coagulation heparin derivative of the present invention can also be used for treating diseases similar to inflammatory bowel disease-related complications and pathogenesis, wherein inflammatory bowel disease-related complications and Diseases with similar pathogenesis include, but are not limited to, irritable bowel syndrome, arthritis and other parenteral complications including ankylosing spondylitis, gangrenous pyoderma, nodular erythema, ulceris, uveitis, scleral outer layer Inflammation and primary sclerosing cholangitis.
  • the anticoagulant heparin derivative can effectively alleviate the abnormal decrease in protein ZO-1 expression
  • the anti-coagulation heparin derivative of the present invention can also be used for the treatment of diseases associated with abnormal expression of protein ZO-1.
  • the de-anti-coagulation heparin derivative of the present invention can significantly reduce the apoptosis rate of intestinal epithelial cells in mice with ulcerative colitis induced by DSS, indicating that the DSS-induced apoptosis of colonic epithelial cells is inhibited, indicating that it can For the treatment of inflammatory bowel disease.
  • the present invention relates to the use of an anticoagulant heparin derivative for the preparation of a medicament for treating inflammatory bowel disease, and inflammatory bowel disease-related complications and diseases having similar pathogenesis, wherein inflammatory bowel disease-related complications and morbidity Mechanisms with similar mechanisms include, but are not limited to, irritable bowel syndrome, arthritis and other parenteral complications including ankylosing spondylitis, gangrenous pyoderma, nodular erythema, ulceris, uveitis, scleral inflammation And primary sclerosing cholangitis.
  • the anti-anthingulin derivative of the present invention After the treatment of a mouse model of ulcerative colitis by administration of the anti-anthingulin derivative of the present invention, it can effectively alleviate intestinal shortening caused by intestinal fistula during the onset of inflammatory bowel disease, and colonic epithelium of mice
  • the mucosal tissue is intact, the structure is clear, the epithelial cells are arranged neatly, the glands are intact, some glands are proliferated, and no abnormalities are found in the submucosa.
  • the apoptosis level can be significantly reduced, indicating that it can effectively alleviate the DSS-induced apoptosis.
  • the de-anti-coagulation heparin derivative of the invention can effectively repair the abnormal expression of tight junction protein ZO-1 in intestinal epithelial cells when inflammatory bowel disease occurs, thereby alleviating the DSS-induced increase in cell membrane permeability, thereby The effect of protecting the intestinal integrity of the intestinal epithelial cells and improving the intestinal epithelial barrier function is achieved.
  • the anti-coagulation heparin derivative of the present invention can effectively reduce inflammatory cell infiltration, and can effectively attenuate spleen enlargement and colon shortening caused by inflammation, intestinal inflammation is remarkably reduced. Therefore, in addition to treating inflammatory bowel disease, the anti-coagulation heparin derivative of the present invention can also be used for treating diseases similar to inflammatory bowel disease-related complications and pathogenesis, wherein inflammatory bowel disease-related complications and Diseases with similar pathogenesis include, but are not limited to, irritable bowel syndrome, arthritis and other parenteral complications including ankylosing spondylitis, gangrenous pyoderma, nodular erythema, ulceris, uveitis, scleral outer layer Inflammation and primary sclerosing cholangitis.
  • the anti-coagulation heparin derivative of the present invention can also be used for the treatment of diseases associated with abnormal expression of protein ZO-1.
  • the de-anti-coagulation heparin derivative of the present invention can significantly reduce the apoptosis rate of intestinal epithelial cells in mice with ulcerative colitis induced by DSS, indicating that the DSS-induced apoptosis of colonic epithelial cells is inhibited, indicating that it can For the treatment of inflammatory bowel disease.
  • the anticoagulant activity of heparin needs to be determined by measuring the activity of the accelerated antithrombin (hereinafter referred to as ATIII) inhibition of factor Xa (hereinafter referred to as anti-factor Xa) and factor IIa (hereinafter referred to as anti-IIa factor) by an in vitro assay.
  • ATIII accelerated antithrombin
  • anti-factor Xa factor Xa
  • factor IIa hereinafter referred to as anti-IIa factor
  • the method for detecting anti-Xa and anti-IIa activity used in the present invention can be referred to the European Pharmacopoeia.
  • the International Unit (IU) against Xa and anti-IIa refers to the activity of a defined amount of heparin or low molecular weight heparin international standard.
  • the anticoagulant activity of the test sample to be tested is obtained by comparison with the corresponding activity of the international standard.
  • Tris-HCl buffer (pH 7.4): Take Tris 6.08g and NaCl 8.77g, add 500mL of water to dissolve it, add 10g of bovine serum albumin, adjust the pH to 7.4 with HCl, and dilute to 1000mL with water.
  • Tris-EDTA buffer (pH 8.4): Take Tris 3.03g, NaCl 5.12g and EDTA ⁇ 2Na 1.4g, add 250 mL of water to dissolve it, adjust the pH to 8.4 with HCl, and dilute to 500 mL with water.
  • Heparin standard and test sample solution heparin activity standard is purchased from EDQM (European Directorate for the Quality of Medicines) heparin low-molecular-mass for assay BRP (Biological Reference Preparation) (H0185000, for detection of anti-factor Xa Activity and anti-factor IIa activity).
  • the standard (S) and the test product (T) were diluted into four different concentrations of solution by using Tris-HCl buffer (pH 7.4), and the ratio of the agent to each dose was controlled at 1:0.7 to 1:0.6. .
  • the concentration should be within the linear range of the dose log-response.
  • the detection of anti-Xa factor is generally 0.025 IU to 0.2 IU per ml, and the detection of anti-IIa factor is generally 0.015 IU to 0.075 IU per ml.
  • ATIII solution ATIII was purchased from Chromogenix (Sweden). When the anti-Xa factor was detected, a solution of 1 IU/mL was prepared in Tris-HCl buffer (pH 7.4); when the anti-IIa factor was detected, a solution of 0.5 IU/mL was prepared in Tris-HCl buffer (pH 7.4).
  • the chromogenic substrate solution when the anti-Xa factor was detected, the chromogenic substrate S-2765 (N- ⁇ -benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline-dihydrochloride) was purchased from Chromogenix ( Sweden).
  • chromogenic substrate S-2238 H-D-phenylalanyl-L-pipecolyl-arginine-p-nitroaniline-dihydrochloride
  • Both chromogenic substrates were stored in a solution of 0.003 M in deionized water and diluted to 0.0005 M with Tris-EDTA buffer (pH 8.4) immediately before use.
  • Anti-Xa factor solution Prepared with Tris-HCl buffer (pH 7.4), the concentration was adjusted to make the absorbance at 405 nm in the anti-Xa experiment with 0.9% NaCl instead of (ultra) low molecular weight heparin. Between 0.7.
  • Anti-IIa factor solution Dissolve and dilute to a 5 IU/mL solution with Tris-HCl buffer (pH 7.4).
  • the absorbance at 405 nm was measured using a 1 cm pathlength semi-micro cuvette with Tris-HCl buffer (pH 7.4) as a blank.
  • the test solution (two tubes in parallel) was replaced with Tris-HCl buffer (pH 7.4) as a blank control tube, and the absorbance of the blank control tube was measured at the beginning and end of the 16 tubes, respectively. There should be no significant difference in absorbance between the two.
  • the standard solution or the test solution is the linear regression of the logarithm of the concentration as the abscissa, according to the 4 ⁇ 4 experimental design of the parallel line principle of the biological reaction statistical method, and calculate the potency and experiment error.
  • the average confidence rate (FL%) must not exceed 15%.
  • the weight average molecular weight (Mw), number average molecular weight (Mn) and distribution coefficient (P) of low molecular weight heparin were determined by gel exclusion high performance liquid chromatography.
  • the column was TSK-GEL G2000SWXL (TOSOH, Japan), the flow rate was controlled at 0.5 mL/min, the column temperature was 35 ° C, and the injection volume was 25 ⁇ L.
  • the UV detector and the differential detector are connected in series to the outlet of the column in a sequential order with a UV detector wavelength of 234 nm.
  • the oligosaccharide analysis of the anti-coagulation heparin derivative of the present invention employs hydrophilic interaction chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS).
  • HILIC hydrophilic interaction chromatography
  • ESI-MS electrospray ionization mass spectrometry
  • the HILIC liquid phase conditions were as follows: loading amount: 10 ⁇ L, mobile phase: phase A: 5 mM ammonium acetate aqueous solution, phase B: 5 mM ammonium acetate solution, 98% acetonitrile solution; flow rate: 0.15 mL/min; elution gradient: 0-5 min , 90% B; 5-45 min, 90-65% B; 45-55 min, 65% B; 55-60 min, 65-20% B; 60-80 min, 20% B; 80-80.01 min, 20-90% B.
  • the ESI-MS mass spectrometry parameters were as follows: negative ion mode spray voltage: 4.2 KV; sheath flow gas flow rate: 20 arb; auxiliary gas flow rate: 5 arb; capillary voltage: -40 V; barrel lens voltage: -50 V; capillary temperature: 275 ° C; : 200 to 2000.
  • the peaks are assigned according to the peak mass-to-charge ratio (m/z) and the theoretical calculated value, and the ratio of the specific peak area to the area of all the oligosaccharide peaks is taken as the relative percentage of the oligosaccharide. .
  • the dialysis bag is used for desalting or the Millipore ultrafiltration device is added with a 1K filter membrane for ultrafiltration concentration and desalting until the filtrate is tested by 0.1 M AgNO 3 without color change.
  • the sample was frozen at -80 ° C, lyophilized in a lyophilizer, and then pulverized into a powder using a mortar or a small pulverizer to obtain anti-coagulated heparin (named NAHP).
  • the anticoagulant activity of the obtained anticoagulated heparin was measured by the above method, and the results showed that the anti-Xa was 5.8 IU/mg and the anti-IIa was 5.8 IU/mg.
  • the molecular weight of the heparin and its distribution were measured by the above methods, and the results are shown in Table 1 or 2 below.
  • enoxaparin purchased from Changshan Biochemical Pharmaceutical Co., Ltd., product name: enoxaparin sodium
  • enoxaparin sodium 20 g was dissolved in 0.6 L of deionized water, and an equivalent volume of 0.2 M was added to 0.6 L of enoxaparin (33 g/L).
  • the sodium periodate solution (now available) was reacted at 300 rpm and 4 ° C for 22 hours in the dark.
  • An excess of sodium periodate was added by adding 80 mL of ethylene glycol, and then 28 g of sodium borohydride was added thereto to react at 4 ° C for 16 hours.
  • the pH was adjusted to 7.0 with HCl.
  • the filtered sample was collected by suction filtration through a 0.22 ⁇ m filter.
  • the dialysis bag is used for desalting or the Millipore ultrafiltration device is added with a 1K filter membrane for ultrafiltration concentration and desalting until the filtrate is tested by 0.1 M AgNO 3 without color change.
  • the sample was frozen at -80 ° C, lyophilized in a lyophilizer, and then pulverized into a powder using a mortar or a small pulverizer to obtain de-anticoagulated enoxaparin (named: NAEno).
  • NAEno de-anticoagulated enoxaparin
  • the anticoagulant activity of the obtained anti-coagulated heparin was measured by the above method, and the results showed that the anti-Xa was 3.5 IU/mg, and the anti-IIa was 5.1 IU/mg.
  • the molecular weight of the heparin and its distribution were measured by the above methods, and the results are shown in Table 1 or 2 below.
  • the fine heparin (purchased from Changshan Biochemical Pharmaceutical Co., Ltd., product name: heparin sodium) was formulated into an aqueous solution with a concentration of 5 mg/ml, and pre-cooled at 4 ° C and then loaded with a 001x7 type cation exchange resin column (H+form) 2.5* 40cm (purchased from Langfang Nanda Resin Co., Ltd.).
  • the effluent was collected by washing with water, immediately neutralized with an excess of pyridine, adjusted to a pH between 6 and 8, and then lyophilized to obtain a heparin pyridinium salt.
  • heparin pyridinium salt was added to 10-fold (w/w) N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MTSTFA) and 100 volumes (v/w) of anhydrous pyridine. Stir at room temperature until completely dissolved, and the reaction mixture was heated at 110 ° C for 2.5 h (the reaction time was adjusted according to the size of the reaction system). The reaction solution was quenched by ice bath, evaporated to 1/10 of the original volume by a rotary evaporator, and MTSTFA was degraded by adding 2 volumes of v/v distilled water, followed by decompression at 35 ° C for 15 min to make the white turbidity of the reaction liquid disappear.
  • MTSTFA N-methyl-N-(trimethylsilyl)-trifluoroacetamide
  • the dialysis bag is used for desalting or the Millipore ultrafiltration device is added with a 1K filter membrane for ultrafiltration concentration and desalting until the filtrate is tested by 0.1 M AgNO 3 without color change.
  • the sample was frozen at -80 ° C, lyophilized in a lyophilizer, and then pulverized into a powder by a mortar or a small pulverizer to obtain de-anti-coagulated heparin (named NAHP-60).
  • NAHP-60 de-anti-coagulated heparin
  • the anticoagulant activity of the obtained anticoagulated heparin was measured by the above method, and the results showed that the anti-Xa was 60.6 IU/mg, and the anti-IIa was 170.8 IU/mg.
  • the molecular weight of the heparin and its distribution were measured by the above methods, and the results are shown in Table 1 or 2 below.
  • the dialysis bag is used for desalting or the Millipore ultrafiltration device is added with a 1K filter membrane for ultrafiltration concentration and desalting until the filtrate is tested by 0.1 M AgNO 3 without color change.
  • the sample was frozen at -80 ° C, lyophilized in a lyophilizer, and then pulverized into a powder using a mortar or a small pulverizer to obtain de-anti-coagulated heparin (designated NAHP-39).
  • the anticoagulant activity of the obtained anticoagulated heparin was measured by the above method, and the results showed that the anti-Xa was 39 IU/mg, and the anti-IIa was 124.9 IU/mg.
  • the molecular weight of the heparin and its distribution were measured by the above methods, and the results are shown in Table 1 or 2 below.
  • the fine heparin (purchased from Changshan Biochemical Pharmaceutical Co., Ltd., product name: heparin sodium) was formulated into an aqueous solution with a concentration of 5 mg/ml, and pre-cooled at 4 ° C and then loaded with a 001x7 type cation exchange resin column (H+form) 2.5* 40cm (purchased from Langfang Nanda Resin Co., Ltd.).
  • the effluent was collected by washing with water, immediately neutralized with an excess of pyridine, adjusted to a pH between 6 and 8, and then lyophilized to obtain a heparin pyridinium salt.
  • heparin pyridinium salt 2 g was added to 25 ml of DMSO containing 5% water, and diluted with water (25 ml) at 50 ° C for 3 h. The pH was adjusted to 9 with NaOH, dialyzed into deionized water and lyophilized to give N-desulfated product. 1.2g N-desulfated product was added 12ml saturated NaHCO 3 (5g NaHCO 3 added to 50ml ddH 2 O, pre-cooled at 4 ° C).
  • N-ace N-reacetylated anti-coagulation heparin derivative
  • Fine heparin was purchased from Changshan Biochemical Co., Ltd., and the weight average molecular weight Mw of the heparin was 17223 (designated HP).
  • the anticoagulant activity was measured by the above method, and the results showed that the anti-Xa was 187.3 IU/mg, and the anti-IIa was 197.2 IU/mg.
  • Enoxaparin was purchased from Hebei Changshan Biochemical Pharmaceutical Co., Ltd., and the heparin had a number average molecular weight of 3,275 and a weight average molecular weight of 4,620 (designated Eno).
  • the anticoagulant activity was measured by the above method, and the results showed that the anti-Xa was 109 IU/mg, and the anti-IIa was 32.7 IU/mg.
  • Example sample anti-Xa (IU/mg) Anti-IIa (IU/mg) Example 1 NAHP 5.8 5.8
  • Example 2 NAEno 3.5 5.1
  • Example 3 6-OdeS 20.3 33.1
  • Example 4 NAHP-60 60.6 170.8
  • Example 5 NAHP-39 39 124.9
  • Example 6 N-ace 1.2 7.5 Comparative example 1 HP 187.3 197.2 Comparative example 2 Eno 109 32.7 Comparative example 4 2-OdeS 75.6 99.6
  • DSS dextran sulfate sodium salt
  • the healthy control group did not do any treatment.
  • the DSS module continued to drink 2.5-3% of DSS drinking water until the end of the experiment.
  • the drug treatment group continued to drink 2.5-3% of DSS drinking water until the end of the experiment.
  • the anticoagulant heparin obtained in Examples 1 and 2, and the substances of Comparative Examples 1 to 3 were dissolved in physiological saline, and administered intragastrically from the first day at a dose of 30 mg/kg for 7 days.
  • the model group showed symptoms such as weight loss, loose stools, and pus and bloody stools.
  • the experiment was completed. After the mice were sacrificed, blood was taken from the eyelids, and the spleen was weighed. The entire length of the colorectal was taken from the cecum end to the anus to measure the length, leaving the proximal end 1/3-1/2 frozen, and the other part Cut, PBS washed and rolled into a paraffin-embedded plastic clip, immersed in 4% paraformaldehyde for fixation.
  • HE staining of colorectal histopathological sections is shown in Figure 1.
  • the results showed that the colonic epithelial erosion, severe glandular structure destruction, and increased mucosal and submucosal inflammatory cell infiltration after DSS induction. After treatment with anticoagulation heparin, the colonic epithelium is protected to a certain extent and the gland structure is relatively intact.
  • the embedded paraffin block can be sliced; the thickness of the slice is about 5 ⁇ m.
  • Hematoxylin staining The hydrated sections were immersed in hematoxylin staining solution for 5-20 min to stain the nuclei. Rinse with tap water for 3 to 5 minutes.
  • Eosin staining The slice after sufficient hydration is directly into the eosin staining solution, and the cytoplasm is stained for about 5 to 15 minutes.
  • NC group normal group: colonic epithelial tissue is intact, structure is clear, epithelial cells are arranged neatly, gland is intact;
  • DSS model colonic mucosal epithelial cells atrophy, necrosis, shedding, gland abnormalities, glandular goblet cells disappear, The inflammatory cells were extensively infiltrated, the basement membrane was broken or disappeared, the gap between the glandular epithelium and the mucosa was increased, the submucosal capillaries proliferated, and the hemorrhage was dilated;
  • HP treatment group colonic mucosal epithelial cells were atrophied, necrotic, shedding, gland incomplete, Inflammatory cells infiltrated, thickened basement membrane, no fracture, increased clearance between glandular epithelium and mucosal muscle layer, submucosal capillaries hyperplasia, and dilated hemorrhage;
  • NAHP treatment group partial exfoliation of colonic
  • Hyperplasia irregular arrangement, visible loss, accompanied by inflammatory cell infiltration, lesions did not involve the submucosa; 5-ASA (Mesalazine) treatment group (positive control treatment group): colonic mucosal epithelial cell necrosis, shedding, gland abnormalities, The arrangement is not neat, the goblet cells proliferate or necrosis, the inflammatory cells infiltrate, and the lesion does not affect the submucosa.
  • 5-ASA Mesalazine treatment group
  • the intestinal muscles of the mice contracted, resulting in a shortened length of the entire colorectal.
  • 3% DSS was administered to mice for 7 consecutive days or after drug treatment, the animals were sacrificed by cervical dislocation, and the whole colorectal was taken and the length was measured.
  • the length of the colon in each group is shown in Fig. 2(a) and (b).
  • the other heparin drugs can alleviate colon shortening to some extent.
  • the NAHP treatment group was better than the 5-ASA treatment group.
  • the effect of anticoagulation of enoxaparin is better than that of enoxaparin.
  • Tight junctions between epithelial cells are important structures that maintain the mechanical barrier and permeability of the mucosal epithelium.
  • ZO-1 protein is one of the important constituent proteins of cell tight junction protein, which not only participates in the maintenance and regulation of epithelial barrier function, but also participates in important processes such as cell material transport and maintaining epithelial polarity.
  • DSS dextran sulfate sodium
  • the ZO-1 protein in NCM460 cells was detected by Western blotting after 48 hours of treatment with 2 mg/ml of the anti-anti-coagulation heparin derivative of Examples 1, 3, and the substances of Comparative Example 1, Comparative Examples 3 and 4. Expression, discussion and evaluation of the protective effect of each heparin derivative on colonic epithelial cells with increased permeability. As shown in Fig. 4, in addition to unfractionated heparin and 2-OdeS derivatives, the expression level of ZO-1 protein in other heparin derivative treatment groups was higher than that in DSS induction group, which proved these. Heparin derivatives have a mitigating effect on DSS-induced cell membrane permeability abnormalities.
  • Apoptosis plays an important role in the development of UC. Under pathological conditions of ulcerative colitis, the normal sequence of proliferation, differentiation, and apoptosis of epithelial cells along the crypt villus may be disrupted. The apoptotic rate of mucosal epithelial cells in the inflammatory zone of UC is significantly increased, which may be another important cause of the destruction of UC epithelial barrier function. Early changes in apoptosis occur on the surface of the cell membrane. One of the changes in the surface of these cell membranes is the transfer of phosphatidylserine (PS) from the cell membrane to the extracellular membrane, exposing PS to the outer surface of the cell membrane.
  • PS phosphatidylserine
  • Annexin V acts as a Ca 2+ -dependent phospholipid-binding protein with a high affinity for PS. Therefore, the protein acts as a sensitive probe to detect PS exposed to the surface of the cell membrane. PS transfer to the cell membrane is not unique to apoptosis, but can also occur in cell necrosis. The difference between the two cell death patterns is that the cell membrane is intact at the initial stage of apoptosis, and cell necrosis is destroyed in its early stages. Therefore, apoptotic cells and necrotic cells can be distinguished by staining the nucleus with propidium iodide (PI).
  • PI propidium iodide
  • DSS dextran sulfate sodium
  • the level of intestinal epithelial cell apoptosis in the anticoagulant heparin-treated group was significantly lower than that in the DSS-induced apoptosis group, which proved Its mitigating effect on DSS-induced apoptosis.
  • the dialysis bag is used for desalting or the Millipore ultrafiltration device is added with a 1K filter membrane for ultrafiltration concentration and desalting until the filtrate is tested by 0.1 M AgNO 3 without color change.
  • the sample was frozen at -80 ° C, lyophilized in a lyophilizer, and then pulverized into a powder using a mortar or a small pulverizer to obtain an anti-coagulated heparin derivative (designated NAHP).
  • NAHP anti-coagulated heparin derivative
  • the anticoagulant activity of the obtained anti-coagulated heparin derivative was measured by the above method, and the results showed that the anti-Xa was 5.8 IU/mg and the anti-IIa was 5.8 IU/mg.
  • the molecular weight and distribution of the de-anti-coagulated heparin derivative were measured by the above methods, and the results are shown in Table 1 below.
  • the P value shown in Table 4 indicates the degree of dispersion of the molecule, which is a value obtained by dividing the weight average molecular weight Mw by the number average molecular weight Mn.
  • the anti-coagulated heparin prepared in Example 7 was dissolved in a reaction buffer, and heparinase I prepared according to the method of ZL200410038098.6 was added to the solution every 0.5 to 1 h, and 20 IU of heparinase I was added each time, and the optical path difference was used.
  • Monitor the 231 nm light absorption A231 for a 1 cm quartz cuvette and UV spectrophotometer (calibrate the instrument with a pH 7.4 buffer. For the accuracy of the test results, the UV spectrophotometer reading A231 is greater than 0.6 When the solution is diluted by a certain multiple, the reading is determined from 0.2 to 0.6).
  • the method of completion is to inactivate the enzyme in the reaction solution in a boiling water bath at 100 ° C for 5 to 10 min, then take out the reaction system and cool to room temperature, add 6 volumes of absolute ethanol to the reaction solution, stir at room temperature for 10 min, then After centrifugation at 4000 r/min for 15 min at room temperature, the precipitate was collected, dissolved in deionized water of 2 to 3 times the mass of the precipitate, filtered through a 0.22 ⁇ m membrane, and the permeate was collected and placed in a freezer at -80 ° C.
  • the solid ice cubes were then lyophilized by feeding into a lyophilizer (cold trap temperature -50 ° C) and then pulverized into a powder using a mortar or a small pulverizer to obtain a low molecular weight anticoagulant heparin (also named: LNAHP).
  • the anticoagulant activity of the obtained anticoagulated heparin was measured by the above method, and the results showed that the anti-Xa was 3.2 IU/mg, and the anti-IIa was 4.0 IU/mg.
  • the molecular weight and distribution of the de-anti-coagulated heparin derivative were measured by the above method, and the results are shown in Table 4 or 5 below.
  • the oligosaccharide distribution of the anti-coagulated heparin derivative was measured by the above method, and the results are shown in Table 7.
  • the anti-coagulated heparin prepared in Example 7 was dissolved in a reaction buffer, and heparinase I prepared according to the method of ZL200410038098.6 was added to the solution every 0.5 to 1 h, and 20 IU of heparinase I was added each time, and the optical path difference was used.
  • Monitor the 231 nm light absorption A231 for a 1 cm quartz cuvette and UV spectrophotometer (calibrate the instrument with a pH 7.4 buffer. For the accuracy of the test results, the UV spectrophotometer reading A231 is greater than 0.6 When the solution is diluted by a certain multiple, the reading is determined from 0.2 to 0.6).
  • the reaction was terminated, and the enzyme activity of the added total heparinase I reached about 340-380 IU.
  • the method of completion is to inactivate the enzyme in the reaction solution in a boiling water bath at 100 ° C for 5 to 10 min, then take out the reaction system and cool to room temperature, add 6 volumes of absolute ethanol to the reaction solution, stir at room temperature for 10 min, then After centrifugation at 4000 r/min for 15 min at room temperature, the precipitate was collected, dissolved in deionized water of 2 to 3 times the mass of the precipitate, filtered through a 0.22 ⁇ m membrane, and the permeate was collected and placed in a freezer at -80 ° C.
  • Solid ice cubes are then lyophilized in a lyophilizer (cold trap temperature -50 ° C) and then pulverized into powder using a mortar or small pulverizer to obtain ultra low molecular weight anticoagulant heparin (also named: ULNAHP). .
  • the anticoagulant activity of the obtained anticoagulated heparin was measured by the above method, and the results showed that the anti-Xa was 3.7 IU/mg, and the anti-IIa was 5.2 IU/mg.
  • the molecular weight and distribution of the de-anti-coagulated heparin derivative were measured by the above method, and the results are shown in Table 4 or 5 below.
  • the oligosaccharide distribution of the anti-coagulated heparin derivative was measured by the above method, and the results are shown in Table 7.
  • the purified heparin is dissolved in the reaction buffer, and heparinase I prepared according to the method of ZL200410038098.6 is added to the solution every 0.5 to 1 hour, 20 IU of heparinase I is added each time, and a quartz cuvette having an optical path difference of 1 cm is used.
  • ultraviolet spectrophotometer to monitor the light absorption of the solution 231nm A231 (using the pH 7.4 buffer to calibrate the instrument to zero, in order to check the accuracy of the results, when the UV spectrophotometer reading A231 is greater than 0.6, the solution is diluted by a certain multiple , so that the reading is measured from 0.2 to 0.6). When it was detected that A231 reached 45, the reaction was terminated.
  • the method of completion is to inactivate the enzyme in the reaction solution in a boiling water bath at 100 ° C for 5 to 10 min, then take out the reaction system and cool to room temperature, add 6 volumes of absolute ethanol to the reaction solution, stir at room temperature for 10 min, then After centrifugation at 4000 r/min for 15 min at room temperature, the precipitate was collected, dissolved in deionized water of 2 to 3 times the mass of the precipitate, filtered through a 0.22 ⁇ m membrane, and the permeate was collected and placed in a freezer at -80 ° C.
  • the solid ice cubes are then lyophilized into a lyophilizer (cold trap temperature -50 ° C) and then pulverized into powder using a mortar or small pulverizer.
  • 20 g of the obtained powder was dissolved in 0.6 L of deionized water, and an equal volume of a 0.2 M sodium periodate solution (currently prepared) was added thereto, and reacted at 300 rpm and 4 ° C for 22 hours in the dark.
  • An excess of sodium periodate was added by adding 80 mL of ethylene glycol, and then 28 g of sodium borohydride was added thereto to react at 4 ° C for 16 hours.
  • the pH was adjusted to 7.0 with HCl.
  • the filtered sample was collected by suction filtration through a 0.22 ⁇ m filter.
  • the dialysis bag is used for desalting or the Millipore ultrafiltration device is added with a 1K filter membrane for ultrafiltration concentration and desalting until the filtrate is tested by 0.1 M AgNO 3 without color change.
  • the sample was frozen at -80 ° C, lyophilized in a lyophilizer, and then pulverized into a powder using a mortar or a small pulverizer to obtain de-anti-coagulated heparin (named NAHep-1).
  • the anticoagulant activity of the obtained anticoagulated heparin was measured by the above method, and the results showed that the anti-Xa was 2 IU/mg, and the anti-IIa was 15.8 IU/mg.
  • the molecular weight and distribution of the heparin derivative were measured by the above method, and the results are shown in Table 4 or 5 below.
  • the oligosaccharide distribution of the heparin derivative was measured by the above method, and the results are shown in Table 7.
  • the purified heparin is dissolved in the reaction buffer, and heparinase I prepared according to the method of ZL200410038098.6 is added to the solution every 0.5 to 1 hour, 20 IU of heparinase I is added each time, and a quartz cuvette having an optical path difference of 1 cm is used.
  • UV spectrophotometer to monitor the light absorption of the solution 231nm A231 (using the pH 7.4 buffer to calibrate the instrument to zero, in order to check the accuracy of the results, when the UV spectrophotometer reading A231 is greater than 0.6, the solution is diluted by a certain multiple , so that the reading is measured from 0.2 to 0.6).
  • the reaction was terminated.
  • the method of completion is to inactivate the enzyme in the reaction solution in a boiling water bath at 100 ° C for 5 to 10 min, then take out the reaction system and cool to room temperature, add 6 volumes of absolute ethanol to the reaction solution, stir at room temperature for 10 min, then After centrifugation at 4000 r/min for 15 min at room temperature, the precipitate was collected, dissolved in deionized water of 2 to 3 times the mass of the precipitate, filtered through a 0.22 ⁇ m membrane, and the permeate was collected and placed in a freezer at -80 ° C.
  • the solid ice cubes are then lyophilized into a lyophilizer (cold trap temperature -50 ° C) and then pulverized into powder using a mortar or small pulverizer.
  • 20 g of the obtained powder was dissolved in 0.6 L of deionized water, and an equal volume of a 0.2 M sodium periodate solution (currently prepared) was added thereto, and reacted at 300 rpm and 4 ° C for 22 hours in the dark.
  • An excess of sodium periodate was added by adding 80 mL of ethylene glycol, and then 28 g of sodium borohydride was added thereto to react at 4 ° C for 16 hours.
  • the pH was adjusted to 7.0 with HCl.
  • the filtered sample was collected by suction filtration through a 0.22 ⁇ m filter.
  • the dialysis bag is used for desalting or the Millipore ultrafiltration device is added with a 1K filter membrane for ultrafiltration concentration and desalting until the filtrate is tested by 0.1 M AgNO 3 without color change.
  • the sample was frozen at -80 ° C, lyophilized in a lyophilizer, and then pulverized into a powder using a mortar or a small pulverizer to obtain de-anti-coagulated heparin (named NAHep-2).
  • the anticoagulant activity of the obtained anti-coagulated heparin was measured by the above method, and the results showed that the anti-Xa was 1.8 IU/mg and the anti-IIa was 8.8 IU/mg.
  • the molecular weight and distribution of the heparin derivative were measured by the above method, and the results are shown in Table 4 or 5 below.
  • the oligosaccharide distribution of the heparin derivative was measured by the above method, and the results are shown in Table 7.
  • Unfractionated heparin was purchased from Changshan Biochemical Co., Ltd., and the weight average molecular weight Mw of the heparin was 17223 (designated HP).
  • the anticoagulant activity was measured by the above method, and the results showed that the anti-Xa was 187.3 IU/mg, and the anti-IIa was 197.2 IU/mg.
  • the molecular weight and its distribution were measured by the above methods, and the results are shown in Table 7 below.
  • the purified heparin is dissolved in the reaction buffer, and heparinase I prepared according to the method of ZL200410038098.6 is added to the solution every 0.5 to 1 hour, 20 IU of heparinase I is added each time, and a quartz cuvette having an optical path difference of 1 cm is used.
  • ultraviolet spectrophotometer to monitor the light absorption of the solution 231nm A231 (using the pH 7.4 buffer to calibrate the instrument to zero, in order to check the accuracy of the results, when the UV spectrophotometer reading A231 is greater than 0.6, the solution is diluted by a certain multiple , so that the reading is measured from 0.2 to 0.6). When it was detected that A231 reached 45, the reaction was terminated.
  • the method of completion is to inactivate the enzyme in the reaction solution in a boiling water bath at 100 ° C for 5 to 10 min, then take out the reaction system and cool to room temperature, add 6 volumes of absolute ethanol to the reaction solution, stir at room temperature for 10 min, then After centrifugation at 4000 r/min for 15 min at room temperature, the precipitate was collected, dissolved in deionized water of 2 to 3 times the mass of the precipitate, filtered through a 0.22 ⁇ m membrane, and the permeate was collected and placed in a freezer at -80 ° C.
  • the solid ice cubes were then lyophilized into a lyophilizer (cold trap temperature -50 ° C) and then pulverized into powder using a mortar or small pulverizer to obtain low molecular weight heparin (designated I-45).
  • the anticoagulant activity of the obtained low molecular weight heparin was measured by the above method, and the results showed that the anti-Xa was 99 IU/mg and the anti-IIa was 43.3 IU/mg.
  • the molecular weight and distribution of the low molecular weight heparin were measured by the above method, and the results are shown in Table 4 or 5 below.
  • the oligosaccharide distribution of the low molecular weight heparin was measured by the above method, and the results are shown in Table 7.
  • the purified heparin is dissolved in the reaction buffer, and heparinase I prepared according to the method of ZL200410038098.6 is added to the solution every 0.5 to 1 hour, 20 IU of heparinase I is added each time, and a quartz cuvette having an optical path difference of 1 cm is used.
  • ultraviolet spectrophotometer to monitor the light absorption of the solution 231nm A231 (using the pH 7.4 buffer to calibrate the instrument to zero, in order to check the accuracy of the results, when the UV spectrophotometer reading A231 is greater than 0.6, the solution is diluted by a certain multiple , so that the reading is measured from 0.2 to 0.6).
  • the reaction was terminated.
  • the method of completion is to inactivate the enzyme in the reaction solution in a boiling water bath at 100 ° C for 5 to 10 min, then take out the reaction system and cool to room temperature, add 6 volumes of absolute ethanol to the reaction solution, stir at room temperature for 10 min, then After centrifugation at 4000 r/min for 15 min at room temperature, the precipitate was collected, dissolved in deionized water of 2 to 3 times the mass of the precipitate, filtered through a 0.22 ⁇ m membrane, and the permeate was collected and placed in a freezer at -80 ° C.
  • the solid ice cubes were then lyophilized in a lyophilizer (cold trap temperature -50 ° C) and then pulverized into powder using a mortar or a small pulverizer to obtain low molecular weight heparin (designated I-98).
  • the anticoagulant activity of the obtained low molecular weight heparin was measured by the above method, and the results showed that the anti-Xa was 42.4 IU/mg, and the anti-IIa was 21.4 IU/mg.
  • the molecular weight and distribution of the low molecular weight heparin were measured by the above method, and the results are shown in Table 4 or 5 below.
  • DSS dextran sulfate sodium salt
  • the healthy control group did not do any treatment.
  • the DSS module continued to drink 2.5-3% of DSS drinking water until the end of the experiment.
  • the drug treatment group continued to drink 2.5-3% of DSS drinking water until the end of the experiment.
  • the anticoagulant heparin obtained in Examples 7, 8, and 9, and the samples of Comparative Examples 7 and 8 were dissolved in physiological saline, and administered intragastrically from the onset of symptoms at a dose of 30 mg/kg/day until the end of the experiment. .
  • mice The changes in body weight of mice are shown in Fig. 6.
  • symptoms such as weight loss, loose stools and pus and bloody stools began to appear.
  • Different medications were given starting from the onset of symptoms, and the (super) low molecular anticoagulation heparin treatment group significantly slowed DSS-induced weight loss.
  • the experiment was completed. After the mice were sacrificed, blood was taken from the eyelids, and the spleen was weighed.
  • the embedded paraffin block can be sliced; the thickness of the slice is about 5 ⁇ m.
  • Hematoxylin staining The hydrated sections were immersed in hematoxylin staining solution for 5-20 min to stain the nuclei. Rinse with tap water for 3 to 5 minutes.
  • Eosin staining The slice after sufficient hydration is directly into the eosin staining solution, and the cytoplasm is stained for about 5 to 15 minutes.
  • NC group normal group: colonic epithelial tissue is intact, structure is clear, epithelial cells are arranged neatly, gland is intact;
  • DSS model colonic mucosal epithelial cells atrophy, necrosis, shedding, gland abnormalities, glandular goblet cells disappear, The inflammatory cells were extensively infiltrated, the basement membrane was broken or disappeared, the gap between the glandular epithelium and the mucosa was increased, the submucosal capillaries proliferated, and the hemorrhage was dilated;
  • HP treatment group colonic mucosal epithelial cells were atrophied, necrotic, shedding, gland incomplete, Inflammatory cells infiltrated, thickened basement membrane, no fracture, increased clearance between glandular epithelium and mucosal muscle layer, submucosal capillaries hyperplasia, and dilated hemorrhage;
  • NAHP treatment group partial exfoliation of colonic
  • the ULNAHP treatment group and the LNAHP treatment group the colonic epithelial tissue was intact, the structure was clear, the epithelial cells were arranged neatly, the glands were intact, and some glands were proliferated. No abnormalities in the submucosa; 5-ASA (Mesalazine) treatment group (positive control treatment group): colonic mucosal epithelial cells were bad Death, shedding, gland abnormalities, irregular alignment, goblet cell hyperplasia or necrosis, inflammatory cell infiltration, lesions do not involve the submucosa.
  • 5-ASA Mesalazine
  • Fig. 7 were histologically scored according to the scoring criteria in Table 8 above, and the results are shown in Fig. 8. It can be seen that the score of the NAHP treatment group is significantly lower than that of the DSS model, while the ULNAHP treatment group and the LNAHP treatment group are more effective than the NAHP treatment group, and the scores are significantly better than the 5-ASA treatment group.
  • the intestinal muscles of the mice contracted, resulting in a shortened length of the entire colorectal.
  • 3% DSS was administered to mice for 7 consecutive days or after drug treatment, the animals were sacrificed by cervical dislocation, and the whole colorectal was taken and the length was measured.
  • the length of the colon in each group is shown in Figure 9.
  • the other heparin drugs can alleviate colon shortening to some extent.
  • the NAHP treatment group the enzymatic degradation of the ULNAHP treatment group and the LNAHP treatment group were better than the 5-ASA treatment group.
  • the enzymatic low molecular and ultra low molecular weight heparin treatment group was superior to the NAHP treatment group.
  • Apoptosis plays an important role in the development of UC. Under the pathological conditions of inflammatory bowel disease, the normal order of proliferation, differentiation and apoptosis of epithelial cells along the crypt villus may be destroyed. The apoptotic rate of mucosal epithelial cells in the inflammatory zone of UC is significantly increased, which may be another important cause of the destruction of UC epithelial barrier function. Early changes in apoptosis occur on the surface of the cell membrane. One of the changes in the surface of these cell membranes is the transfer of phosphatidylserine (PS) from the cell membrane to the extracellular membrane, exposing PS to the outer surface of the cell membrane.
  • PS phosphatidylserine
  • Annexin V acts as a Ca 2+ -dependent phospholipid-binding protein with a high affinity for PS. Therefore, the protein acts as a sensitive probe to detect PS exposed to the surface of the cell membrane. PS transfer to the cell membrane is not unique to apoptosis, but can also occur in cell necrosis. The difference between the two cell death patterns is that the cell membrane is intact at the initial stage of apoptosis, and cell necrosis is destroyed in its early stages. Therefore, apoptotic cells and necrotic cells can be distinguished by staining the nucleus with propidium iodide (PI).
  • PI propidium iodide
  • 3% dextran sulfate sodium was used to induce apoptosis of human normal colonic epithelial cells NCM460 (purchased from ATCC (Rockefeller, MD, USA)) to simulate DSS-induced ulcerative colitis in mice.
  • DSS dextran sulfate sodium
  • the level of intestinal epithelial cell apoptosis in the (super) low-molecular anticoagulant heparin-treated group was significantly lower than that in the DSS-induced apoptosis group, effectively demonstrating the pair of ULNAHP and LNAHP.
  • the mitigating effect of DSS-induced apoptosis was superior to that of the 5-ASA group and the NAHP group.
  • Tight junctions between epithelial cells are important structures that maintain the mechanical barrier and permeability of the mucosal epithelium.
  • ZO-1 protein is one of the important constituent proteins of cell tight junction protein, which not only participates in the maintenance and regulation of epithelial barrier function, but also participates in important processes such as cell material transport and maintaining epithelial polarity.
  • DSS dextran sulfate sodium
  • the expression level of ZO-1 protein in the derivative treatment group was significantly higher than that in the DSS-induced group, but NAHep-1 (Comparative Example 5) and NAHep- obtained by anticoagulant modification after enzymatic hydrolysis. 2 (Comparative Example 6), and enzymatic hydrolysis of low molecular weight heparin (Comparative Examples 9, 10) did not have the effect of alleviating the decrease in ZO-1.
  • This experiment effectively proved that the anticoagulant heparin derivative obtained by anticoagulation modification and re-enzymatic hydrolysis had the effect of alleviating DSS-induced cell membrane permeability abnormality.

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Abstract

本发明公开了一种去抗凝肝素衍生物及其用于炎症性肠病的治疗，该去抗凝肝素衍生物的抗Xa因子小于等于70 IU/mg，优选小于等于60 IU/mg，优选抗小于等于50 IU/mg，优选小于等于40 IU/mg，优选小于等于30 IU/mg，优选小于等于20 IU/mg，优选小于等于10 IU/mg，其抗IIa因子小于等于175 IU/mg，优选小于等于170 IU/mg，优选小于等于160 IU/mg，优选小于等于150 IU/mg，优选小于等于140 IU/mg，优选小于等于130 IU/mg，优选小于等于120 IU/mg，优选小于等于110 IU/mg，优选小于等于100 IU/mg，优选小于等于90 IU/mg，优选小于等于80 IU/mg，优选小于等于70 IU/mg，优选小于等于60 IU/mg，优选小于等于50 IU/mg，优选小于等于40 IU/mg，优选小于等于30 IU/mg，优选小于等于20 IU/mg，优选小于等于10 IU/mg。

Description

去抗凝肝素衍生物及其用于炎症性肠病的治疗 技术领域
本发明涉及制备去抗凝肝素衍生物，以及其用于预防和/或治疗炎症性肠病的用途。
背景技术
炎症性肠病(Inflammatory Bowel Disease,IBD)是一组发病非常广泛、无法治愈的慢性炎性病症，包括溃疡性结肠炎(Ulcerative Colitis,UC)和克罗恩病(Crohn's disease,CD)。据报道，在西方国家IBD患病率最高达到0.8％，在亚洲地区IBD的发病率和患病率均呈持续增长趋势并且增速迅猛，成为全球广泛关注的疾病。预计在未来十年内很多国家的患病率增长幅度将超过40％。IBD相关药物需求将有大幅度增长(Bernstein C N,et al.World Gastroenterology Organisation Global Guidelines Inflammatory Bowel Disease:Update August 2015.Journal of Clinical Gastroenterology,2016,50(10):p.803-818.)。在东方国家，IBD的发病以UC为主。在印度，UC/CD的发病率比例是8-10:1(Bernstein,C.N.,et al.,World Gastroenterology Organization Practice Guidelines for the diagnosis and management of IBD in 2010.Inflamm Bowel Dis,2010.16(1):p.112-24.)。目前UC的病因和发病机制尚不明确且无法治愈(Kornbluth,A.and D.B.Sachar,Ulcerative colitis practice guidelines in adults:American College Of Gastroenterology,Practice Parameters Committee.Am J Gastroenterol,2010.105(3):p.501-23；quiz 524.)。
UC的治疗药物在整体动物模型和临床实验中已研究了40多年时间。UC患者的传统治疗，一般采用巯基嘌呤类免疫抑制剂，皮质类固醇消炎药等(Chen,Y.,et al.,PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function.J Biol Chem,2015.290(33):p.20580-9.Ordas,I.,et al.,Ulcerative colitis.Lancet,2012.380(9853):p.1606-19.Zhang,M.,et al.,The proinflammatory effect and molecular mechanism of IL-17in the intestinal epithelial cell line HT-29.J BUON,2015.20(1):p.120-7.)。但是由于药物 特异性较差，副作用较强，临床治疗仍然无法得到令人满意的效果(Rosenberg,L.N.and M.A.Peppercorn,Efficacy and safety of drugs for ulcerative colitis.Expert Opin Drug Saf,2010.9(4):p.573-92.)。在药物治疗无效，或炎症进一步发展的情况下，一般只有采用手术治疗对病变部位进行切除(Akiho,H.,et al.,Promising biological therapies for ulcerative colitis:A review of the literature.World J Gastrointest Pathophysiol,2015.6(4):p.219-27.)。
UC病人本身具有高凝的风险，常用肝素或低分子量肝素(Low Molecular Weight Heparins,LMWHs)来进行预防及治疗。
肝素是一类硫酸化、多分散、线性的糖胺聚糖(Glycosaminoglycans，GAGs)，是最重要的抗凝药物之一。在临床上广泛应用于防治血栓栓塞性疾病。此外，肝素及其衍生物具有广泛的生物学活性，包括协调细胞黏附、调控细胞生长和增殖、发育过程、细胞表面结合脂蛋白脂肪酶和其它蛋白质、新血管发生、病毒入侵和肿瘤转移等等。
LMWHs的抗炎等生物学活性也发挥治疗UC的作用，很多临床UC患者使用LMWHs后症状得到缓解(Lean,Q.Y.,et al.,Heparins in ulcerative colitis:proposed mechanisms of action and potential reasons for inconsistent clinical outcomes.Expert Rev Clin Pharmacol,2015.8(6):p.795-811.)。乙酰肝素酶是肝素类药物的重要靶点，抑制乙酰肝素酶的活性，防止肠黏膜的进一步损伤是其发挥治疗作用机制之一(Waterman,M.,et al.,Heparanase upregulation by colonic epithelium in inflammatory bowel disease.Mod Pathol,2007.20(1):p.8-14.)。但目前LMWHs在治疗UC上，存在着构效关系尚不明确等问题。
探索利用LMWHs治疗溃疡性结肠炎(UC)已有20多年时间。一些病人皮下注射LMWHs(如达肝素，那曲肝素)后症状得到明显缓解。另外一部分研究考察了口服肝素对溃疡性结肠炎的治疗效果，70％-90％的临床病人得到了完全缓解。然而，也有某些研究得到的结论是相矛盾的。例如，Elan在2001年开发了Deligoparin(OP-2000)来作为UC的潜在治疗药物。虽然安全性评价数据结果很好，但临床II期和III期实验结果却十分令人失望(Korzenik,J.,et al.,Multicenter,randomized,double-blind,placebo-controlled trial of deligoparin(ultra low molecular weight heparin)for active ulcerative colitis.Gastroenterology,2003.124(4):p.A67.)。导致这些矛盾的可能原因有很多，譬如给药剂量不同，病情进展和严重 程度不一，临床终点不一致等。
发明内容
本发明的目的是要提供一类具有预防和/或治疗炎症性肠病的去抗凝肝素衍生物类药物。肝素类药物在临床上常用于伴有高凝状态的炎症性肠病病人，一方面发挥肝素本身的抗凝血功能，另外肝素也具有抗炎效果，但抗凝血活性的保留始终存在诱发出血的风险，这些副作用制约了肝素在与凝血相关性不强的炎症性疾病中的应用。
去抗凝肝素衍生物用于炎症性肠病的治疗还未见报道，目前主要有利用高碘酸氧化法对肝素去抗凝的方法，还有利用硅烷化试剂、碱催化或溶剂解等化学方法对肝素进行去硫酸化修饰达到去抗凝效果。
在本发明中，本发明人尝试对市场上购买的肝素、可商购的低分子量肝素依诺肝素等利用后述的几种不同的方法进行肝素的去抗凝处理，研究结果发现去抗凝修饰对于提高炎症性肠病治疗效果十分关键。相对于具有抗凝活性的各种肝素或(超)低分子量肝素而言，其各自经过处理后的去抗凝肝素衍生物都显示出良好的IBD治疗效果。
本发明的发明人致力于肝素产业技术的创新研究，利用麦芽糖结合蛋白(Maltose Binding Protein，MBP)融合表达技术，实现了系列肝素酶(Heparinase，分别为MBP-HepI、MBP-HepII、MBP-HepIII，可以分别参见中国专利ZL200410038098.6、ZL201010259905.2，以及ZL201010259913.7)的高活性、可溶性表达和工业化生产，系列肝素酶被列为中国药典标准酶。
在本发明中，首先对肝素进行修饰得到去抗凝肝素衍生物，然后利用肝素酶降解去抗凝肝素衍生物得到经酶降解的低分子量和/或超低分子量去抗凝肝素衍生物。
具体来说，在本发明中，本发明人尝试对市场上购买的肝素利用后述几种不同的方法进行去抗凝处理，再利用肝素酶I进行可控降解。研究结果发现相对于高分子量的去抗凝肝素而言，肝素酶降解得到的低分子量和超低分子量的去抗凝肝素衍生物能明显提高炎症性肠病治疗效果。
具体来说，本发明涉及以下内容：
(1).一种去抗凝肝素衍生物，其抗Xa因子小于等于70IU/mg，优选抗Xa 因子小于等于60IU/mg，优选抗Xa因子小于等于50IU/mg，优选抗Xa因子小于等于40IU/mg，优选抗Xa因子小于等于30IU/mg，优选抗Xa因子小于等于20IU/mg，优选抗Xa因子小于等于10IU/mg，并且
其抗IIa因子小于等于175IU/mg，优选抗IIa因子小于等于170IU/mg，优选抗IIa因子小于等于160IU/mg，优选抗IIa因子小于等于150IU/mg，优选抗IIa因子小于等于140IU/mg，优选抗IIa因子小于等于130IU/mg，优选抗IIa因子小于等于120IU/mg，优选抗IIa因子小于等于110IU/mg，优选抗IIa因子小于等于100IU/mg，优选抗IIa因子小于等于90IU/mg，优选抗IIa因子小于等于80IU/mg，优选抗IIa因子小于等于70IU/mg，优选抗IIa因子小于等于60IU/mg，优选抗IIa因子小于等于50IU/mg，优选抗IIa因子小于等于40IU/mg，优选抗IIa因子小于等于30IU/mg，优选抗IIa因子小于等于20IU/mg，优选抗IIa因子小于等于10IU/mg。
(2).根据(1)所述的去抗凝肝素衍生物，其中，所述去抗凝肝素衍生物的重均分子量为8000以上。
(3).根据(1)所述的去抗凝肝素，其中，所述去抗凝肝素衍生物的重均分子量为小于8000。
(4).一种去抗凝肝素衍生物在用于制备治疗炎症性肠病，以及炎症性肠病相关并发症及发病机理相似的疾病的药物中的用途，其中，炎症性肠病相关并发症及发病机理相似的疾病包括但不限于肠易激综合征、关节炎和其他肠外并发症包括强直性脊柱炎、坏疽性脓皮病、结节性红斑、虹膜炎、葡萄膜炎、巩膜外层炎和原发性硬化性胆管炎。
(5).根据(4)所述的用途，其中，所述去抗凝肝素衍生物的抗Xa因子小于等于70IU/mg，优选抗Xa因子小于等于60IU/mg，优选抗Xa因子小于等于50IU/mg，优选抗Xa因子小于等于40IU/mg，优选抗Xa因子小于等于30IU/mg，优选抗Xa因子小于等于20IU/mg，优选抗Xa因子小于等于10IU/mg，并且
其抗IIa因子小于等于175IU/mg，优选抗IIa因子小于等于170IU/mg，优选抗IIa因子小于等于160IU/mg，优选抗IIa因子小于等于150IU/mg，优选抗IIa因子小于等于140IU/mg，优选抗IIa因子小于等于130IU/mg，优选抗IIa因子小于等于120IU/mg，优选抗IIa因子小于等于110IU/mg，优选抗IIa因子小于等于100IU/mg，优选抗IIa因子小于等于90IU/mg，优选抗IIa因子小于等于 80IU/mg，优选抗IIa因子小于等于70IU/mg，优选抗IIa因子小于等于60IU/mg，优选抗IIa因子小于等于50IU/mg，优选抗IIa因子小于等于40IU/mg，优选抗IIa因子小于等于30IU/mg，优选抗IIa因子小于等于20IU/mg，优选抗IIa因子小于等于10IU/mg。
(6).根据(4)或(5)所述的用途，其中，所述去抗凝肝素衍生物的重均分子量为8000以上。
(7).根据(4)或(5)所述的用途，其中，所述去抗凝肝素衍生物的重均分子量为小于8000。
(8).根据(1)所述的去抗凝肝素衍生物，其重均分子量为600～8000，优选重均分子量为1000～7800，进一步优选重均分子量为1500～7500，进一步优选重均分子量为2000～7000，其数均分子量为600～6000，进一步优选数均分子量为1200～5800，进一步优选数均分子量为1500～5500，进一步优选数均分子量为1800～5000。
(9).根据(1)或(8)所述的去抗凝肝素衍生物，其2糖单元占全部糖成分的含量在40重量％以上，进一步优选在45重量％以上，进一步优选在50重量％以上，进一步优选在55重量％以上。
(10).根据(1)、(8)或(9)中任一项所述的去抗凝肝素衍生物，其4糖单元占全部糖成分的含量为10～30重量％，进一步优选为15～25重量％。
(11).根据(1)或(8)～(10)中任一项所述的去抗凝肝素衍生物，其6糖单元占全部糖成分的含量在15重量％以下，优选在12重量％以下，进一步优选在10重量％以下，进一步优选在8重量％以下。
(12).根据(1)或(8)～(11)中任一项所述的去抗凝肝素衍生物，其重均分子量与数均分子量之比(重均分子量/数均分子量)在1.4以上，优选在1.45以上，进一步优选在1.50以上。
(13).一种制备去抗凝肝素衍生物的方法，其包括：
对原料肝素进行去抗凝处理，得到去抗凝处理的产物，以及
随后利用酶对经去抗凝处理的产物进行酶解以获得去抗凝肝素衍生物。
(14).根据(13)所述的方法，其中，所述酶为肝素酶。
(15).根据(14)所述的方法，其中，所述肝素酶为肝素酶I。
(16).根据(13)～(15)中任一项所述的方法，其中，所述对原料肝素进行去抗凝 处理是利用高碘酸氧化法对原料肝素进行处理。
(17).根据项(13)～(16)中任一项所述的方法，其中，所述去抗凝肝素衍生物是(1)或(8)～(12)中任一项所述的去抗凝肝素衍生物。
(18).根据(4)所述的用途，其中，所述去抗凝肝素衍生物是(1)或(8)～(12)中任一项所述的去抗凝肝素衍生物。
(19).根据(18)所述的用途，其中，所述去抗凝肝素衍生物是利用(13)～(17)中任一项所述的方法制备的去抗凝肝素衍生物。
(20).一种治疗炎症性肠病，以及炎症性肠病相关并发症及发病机理相似的疾病的方法，其包括：
向有需要的受试者给药去抗凝肝素衍生物，其中该去抗凝肝素衍生物为发明所述的去抗凝肝素衍生物。
附图说明
图1实验例1-3中结直肠组织病理切片HE染色。
图2实验例1-3中的全段结直肠长度结果，其中(a)是结直肠的照片，(b)是显示各组结直肠长度的柱状图。
图3实验例1-3试验的脾脏指数。
图4实验例1-3中的肠上皮细胞紧密连接蛋白ZO-1表达水平。
图5实验例1-3的肠上皮细胞凋亡水平，其中(a)是AnnexinV/PI双染的流式四象限图，(b)是凋亡细胞百分比图。
图6实验例4-6中的小鼠体重变化百分比图。
图7实验例4-6中结直肠组织病理切片HE染色结果。
图8实验例4-6中结直肠组织病理切片HE染色结果的组织学评分。
图9实验例4-6中的全段结直肠长度结果，其中(a)是结直肠的照片，(b)是显示各组结直肠长度统计结果的柱状图。
图10实验例4-6中的肠上皮细胞凋亡水平，其中(a)是AnnexinV/PI双染的流式四象限图，(b)是凋亡细胞百分比图。
图11实验例4-6中的肠上皮细胞紧密连接蛋白ZO-1表达水平。
具体实施方式
以下，对本文的实施方式进行具体说明。
<本发明的去抗凝肝素衍生物(以下也称为去抗凝肝素)>
本发明涉及的去抗凝肝素衍生物是对肝素或(超)低分子量肝素进行去抗凝处理后得到的物质。
本发明涉及的去抗凝肝素衍生物还可以是在去抗凝修饰基础上再利用肝素酶控制降解得到的物质。
通常来说肝素、低分子量肝素、戊糖都是通过加快抗凝血酶Ⅲ灭活凝血因子的速度而起到抗凝作用，该类药物的主要作用是抗Xa和抗IIa因子活性。通过对肝素类药物抗Xa活性与抗IIa活性的研究发现，抗Xa活性对分子质量不敏感，抗IIa活性则依赖分子质量的大小。分子质量越大，抗IIa活性越强。肝素对IIa因子灭活有赖于肝素-抗凝血酶-IIa因子三联复合物的形成，此时肝素同时结合于抗凝血酶和因子IIa，要实现这种连接肝素至少要含有18个糖单位，其中起“桥梁”作用需要13个单糖，另需5个单糖作为识别片段。每个单糖平均分子质量为300Da，因此分子质量一定要达到5400Da以上才具有抗IIa活性。普通肝素平均分子质量15000-19000Da，绝大多数分子在5400Da以上，其抗Xa与抗IIa活性比值约为1。低分子肝素平均分子质量为4000-5000Da，分子质量在5400Da以上的分子片段所占比例较小，一般情况下其抗Xa:抗IIa活性约1.5:1～5:1。
在本发明的去抗凝肝素的抗Xa因子小于等于70IU/mg，优选抗Xa因子小于等于60IU/mg，优选抗Xa因子小于等于50IU/mg，优选抗Xa因子小于等于40IU/mg，优选抗Xa因子小于等于30IU/mg，优选抗Xa因子小于等于20IU/mg，优选抗Xa因子小于等于10IU/mg。
在本发明的去抗凝肝素的抗IIa因子小于等于175IU/mg，优选抗IIa因子小于等于170IU/mg，优选抗IIa因子小于等于160IU/mg，优选抗IIa因子小于等于150IU/mg，优选抗IIa因子小于等于140IU/mg，优选抗IIa因子小于等于130IU/mg，优选抗IIa因子小于等于120IU/mg，优选抗IIa因子小于等于110IU/mg，优选抗IIa因子小于等于100IU/mg，优选抗IIa因子小于等于90IU/mg，优选抗IIa因子小于等于80IU/mg，优选抗IIa因子小于等于70IU/mg，优选抗IIa因子小于等于60IU/mg，优选抗IIa因子小于等于50IU/mg，优选抗IIa因子小于等于40IU/mg，优选抗IIa因子小于等于30IU/mg，优选抗IIa因子小于等于20IU/mg，优选抗IIa因子小于等于10IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于175IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于175IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于175IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于175IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于175IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于175IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于175IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于170IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于170IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于170IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于170IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于170IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于170IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于170IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于160IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于160IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于160IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于160IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于160IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于160IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于160IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于150IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于150IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等 于50IU/mg，且抗IIa因子小于等于150IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于150IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于150IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于150IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于150IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于140IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于140IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于140IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于140IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于140IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于140IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于140IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于130IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于130IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于130IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于130IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于130IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于130IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于130IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于120IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于120IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于120IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于120IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于120IU/mg。本 发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于120IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于120IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于110IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于110IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于110IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于110IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于110IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于110IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于110IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于100IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于100IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于100IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于100IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于100IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于100IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于100IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于90IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于90IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于90IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于90IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于90IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于90IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于90IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于80IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于80IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于80IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于80IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于80IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于80IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于80IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于70IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于70IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于70IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于70IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于70IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于70IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于70IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于60IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于60IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于60IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于60IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于60IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于60IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于60IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于50IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于50IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于 50IU/mg，且抗IIa因子小于等于50IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于50IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于50IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于50IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于50IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于40IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于40IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于40IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于40IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于40IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于40IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于40IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于30IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于30IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于30IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于30IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于30IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于30IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于30IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于20IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于20IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于20IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于20IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于20IU/mg。本发明 的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于20IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于20IU/mg。
本发明的一种去抗凝肝素的抗Xa因子小于等于70IU/mg，且抗IIa因子小于等于10IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于60IU/mg，且抗IIa因子小于等于10IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于50IU/mg，且抗IIa因子小于等于10IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于40IU/mg，且抗IIa因子小于等于10IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于30IU/mg，且抗IIa因子小于等于10IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于20IU/mg，且抗IIa因子小于等于10IU/mg。本发明的一种去抗凝肝素的抗Xa因子小于等于10IU/mg，且抗IIa因子小于等于10IU/mg。
在本发明的一个具体的实施方式中，一种去抗凝肝素衍生物的抗Xa因子为5.8IU/mg，抗IIa因子为5.8IU/mg，并且该去抗凝肝素衍生物的重均分子量为15158Da，数均分子量为13224Da，其重均分子量分布为，大于24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为8.57％，16k～24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为28％，8k～16k的肝素分子所占整个去抗凝肝素衍生物的比例为53.8％，小于8k Da的肝素分子所占整个去抗凝肝素衍生物的比例为9.63％。该去抗凝肝素衍生物是通过对市场上购买的普通肝素进行去抗凝处理之后得到的。
在本发明的一个具体的实施方式中，一种去抗凝肝素衍生物的抗Xa因子为3.5IU/mg，抗IIa因子为5.1IU/mg，并且该去抗凝肝素衍生物的重均分子量为4326Da，数均分子量为3254Da，其重均分子量分布为，小于3k Da的肝素分子所占整个去抗凝肝素衍生物的比例为35.55％，3k～5k Da的肝素分子所占整个去抗凝肝素衍生物的比例为31.94％，5k～8k的肝素分子所占整个去抗凝肝素衍生物的比例为24.07％，大于8k Da的肝素分子所占整个去抗凝肝素衍生物的比例为8.44％。该去抗凝肝素衍生物是通过对市场上购买的低分子肝素，即伊诺肝素进行去抗凝处理之后得到的。
在本发明的一个具体的实施方式中，一种去抗凝肝素衍生物的抗Xa因子为20.3IU/mg，抗IIa因子为33.1IU/mg，并且该去抗凝肝素衍生物的重均分子量为 16427Da，数均分子量为13117Da，其重均分子量分布为，大于24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为20.62％，16k～24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为20.00％，8k～16k的肝素分子所占整个去抗凝肝素衍生物的比例为36.71％，小于8k Da的肝素分子所占整个去抗凝肝素衍生物的比例为22.67％。该去抗凝肝素衍生物是通过对市场上购买的普通肝素进行去抗凝处理之后得到的。
在本发明的一个具体的实施方式中，一种去抗凝肝素衍生物的抗Xa因子为60.6IU/mg，抗IIa因子为170.8IU/mg，并且该去抗凝肝素衍生物的重均分子量为15793Da，数均分子量为13223Da，其重均分子量分布为，大于24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为13.44％，16k～24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为26.04％，8k～16k的肝素分子所占整个去抗凝肝素衍生物的比例为44.20％，小于8k Da的肝素分子所占整个去抗凝肝素衍生物的比例为16.32％。该去抗凝肝素衍生物是通过对市场上购买的普通肝素进行去抗凝处理之后得到的。
在本发明的一个具体的实施方式中，一种去抗凝肝素衍生物的抗Xa因子为39IU/mg，抗IIa因子为124.9IU/mg，并且该去抗凝肝素衍生物的重均分子量为15212Da，数均分子量为12791Da，其重均分子量分布为，大于24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为10.91％，16k～24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为24.46％，8k～16k的肝素分子所占整个去抗凝肝素衍生物的比例为46.95％，小于8k Da的肝素分子所占整个去抗凝肝素衍生物的比例为17.68％。该去抗凝肝素衍生物是通过对市场上购买的普通肝素进行去抗凝处理之后得到的。
在本发明的一个具体的实施方式中，一种去抗凝肝素衍生物的抗Xa因子为1.2IU/mg，抗IIa因子为7.5IU/mg，并且该去抗凝肝素衍生物的重均分子量为16706Da，数均分子量为13915Da，其重均分子量分布为，大于24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为17.10％，16k～24k Da的肝素分子所占整个去抗凝肝素衍生物的比例为28.55％，8k～16k的肝素分子所占整个去抗凝肝素衍生物的比例为40.67％，小于8k Da的肝素分子所占整个去抗凝肝素衍生物的比例为13.68％。该去抗凝肝素衍生物是通过对市场上购买的普通肝素进行去抗凝处理之后得到的。
用于生产本发明的去抗凝肝素衍生物的原料肝素可以是普通肝素，也可以是(超)低分子肝素，既可以是重均分子量大于8000Da的肝素，也可以是重均分子量在8000Da以下的肝素。
本发明所述的去抗凝肝素衍生物的抗Xa因子和抗IIa因子的活性同时满足上述要求。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的抗Xa因子在0～70IU/mg的范围，其抗IIa因子在0～175IU/mg的范围。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的抗Xa因子在0～60IU/mg的范围，其抗IIa因子在0～140IU/mg的范围。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的抗Xa因子在0～50IU/mg的范围，其抗IIa因子在0～110IU/mg的范围。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的抗Xa因子在0～40IU/mg的范围，其抗IIa因子在0～80IU/mg的范围。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的抗Xa因子在0～30IU/mg的范围，其抗IIa因子在0～50IU/mg的范围。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的抗Xa因子在0～20IU/mg的范围，其抗IIa因子在0～30IU/mg的范围。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的抗Xa因子在0～10IU/mg的范围，其抗IIa因子在0～10IU/mg的范围。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的重均分子量为1000～8000，其重均分子量为600～8000，优选重均分子量为1000～7800，进一步优选重均分子量为1500～7500，进一步优选重均分子量为2000～7000，其数均分子量为600～6000，进一步优选数均分子量为1200～5800，进一步优选数均分子量为1500～5500，进一步优选数均分子量为1800～5000。
例如，优选重均分子量为2000～7500，进一步优选为2500～7300，进一步优选为2800～7100，其数均分子量为600～6000，优选数均分子量为1500～5000，进一步优选为1800～4800，进一步优选为1900～4600。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的重均分子量与数均分子量之比(重均分子量/数均分子量)在1.40以上，优选在1.41以上，进一步优选在1.42以上，进一步优选在1.43以上，进一步优选在1.44以上，进一步优选 在1.45以上，进一步优选在1.46以上，进一步优选在1.47以上，进一步优选在1.48以上，进一步优选在1.49以上，进一步优选在1.50以上。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的2糖单元占全部糖成分的含量在40重量％以上，进一步优选在45重量％以上，进一步优选在50重量％以上，进一步优选在55重量％以上。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的4糖单元占全部糖成分的含量为10～30重量％，进一步优选为15～25重量％。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的6糖单元占全部糖成分的含量在15重量％以下，优选在12重量％以下，进一步优选在10重量％以下，进一步优选在8重量％以下。
在一个具体的实施方式中，本发明的去抗凝肝素衍生物的8糖单元占全部糖成分的含量在10重量％以下，优选在7重量％以下，进一步优选在6重量％以下，进一步优选在5重量％以下，进一步优选在4.5重量％以下，进一步优选在4重量％以下。
本发明中的全部糖成分是指利用下述实施例中列举的方法检测时检测到全部糖成分，而2糖单元占全部糖成分的含量是指能够检测出来的全部2糖单元的合计在全部糖成分中所占的比例，4糖单元占全部糖成分的含量是指能够检测出来的全部4糖单元的合计在全部糖成分中所占的比例，6糖单元占全部糖成分的含量是指能够检测出来的全部6糖单元的合计在全部糖成分中所占的比例，8糖单元占全部糖成分的含量是指能够检测出来的全部8糖单元的合计在全部糖成分中所占的比例。
<本发明中使用的生产去抗凝肝素衍生物的方法>
通过高碘酸氧化法能得到去除抗凝活性的肝素分子，并且其他生物学活性能很大程度保留，硫酸化程度与形式基本保持不变。高碘酸可以选择性的氧化含有未取代羟基或氨基的邻位碳原子，使得无硫酸化的糖醛酸C(2)-C(3)键断裂，肝素分子内的抗凝血酶结合五糖中的葡萄糖醛酸因而遭到破坏，失去抗凝活性；高碘酸氧化得到的聚醛类氧化肝素通过硼氢化物的还原以保持稳定(Islam,T.,et al.,Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site.Carbohydrate Research,2002.337(21–23):p.2239-2243.)。经过过去二三十年的研究发现，不同去抗凝程度和不同分子量大小的氧化去抗凝 肝素具有抗肿瘤转移、抗炎、抗疟等多种生物学活性，已被应用于急性心肌梗死、肿瘤转移、血管生成等领域的研究之中(Cassinelli,G.and A.Naggi,Old and new applications of non-anticoagulant heparin.International Journal of Cardiology,2016.212,Supplement 1:p.S14-S21.)。部分药物已进入临床研究阶段，如分子量与肝素相近的SST0001(Cassinelli,G.,et al.,Antitumor efficacy of the heparanase inhibitor SST0001alone and in combination with antiangiogenic agents in the treatment of human pediatric sarcoma models.Biochemical Pharmacology,2013.85(10):p.1424-1432.)，还有低分子量的Vasoflux，M402等(Zhou,H.,et al.,M402,a novel heparan sulfate mimetic,targets multiple pathways implicated in tumor progression and metastasis.PloS one,2011.6(6):p.e21106.Weitz,J.I.,et al.,Vasoflux,a new anticoagulant with a novel mechanism of action.Circulation,1999.99(5):p.682-689.)。
利用硅烷化试剂如N,O-双(三甲基硅基)乙酰胺(BTSA)或N-甲基-N-(三甲基硅基)-三氟乙酰胺(MTSTFA)与肝素吡啶盐反应可以选择性地去除肝素的6-O-硫酸基，BTSA在温和条件下能部分去除肝素的6-O-硫酸基，而剧烈条件下往往有N-去硫酸化的副反应产生。MTSTFA可以完全去除肝素的6-O-硫酸基并且副反应很少，仅有少量的2-O-硫酸基会被影响(Kariya,Y.,et al.,Preparation of completely 6-O-desulfated heparin and its ability to enhance activity of basic fibroblast growth factor.Journal of Biological Chemistry,2000.275(34):p.25949-25958.)。
碱催化法是去除肝素部分O-硫酸基的常用修饰手段。当肝素在碱性条件下冻干(pH为11-14)，2-O-硫酸-α-L-艾杜糖醛酸残基发生去硫酸化形成2,3-环氧化合物中间体，进一步水解形成去硫酸化的α-L-艾杜糖醛酸，得到2-O去硫酸化的肝素。肝素链中D-葡萄糖胺残基上较为罕见的3-O硫酸基也会受到影响部分脱落，而其余硫酸基则保持完整。不同的碱性条件(如在不同浓度的氢氧化钠溶液中)可能会导致2-O去硫酸化程度不一。反应过程也可以添加还原剂如硼氢化钠以保护肝素链不会降解片段化(Fryer,A.,et al.,Selective O-desulfation produces nonanticoagulant heparin that retains pharmacological activity in the lung.Journal of Pharmacology and Experimental Therapeutics,1997.282(1):p.208-219.)。
肝素葡萄糖胺残基上N-硫酸基的去除常采用溶剂解法。水解去N位硫酸基 可能伴随糖苷键的断裂和O-硫酸基的去除，而肝素吡啶盐在含少量水的DMSO中能实现N-硫酸基的去除并且无糖苷键断裂(Inoue,Y.,&Nagasawa,K.Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol.Carbohydrate research,1976.46(1):p.87-95.)。随后利用乙酰化试剂如乙酸酐可以实现N-再乙酰化(Purkerson M L,Tollefsen D M,Klahr S.N-desulfated/acetylated heparin ameliorates the progression of renal disease in rats with subtotal renal ablation.Journal of Clinical Investigation,1988,81(1):p.69.)。
在本发明中，分别采用上述四种方法获得了不同的去抗凝肝素衍生物。下述实施例1、2、4、5和7-9中采用高碘酸氧化法破坏五糖结构从而去除抗凝活性。下述实施例3采用硅烷化试剂法去除6-O硫酸基从而去除抗凝活性。下述实施例6采用溶剂解法去除N-硫酸基从而去除抗凝活性。
在本发明的一个具体的实施方式中，将底物肝素(例如，肝素钠)溶于水中，并加入高碘酸钠溶液，进行反应。反应一段时间后，加入乙二醇以中和过量的高碘酸钠，再添加硼氢化钠于反应。调节pH值后，经过滤，收集过滤样品。再利用透析袋等进行浓缩和除盐，最终得到去除抗凝活性的去抗凝肝素衍生物。
本发明中使用的经去抗凝处理后的底物肝素的浓度可以由本领域技术人员来确定，没有特定地限制，优选为1～100g/L。本发明中使用的底物肝素是大分子量的未分级的肝素，其分子量例如可以是分布在5000～30000，平均分子量为20000。
<本发明中使用的对去抗凝肝素衍生物进行酶解的方法>
在一个具体的实施方式中，生产本发明的去抗凝肝素衍生物的过程中使用了肝素酶，例如肝素酶I。现有技术中，肝素酶I的E.C.号为E.C.4.2.2.7。也可以采用购买的肝素酶I，例如购自Sigma公司或IBEX公司买到的肝素酶I。肝素酶也可以是通过分子生物学方法构建的重组肝素酶I或者肝素酶I与任何融合伴侣形成的融合蛋白。根据本发明一个优选的实施方案，肝素酶I是肝素酶I的融合蛋白，尤其是包含MBP的肝素酶I的融合蛋白。
肝素酶I也可以是与任何融合伴侣形成的融合蛋白，只要具有肝素酶I的活性即可。根据本文一个优选的实施方案，肝素酶I是肝素酶I和融合伴侣形成的融合蛋白，尤其是麦芽糖结合蛋白(MBP)和肝素酶I的融合蛋白。肝素酶I和MBP的融合蛋白在下文中有时被称为MBP-HepA(参见中国专利ZL 200410038098.6，授权公告号CN1312183C)。
在生产本发明的去抗凝肝素衍生物时，肝素酶I与底物肝素(或去抗凝处理后的底物肝素)进行反应的方式可以分批的、连续的或半连续的，本领域普通技术人员可以根据生产的需要适当地选择。对于反应的时间、反应装置而言，只要是可以得到目标的低分子量肝素即可，可以由本领域普通技术人员适当确定。
在一个具体的实施方案中，在生产本发明的去抗凝肝素衍生物的方法中，向反应器中加入经上文描述的方法得到的去抗凝底物肝素溶液，然后加入肝素酶I，与去抗凝处理后的底物肝素进行反应。随着反应的进行，去抗凝肝素底物逐渐被降解，每隔一段时间对反应液进行监测，在适当的时候终止反应。将上述反应终止了的混合溶液用纤维素膜真空初滤装置进行初滤，再利用超滤装置进行超滤得到次滤液。然后加入乙醇混合均匀后，离心弃上清收集沉淀，然后往沉淀中加入丙酮洗涤并用旋转蒸发仪进行减压蒸干即得到(超)低分子量肝素产品粉末。本领域技术人员可以理解上述方法仅仅为示例性的，也可以采用其他方法获得通过酶反应降解后的(超)低分子量肝素。
肝素酶I的用量，本领域普通技术人员可以参考不同酶的活性根据生产所需来适当确定，优选每种酶的用量为肝素酶I在每升反应液50IU～500IU的范围，优选在100IU～250IU的范围。其中IU表示：在温度为30℃，pH7.4条件下，每分钟产生1μmol 4,5不饱和末端产物的酶量。
用于生产本发明中使用的去抗凝肝素衍生物的底物肝素可以商购，也可以从动物中直接提取，例如可以从猪小肠粘膜提取。肝素二糖单位主要为L-艾杜糖醛酸和N-硫酸化的氨基葡萄糖通过α(1→4)糖苷键连接。
用于生产本发明的去抗凝肝素衍生物的底物可以购买自例如河北常山生化药业股份有限公司的肝素、烟台东诚生化股份有限公司、深圳市海普瑞药业股份有限公司、常州千红生化制药股份有限公司、美药星(南京)制药有限公司等。
本发明中使用的经去抗凝处理后的底物肝素的浓度可以由本领域技术人员来确定，没有特定地限制，优选为1～100g/L。
在进行本发明的生产方法之前，可以将经去抗凝处理后的底物肝素添加到缓冲液中，配制到合适的浓度。所使用的缓冲液只要不损害肝素酶I的酶活即可。在一个具体的生产方法中，采用的是20mM Tris、20mM CaCl 2、50mM NaCl，并用1mM盐酸调节pH为7左右，例如7.4～7.6的缓冲液。在另一个具体的生产方法中，采用的是5.0mM CaCl 2和200mM NaCl的去离子水溶液中，然后用1M  HCl溶液调节pH至7.0的缓冲溶液。
对肝素酶I与经去抗凝处理后的底物肝素进行反应时的温度没有具体限定，只要是不会使肝素酶I失活的温度即可，例如可以设定为10～45℃，最优选30℃。
对于肝素酶I与经去抗凝处理后的底物肝素进行反应的时间没有具体限定，本领域技术人员可以根据所添加的肝素酶的酶活、底物的浓度和反应的温度可以适当选择，在一个具体的方法中，肝素酶与底物反应的时间可以5分钟～10小时，也可以为10分钟～4小时。
在肝素酶I与经去抗凝处理后的底物肝素进行反应的过程中，对反应的溶液进行监测的方式可以根据反应体系来适当选择，在一个具体的方法中，利用紫外分光光度计检测231nm处的吸光度的变化，随着反应的进行231nm处的吸光度A 231不断增加，从而通过吸光度的增加来确定反应进行的程度。
在反应的过程中，当按照上述方法测定反应进行到所期望的程度时，可以终止反应，以进一步分离以获得超低分子量肝素或低分子量肝素。其中对于终止反应的方法，本领域技术人员可以根据其掌握的知识来选择，例如加入终止反应的试剂，或提高温度以使酶失活。在一个具体的实施方式中，终止反应时加盐酸调节pH值至2.0后停留3分钟，再用2.0M的NaOH将pH值调回7.0。从不添加其它的杂质的观点来看，优选提高反应体系的温度使酶失活从而终止反应。在一个具体的实施方式中，将整个反应体系放置在100℃水浴中10分钟，从而使酶降解底物的反应终止。
<本发明中去抗凝肝素衍生物的用途>
在本发明涉及的去抗凝肝素衍生物可用于治疗炎症性肠病，例如可以用于治疗溃疡性结肠炎和克罗恩病。
利用本发明的去抗凝肝素衍生物给药治疗诱导了结肠炎的小鼠模型之后，其可以有效地缓解炎症性肠病发病过程中肠痉挛引发的结肠缩短，并且小鼠的结肠上皮粘膜组织与发病组相比较为完整，腺体结构清晰，炎细胞浸润有所减少，使得给药治疗组的肠道炎症明显减轻，并且能够有效减弱炎症引起的脾脏增大。利用本发明的去抗凝肝素衍生物能够有效地修复炎症性肠病发生时结肠上皮细胞中紧密连接蛋白ZO-1表达异常降低，起到了对DSS诱导的细胞膜通透性增加的缓解作用，从而达到保护肠上皮细胞结构完整性，改善肠上皮屏障功能的效果。如上所述，由于本发明的去抗凝肝素衍生物能够有效地减少炎细胞浸润，并且能 够有效减弱炎症引起的脾脏增大，结肠缩短，使肠道炎症明显减轻。因此本发明的去抗凝肝素衍生物除了可以治疗炎症性肠病之外，还可以用于治疗与炎症性肠病相关并发症及发病机理相似的疾病，其中，炎症性肠病相关并发症及发病机理相似的疾病包括但不限于肠易激综合征、关节炎和其他肠外并发症包括强直性脊柱炎、坏疽性脓皮病、结节性红斑、虹膜炎、葡萄膜炎、巩膜外层炎和原发性硬化性胆管炎。
此外，由于去抗凝肝素衍生物可以有效缓解蛋白ZO-1表达的异常降低，因此本发明的去抗凝肝素衍生物还可以用于治疗与蛋白ZO-1表达异常相关的疾病。
此外，本发明涉及的去抗凝肝素衍生物可以显著降低利用DSS诱导的溃疡性结肠炎小鼠肠上皮细胞的凋亡率，表明其DSS诱导的结肠上皮细胞凋亡的缓解效果，显示其可以用于治疗炎症性肠病。
本发明涉及去抗凝肝素衍生物在用于制备治疗炎症性肠病，以及炎症性肠病相关并发症及发病机理相似的疾病的药物中的用途，其中，炎症性肠病相关并发症及发病机理相似的疾病包括但不限于肠易激综合征、关节炎和其他肠外并发症包括强直性脊柱炎、坏疽性脓皮病、结节性红斑、虹膜炎、葡萄膜炎、巩膜外层炎和原发性硬化性胆管炎。
利用本发明的去抗凝肝素衍生物给药治疗诱导了溃疡性结肠炎的小鼠模型之后，其可以有效地缓解炎症性肠病发病过程中肠痉挛引发的结肠缩短，并且小鼠的结肠上皮粘膜组织完整，结构清晰，上皮细胞排列整齐，腺体完整，部分腺体增生，粘膜下层未见异常。
利用本发明的去抗凝肝素衍生物处理DSS诱导的肠上皮细胞凋亡模型之后，其可以明显降低细胞凋亡水平，说明其对DSS诱导的细胞凋亡的有效缓解效果。
利用本发明的去抗凝肝素衍生物能够有效地修复炎症性肠病发生时肠上皮细胞中紧密连接蛋白ZO-1表达异常降低，起到了对DSS诱导的细胞膜通透性增加的缓解作用，从而达到保护肠上皮细胞结构完整性，改善肠上皮屏障功能的效果。
如上所述，由于本发明的去抗凝肝素衍生物能够有效地减少炎性细胞浸润，并且能够有效减弱炎症引起的脾脏增大、结肠缩短，对于肠道炎症明显减轻。因此本发明的去抗凝肝素衍生物除了可以治疗炎症性肠病之外，还可以用于治疗与炎症性肠病相关并发症及发病机理相似的疾病，其中，炎症性肠病相关并发症及 发病机理相似的疾病包括但不限于肠易激综合征、关节炎和其他肠外并发症包括强直性脊柱炎、坏疽性脓皮病、结节性红斑、虹膜炎、葡萄膜炎、巩膜外层炎和原发性硬化性胆管炎。
此外，由于去抗凝肝素衍生物可以有效缓解蛋白ZO-1表达的异常，因此本发明的去抗凝肝素衍生物还可以用于治疗与蛋白ZO-1表达异常相关的疾病。此外，本发明涉及的去抗凝肝素衍生物可以显著降低利用DSS诱导的溃疡性结肠炎小鼠肠上皮细胞的凋亡率，表明其DSS诱导的结肠上皮细胞凋亡的缓解效果，显示其可以用于治疗炎症性肠病。
实施例
1.抗Xa、IIa因子活性检测
肝素的抗凝活性需要通过体外试验测定其加速抗凝血酶(以下简称ATIII)抑制Xa因子(以下简称抗Xa因子)和IIa因子(以下简称抗IIa因子)的活性来决定。本发明中采用的抗Xa和抗IIa因子活性检测方法可参照欧洲药典。抗Xa和抗IIa的国际单位(International Unit，IU)是指确定量的肝素或低分子肝素国际标准品所含的活性。待测肝素供试品的抗凝活性是通过与国际标准品相应活性进行对比计算得到的。
(1)溶液配制：
Tris-HCl缓冲液(pH7.4)：取Tris 6.08g和NaCl 8.77g，加水500mL使之溶解，加牛血清蛋白10g，用HCl调节pH值至7.4，加水稀释至1000mL。
Tris-EDTA缓冲液(pH8.4)：取Tris 3.03g、NaCl 5.12g和EDTA·2Na1.4g，加水250mL使之溶解，用HCl调节pH值至8.4，加水稀释至500mL。
肝素标准品及供试样品溶液：肝素活性标准品购自EDQM(European Directorate for the Quality of Medicines)的heparin low-molecular-mass for assay BRP(Biological Reference Preparation)(H0185000,for detection of anti-factor Xa activity and anti-factor IIa activity)。用Tris-HCl缓冲液(pH7.4)分别将标准品(S)和供试品(T)稀释成4个不同浓度的溶液，各剂量间的剂距比控制在1:0.7～1:0.6。该浓度应在剂量对数～反应的线性范围内，检测抗Xa因子时一般为每毫升0.025IU～0.2IU，检测抗IIa因子时一般为每毫升0.015IU～0.075IU。
ATIII溶液：ATIII购自Chromogenix公司(Sweden)。检测抗Xa因子时以 Tris-HCl缓冲液(pH7.4)配制成1IU/mL的溶液；检测抗IIa因子时以Tris-HCl缓冲液(pH7.4)配制成0.5IU/mL的溶液。
发色底物溶液：检测抗Xa因子时用发色底物S-2765(N-α-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline-dihydrochloride)，购自Chromogenix公司(Sweden)。检测抗IIa因子时用发色底物S-2238(H-D-phenylalanyl-L-pipecolyl-arginine-p-nitroaniline-dihydrochloride)，购自Chromogenix公司(Sweden)。两种发色底物均用去离子水制成0.003M的溶液储存，临用前用Tris-EDTA缓冲液(pH8.4)稀释至0.0005M。
抗Xa因子溶液：用Tris-HCl缓冲液(pH7.4)配制，调试浓度，使之在用0.9％NaCl替代(超)低分子量肝素的抗Xa实验中，在405nm处的吸光度值处于0.6～0.7之间。
抗IIa因子溶液：用Tris-HCl缓冲液(pH7.4)溶解并稀释成5IU/mL的溶液。
测定方法：
取1.5mL离心管16支，分别标记T 1，T 2，T 3，T 4及S 1，S 2，S 3，S 4。各浓度平行做两管。每管分别加入4种浓度的供试品(T)或标准品(S)稀释液50μl，以及50μl ATIII溶液，混匀，注意不要有气泡。按S 1，S 2，S 3，S 4，T 1，T 2，T 3，T 4，T 1，T 2，T 3，T 4，S 1，S 2，S 3，S 4顺序排列，37℃水浴平衡1分钟后每管中加入100μl抗Xa(或抗IIa)因子溶液，37℃准确孵育1min后加入发色底物溶液250μl，混合，37℃水浴保温4分钟立即各加30％的醋酸溶液375μl终止反应。用1cm光程的半微量比色皿，以Tris-HCl缓冲液(pH7.4)为空白，测定405nm处的吸光度。以Tris-HCl缓冲液(pH7.4)代替供试品溶液(平行做两管)同法操作作为空白对照管，在16管开始和结尾时，分别测定空白对照管的吸光度。两者吸光度不得有显著性差异。以吸光度为纵坐标，标准品溶液(或供试品溶液)为浓度对数值为横坐标做线性回归，按生物检定统计法中的量反应平行线原理4×4法实验设计，计算效价及实验误差。平均信限率(FL％)不得大于15％。
2.分子量及其分布测定方法。
采用凝胶排阻高效液相色谱法测定低分子量肝素的重均分子量(Mw)，数均分子量(Mn)和分布系数(P)。色谱柱为TSK-GEL G2000SWXL(TOSOH，日本)，控制流速为0.5mL/min，柱温35℃，进样体积为25μL。采用WATERS(1525，美 国)色谱系统，紫外检测器和示差检测器以先后次序串联连接于色谱柱的出口，紫外检测器波长为234nm。分子量及其分布测定方法可以参考Wu,Jingjun等人"Controllable production of low molecular weight heparins by combinations of heparinase I/II/III."Carbohydrate polymers 101(2014):484-492中所记载的方法。
3.寡糖分析结果
本发明涉及的去抗凝肝素衍生物的寡糖分析采用亲水相互作用色谱(HILIC)与电喷雾质谱联用(ESI-MS)方法。测试步骤如下：称取一定质量的下述实施例中制备的待测样品的粉末，配制成浓度1μg/μL的待测样品溶液。HILIC液相条件如下：上样量：10μL，流动相：A相：5mM醋酸铵水溶液，B相：5mM醋酸铵溶液，98％乙腈溶液；流速：0.15mL/min；洗脱梯度：0-5min,90％B；5-45min,90-65％B；45-55min,65％B；55-60min，65-20％B；60-80min,20％B；80-80.01min,20-90％B。ESI-MS质谱参数如下：负离子模式喷雾电压：4.2KV；鞘流气流速：20arb；辅助气流速：5arb；毛细管电压：-40V；镜筒透镜电压：-50V；毛细管温度：275℃；扫描质量范围：200～2000。
得到色谱图后根据出峰质荷比(m/z)与理论计算值比对进行各峰归属，并以特定峰面积占所有解得寡糖峰面积的比作为该寡糖的相对百分含量。
实施例1
将20g精品肝素(购自常山生化药业股份有限公司，产品名称：肝素钠)溶于0.6L去离子水，在0.6L精品肝素(33g/L)中添加同等体积的0.2M高碘酸钠溶液(现配)，300rpm、4℃避光反应22小时。加入80mL乙二醇中和过量高碘酸钠，再添加28g硼氢化钠于4℃反应16小时。用HCl调节pH至7.0。经0.22μm滤膜抽滤，收集过滤样品。再利用透析袋除盐或者通过Millipore超滤装置加1K滤膜进行超滤浓缩和除盐，直至滤出液经0.1M AgNO 3检验无颜色变化即认为除盐完成。样品于-80℃冷冻后置于冻干机中冻干，然后用研钵或小型粉碎机粉碎成粉末保存，得到去抗凝肝素(命名为NAHP)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为5.8IU/mg，抗IIa为5.8IU/mg。并利用上述方法测定了该肝素的分子量及其分布，结果如下表1或2所示。
实施例2
将20g依诺肝素(购自常山生化药业股份有限公司，产品名称：依诺肝素钠)溶于0.6L去离子水，在0.6L依诺肝素(33g/L)中添加同等体积的0.2M高碘酸钠溶液(现配)，300rpm、4℃避光反应22小时。加入80mL乙二醇中和过量高碘酸钠，再添加28g硼氢化钠于4℃反应16小时。用HCl调节pH至7.0。经0.22μm滤膜抽滤，收集过滤样品。再利用透析袋除盐或者通过Millipore超滤装置加1K滤膜进行超滤浓缩和除盐，直至滤出液经0.1M AgNO 3检验无颜色变化即认为除盐完成。样品于-80℃冷冻后置于冻干机中冻干，然后用研钵或小型粉碎机粉碎成粉末保存，得到去抗凝依诺肝素(命名为：NAEno)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为3.5IU/mg，抗IIa为5.1IU/mg。并利用上述方法测定了该肝素的分子量及其分布，结果如下表1或2所示。
实施例3
将精品肝素(购自常山生化药业股份有限公司，产品名称：肝素钠)配成浓度为5mg/ml的水溶液，在4℃预冷后上001x7型阳离子交换树脂柱(H+form)2.5*40cm(购自廊坊南大树脂有限公司)。利用水洗收集流出物，立即用过量的吡啶中和，调pH值至6～8之间，然后冻干得到肝素吡啶盐。将6g肝素吡啶盐加入10倍(w/w)N-甲基-N-(三甲基硅基)-三氟乙酰胺(MTSTFA)和100倍体积(v/w)的无水吡啶中。室温下搅拌直至完全溶解，反应混合物于110℃加热2.5h(根据反应体系大小可调整反应时间)。反应液冰浴终止反应，利用旋转蒸发仪蒸发至原体积的1/10，加入2体积v/v的蒸馏水降解MTSTFA，随后35℃减压15min使反应液白色浑浊物消失。
产物纯化方法：将反应产物用蒸馏水透析3天，透析液上蒸馏水平衡过的阳离子交换树脂(H+form,3*13cm)，用水洗脱。酸性洗脱液合并在一起后用1N NaOH调节pH至9.5，之后蒸馏水透析过夜。最后的透析液冻干，从而得到去除了6-O-硫酸基的去抗凝肝素，将其命名为6-OdeS。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为20.3IU/mg，抗IIa为33.1IU/mg。
实施例4
将20g精品肝素(购自常山生化药业股份有限公司，产品名称：肝素钠)溶于 0.6L去离子水，在0.6L精品肝素(33g/L)中添加同等体积的0.2M高碘酸钠溶液(现配)，300rpm、4℃避光反应1小时。加入80mL乙二醇中和过量高碘酸钠，再添加28g硼氢化钠于4℃反应16小时。用HCl调节pH至7.0。经0.22μm滤膜抽滤，收集过滤样品。再利用透析袋除盐或者通过Millipore超滤装置加1K滤膜进行超滤浓缩和除盐，直至滤出液经0.1M AgNO 3检验无颜色变化即认为除盐完成。样品于-80℃冷冻后置于冻干机中冻干，然后用研钵或小型粉碎机粉碎成粉末保存，得到去抗凝肝素(命名为NAHP-60)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为60.6IU/mg，抗IIa为170.8IU/mg。并利用上述方法测定了该肝素的分子量及其分布，结果如下表1或2所示。
实施例5
将20g精品肝素(购自常山生化药业股份有限公司，产品名称：肝素钠)溶于0.6L去离子水，在0.6L精品肝素(33g/L)中添加同等体积的0.2M高碘酸钠溶液(现配)，300rpm、4℃避光反应4小时。加入80mL乙二醇中和过量高碘酸钠，再添加28g硼氢化钠于4℃反应16小时。用HCl调节pH至7.0。经0.22μm滤膜抽滤，收集过滤样品。再利用透析袋除盐或者通过Millipore超滤装置加1K滤膜进行超滤浓缩和除盐，直至滤出液经0.1M AgNO 3检验无颜色变化即认为除盐完成。样品于-80℃冷冻后置于冻干机中冻干，然后用研钵或小型粉碎机粉碎成粉末保存，得到去抗凝肝素(命名为NAHP-39)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为39IU/mg，抗IIa为124.9IU/mg。并利用上述方法测定了该肝素的分子量及其分布，结果如下表1或2所示。
实施例6
将精品肝素(购自常山生化药业股份有限公司，产品名称：肝素钠)配成浓度为5mg/ml的水溶液，在4℃预冷后上001x7型阳离子交换树脂柱(H+form)2.5*40cm(购自廊坊南大树脂有限公司)。利用水洗收集流出物，立即用过量的吡啶中和，调pH值至6～8之间，然后冻干得到肝素吡啶盐。
2g肝素吡啶盐加入25ml含5％水的DMSO，50℃水浴3h后25ml水稀释。用NaOH调pH至9，透析至去离子水中，冻干得到N-去硫酸化产物。1.2g N-去硫酸化产物加入12ml饱和NaHCO 3(5g NaHCO 3加入50ml ddH 2O，4℃预冷) 置于冰上，加入1.2ml醋酐pH下降，加入NaOH调至8左右,每30min-1h重复此步骤，共反应3h，4℃透析至去离子水中，冻干即可得到N-再乙酰化去抗凝肝素衍生物(命名为N-ace)，利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为1.2IU/mg，抗IIa为7.5IU/mg。并利用上述方法测定了该肝素的分子量及其分布，结果如下表1或2所示。
对比例1
从河北常山生化购买精品肝素，该肝素的重均分子量Mw为17223(命名为HP)。利用上述方法检测抗凝活性，结果显示抗Xa为187.3IU/mg，抗IIa为197.2IU/mg。
对比例2
从河北常山生化药业购买依诺肝素，该肝素的数均分子量为3275，重均分子量为4620(命名为Eno)。利用上述方法检测抗凝活性，结果显示抗Xa为109IU/mg，抗IIa为32.7IU/mg。
表1实施例和对比例中的肝素及去抗凝肝素的分子量分布
Figure PCTCN2018075359-appb-000001
Figure PCTCN2018075359-appb-000002
表2实施例和对比例中的低分子肝素及去抗凝低分子肝素的分子量分布
Figure PCTCN2018075359-appb-000003
表3实施例和比较例中的肝素分子量及去抗凝肝素的抗Xa和抗IIa结果
实施例 样品 抗Xa(IU/mg) 抗IIa(IU/mg)
实施例1 NAHP 5.8 5.8
实施例2 NAEno 3.5 5.1
实施例3 6-OdeS 20.3 33.1
实施例4 NAHP-60 60.6 170.8
实施例5 NAHP-39 39 124.9
实施例6 N-ace 1.2 7.5
对比例1 HP 187.3 197.2
对比例2 Eno 109 32.7
对比例4 2-OdeS 75.6 99.6
对比例3
从药店购买美沙拉秦缓释颗粒(5-Amino Salicylic Acid，5-ASA)。适应症为：溃疡性结肠炎，用于溃疡性结肠炎的急性发作，防止复发；克罗恩病，用于频繁发病的克罗恩病病人，预防急性发作。
对比例4
将2g精品肝素(购自常山生化药业股份有限公司，产品名称：肝素钠)加入到100ml 0.4N氢氧化钠溶液中，可同时添加0.2-1g硼氢化钠(NaBH 4)防止肝素糖链在碱性条件下断裂，将反应液冻干。然后将带黄色的冻干产物溶解于50ml水中，用20％醋酸溶液中和至pH 7，并彻底透析至超纯水中然后冻干，得到去除糖醛酸残基上2-O-硫酸基的去抗凝肝素，将其命名为2-OdeS。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为75.6IU/mg，抗IIa为99.6 IU/mg。
实验例1硫酸葡聚糖钠诱导小鼠溃疡性结肠炎的试验数据
2.5-3％硫酸葡聚糖钠盐(DSS，Mw:36,000-50,000,MP biomedicals,LLC)溶于C57BL/6J品系小鼠的饮用水中，同时设置DSS造模组、健康对照组以及药物治疗组。健康对照组不做任何处理，DSS造模组持续自由饮用含2.5-3％的DSS饮用水直至实验结束，药物治疗组持续自由饮用含2.5-3％的DSS饮用水直至实验结束，并采用实施例1和2得到的去抗凝肝素，以及对比例1～3的物质用生理盐水溶解，从第一天起给予灌胃给药，给药剂量为30mg/kg，持续7天。
在诱导第三天开始模型组出现体重下降、稀便和脓血便等症状。第七天结束实验，处死小鼠后眼眶取血，取脾脏称重，从盲肠端到肛门处剪取全段结直肠拍照测量长度，留近端1/3-1/2冻存，另外部分剪开，PBS洗净并卷好置于石蜡包埋用塑料夹中，浸没入4％多聚甲醛中固定。
结直肠组织病理切片HE染色如图1所示，结果表明经过DSS诱导之后结肠上皮糜烂，腺体结构严重破坏，黏膜和黏膜下炎性细胞浸润增多。在给予去抗凝肝素治疗后，结肠上皮得到一定程度保护，腺体结构相对完整。
结肠病理组织切片：
(一)常规石蜡切片的制备：
(1)取材与固定：切取组织时应使用锋利的刀、剪，将结肠组织用剪刀剖开，用灭菌的PBS溶液洗净，截取约1.5-2.0cm远端结肠并从肛门侧向近端卷好放入蜡块包埋夹。结直肠组织块用10％福尔马林溶液固定24～48h。
(2)包埋：先经梯度乙醇脱水后用二甲苯透明，然后入溶融的石蜡中浸透每次30min，共3次；再包埋。
(3)切片：包埋好的石蜡块即可进行切片；切片的厚度为5μm左右。
(二)苏木精-伊红(hematoxylin and eosin,HE)染色方法：
(1)脱蜡：主要用二甲苯脱蜡。
(2)梯度乙醇水化。
(3)自来水冲洗。
(4)苏木精染色：水化后的切片放入苏木精染液中浸5～20min，染细胞核。自来水冲洗3～5min。
(5)1％盐酸乙醇分化5～30s。自来水冲洗1～3min。
(6)弱碱性水溶液返蓝30s～1min。自来水充分冲洗5～10分钟。
(7)伊红染色：充分水化后的切片直接入伊红染色液中，染细胞质5～15min左右。
(8)梯度乙醇脱水。
(9)二甲苯透明。
(10)中性树胶封片。显微镜下观察组织结构的变化。
结直肠HE染色组织学变化。NC组(正常组)：结肠上皮组织完整，结构清晰，上皮细胞排列整齐，腺体完整；DSS造模组：结肠粘膜上皮细胞萎缩、坏死、脱落，腺体异常，腺体杯状细胞消失，炎症细胞广泛浸润，基底膜断裂或消失，腺上皮与粘膜肌层间隙增大，粘膜下层毛细血管增生，并扩张出血；HP治疗组：结肠粘膜上皮细胞萎缩、坏死、脱落，腺体不完整，可见炎症细胞浸润，基底膜增厚，未见断裂，腺上皮与粘膜肌层间隙增大，粘膜下层毛细血管增生，并扩张出血；NAHP治疗组：结肠粘膜上皮见部分脱落，腺体轻度增生，少数腺体结构不完整，可见少量炎症细胞浸润，粘膜下层未见异常；Eno治疗组：结肠粘膜上皮细胞部分萎缩、坏死、脱落，腺体异常，杯状细胞增生，伴有炎症细胞浸润，病变未累及粘膜下层；NAEno治疗组：结肠上皮组织较完整，上皮细胞可见部分脱落，腺体增生，排列不整齐，可见缺失，伴有炎症细胞浸润，病变未累及粘膜下层；5-ASA(美沙拉秦)治疗组(阳性对照治疗组)：结肠粘膜上皮细胞坏死、脱落，腺体异常，排列不整齐，杯状细胞增生或坏死，炎症细胞浸润，病变未累及粘膜下层。
溃疡性结肠炎诱导成功后，小鼠肠道肌肉痉挛收缩，导致全结直肠长度缩短。连续7天给予小鼠3％DSS或同时给予药物治疗后，颈椎脱臼法处死动物，取全结直肠，测量长度。各组全段结肠长度如图2(a)和(b)所示，除未分级肝素外，其余肝素类药物都能一定程度缓解结肠缩短。其中，NAHP治疗组较5-ASA治疗组效果更佳。去抗凝依诺肝素的效果较依诺肝素更优。
脾脏是重要的外周免疫器官之一。炎性细胞的浸润和异常的免疫反应可导致脾肿大。连续7天给予小鼠3％DSS或同时给予药物治疗后，颈椎脱臼法处死动物，取脾脏，称重并计算脾脏指数。各组脾脏指数如图3所示，脾脏指数＝脾重(mg)/体重(g)x10。给予去抗凝肝素能有效减弱炎症引起的脾脏增大，NAHP的效 果明显优于未分级肝素，去抗凝依诺肝素优于依诺肝素。
实验例2肝素衍生物缓解硫酸葡聚糖钠诱导的人正常结肠上皮细胞NCM460细胞膜通透性异常的试验数据。
上皮细胞间紧密连接是维持粘膜上皮机械屏障和通透性的重要结构。ZO-1蛋白是细胞紧密连接蛋白的重要组成蛋白之一，不但参与维持和调节上皮屏障功能，还参与细胞物质转运和维持上皮极性等重要过程。在本实验中应用3％硫酸葡聚糖钠盐(DSS)诱导人正常结肠上皮细胞NCM460(购自ATCC(Rockefeller,MD,USA))的细胞膜通透性增大，来模拟DSS诱导的溃疡性结肠炎小鼠肠上皮细胞通透性增大的细胞破坏作用。在给予细胞2mg/ml的实施例1、3的去抗凝肝素衍生物、以及对比例1、对比例3和4的物质处理48小时后，通过蛋白质印迹法检测NCM460细胞中ZO-1蛋白的表达情况，探讨和评估各肝素衍生物对通透性增大的结肠上皮细胞的保护作用。如图4所示，除未分级肝素和2-OdeS衍生物外，其他肝素衍生物治疗组中ZO-1蛋白表达水平均高于DSS诱导组细胞ZO-1蛋白的表达水平，有效证明了这些肝素衍生物对DSS诱导的细胞膜通透性异常的缓解效果。
实验例3肝素衍生物缓解硫酸葡聚糖钠诱导的人正常结肠上皮细胞NCM460细胞凋亡的试验数据。
细胞凋亡在UC的发生发展中起到重要作用。在溃疡性结肠炎病理条件下，上皮细胞沿隐窝绒毛轴增殖一分化一凋亡的正常顺序可能被破坏。UC炎症活动区域的粘膜上皮细胞凋亡速率明显增加，可能是UC上皮屏障功能的破坏的另一大重要原因。细胞凋亡早期改变发生在细胞膜表面，这些细胞膜表面的改变之一是磷脂酰丝氨酸(PS)从细胞膜内转移到细胞膜外，使PS暴露在细胞膜外表面。Annexin V作为一种Ca 2+依赖的磷脂结合蛋白，对PS有高度的亲和性。因此，该蛋白可充当一敏感的探针检测暴露在细胞膜表面的PS。PS转移到细胞膜外不是凋亡所独特的，也可发生在细胞坏死中。两种细胞死亡方式间的差别是在凋亡的初始阶段细胞膜是完好的，而细胞坏死在其早期阶段细胞膜的完整性就破坏了。因此结合碘化丙啶(PI)染细胞核可以区分凋亡细胞和坏死细胞。
在本实验中应用3％硫酸葡聚糖钠盐(DSS)诱导人正常结肠上皮细胞NCM460(购自ATCC(Rockefeller,MD,USA))凋亡，来模拟DSS诱导的溃疡性结肠炎小鼠肠上皮细胞凋亡过程。在给予细胞2mg/ml的实施例1、2、4、5和6的去抗凝肝素衍生物、和对比例1和3的物质处理48小时后，通过FITC-Annexin/PI染色并进行流式细胞仪分选检测NCM460细胞凋亡情况，探讨和评估各肝素衍生物缓解结肠上皮细胞凋亡效果。图5(a)中，Annexin V阴性-PI阴性代表正常细胞；Annexin V阳性-PI阴性代表凋亡早期的细胞；Annexin V阳性-PI阳性代表凋亡晚期的细胞或坏死的细胞。经过流式细胞图像分析，可得到各细胞群所占总细胞数的比例。经过统计分析凋亡早期的细胞群比例得到图5(b)结果。如图5(a)、(b)所示，去抗凝肝素衍生物治疗组(NAHP组和N-ace组)中的肠上皮细胞凋亡水平明显低于DSS诱导凋亡组，有效证明了其对DSS诱导的细胞凋亡作用的缓解效果。
实施例7
将20g精品肝素(购自常山生化药业股份有限公司，产品名称：肝素钠)溶于0.6L去离子水，在0.6L精品肝素(33g/L)中添加同等体积的0.2M高碘酸钠溶液(现配)，300rpm、4℃避光反应22小时。加入80mL乙二醇中和过量高碘酸钠，再添加28g硼氢化钠于4℃反应16小时。用HCl调节pH至7.0。经0.22μm滤膜抽滤，收集过滤样品。再利用透析袋除盐或者通过Millipore超滤装置加1K滤膜进行超滤浓缩和除盐，直至滤出液经0.1M AgNO 3检验无颜色变化即认为除盐完成。样品于-80℃冷冻后置于冻干机中冻干，然后用研钵或小型粉碎机粉碎成粉末保存，得到去抗凝肝素衍生物(命名为NAHP)。利用上述方法检测得到的去抗凝肝素衍生物的抗凝活性，结果显示抗Xa为5.8IU/mg，抗IIa为5.8IU/mg。并利用上述方法测定了该去抗凝肝素衍生物的分子量及其分布，结果如下表1所示。
另外，表4中显示的P值表示该分子的分散度，是利用重均分子量Mw除以数均分子量Mn之后得出的数值。
实施例8
将实施例7制备的去抗凝肝素溶于反应缓冲液，每隔0.5～1h向该溶液添加按照ZL200410038098.6方法制备的肝素酶I，每次添加20IU肝素酶I，使用光程 差为1cm的石英比色皿和紫外分光光度计监测溶液231nm的光吸收A231(使用pH7.4的缓冲液对仪器进行校准调零，为了检测结果的准确性，当紫外分光光度计读数A231大于0.6时，将溶液稀释一定倍数，使读数在0.2～0.6来测定)。当检测到A231达到46时，使反应结束，此时添加的总肝素酶I的酶活达到约220-250IU。结束的方法为在100℃沸水浴中对反应溶液中的酶灭活5～10min，然后取出反应体系冷却至室温，向反应溶液中添加6倍体积的无水乙醇，在室温下搅拌10min，然后在室温下以4000r/min的速度离心15min，收集沉淀，加入质量是沉淀2～3倍的去离子水溶解，使用0.22μm的膜过滤，收集透过液并放置在-80℃低温冰箱冻成坚实的冰块，然后送入冻干机(冷阱温度为-50℃)冻干，然后用研钵或者小型粉碎机粉碎成粉末，得到低分子量去抗凝肝素(也命名为：LNAHP)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为3.2IU/mg，抗IIa为4.0IU/mg。并利用上述方法测定了该去抗凝肝素衍生物的分子量及其分布，结果如下表4或5所示。利用上述方法测定了该去抗凝肝素衍生物的寡糖分布，其结果如表7所示。
实施例9
将实施例7制备的去抗凝肝素溶于反应缓冲液，每隔0.5～1h向该溶液添加按照ZL200410038098.6方法制备的肝素酶I，每次添加20IU肝素酶I，使用光程差为1cm的石英比色皿和紫外分光光度计监测溶液231nm的光吸收A231(使用pH7.4的缓冲液对仪器进行校准调零，为了检测结果的准确性，当紫外分光光度计读数A231大于0.6时，将溶液稀释一定倍数，使读数在0.2～0.6来测定)。当检测到A231达到106时，使反应结束，此时添加的总肝素酶I的酶活达到约340-380IU。结束的方法为在100℃沸水浴中对反应溶液中的酶灭活5～10min，然后取出反应体系冷却至室温，向反应溶液中添加6倍体积的无水乙醇，在室温下搅拌10min，然后在室温下以4000r/min的速度离心15min，收集沉淀，加入质量是沉淀2～3倍的去离子水溶解，使用0.22μm的膜过滤，收集透过液并放置在-80℃低温冰箱冻成坚实的冰块，然后送入冻干机(冷阱温度为-50℃)冻干，然后用研钵或者小型粉碎机粉碎成粉末，得到超低分子量去抗凝肝素(也命名为：ULNAHP)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为3.7IU/mg，抗IIa为5.2IU/mg。并利用上述方法测定了该去抗凝肝素衍生物的分子量及其分 布，结果如下表4或5所示。利用上述方法测定了该去抗凝肝素衍生物的寡糖分布，其结果如表7所示。
对比例5
将精品肝素溶于反应缓冲液，每隔0.5～1h向该溶液添加按照ZL200410038098.6方法制备的肝素酶I，每次添加20IU肝素酶I，使用光程差为1cm的石英比色皿和紫外分光光度计监测溶液231nm的光吸收A231(使用pH7.4的缓冲液对仪器进行校准调零，为了检测结果的准确性，当紫外分光光度计读数A231大于0.6时，将溶液稀释一定倍数，使读数在0.2～0.6来测定)。当检测到A231达到45时，使反应结束。结束的方法为在100℃沸水浴中对反应溶液中的酶灭活5～10min，然后取出反应体系冷却至室温，向反应溶液中添加6倍体积的无水乙醇，在室温下搅拌10min，然后在室温下以4000r/min的速度离心15min，收集沉淀，加入质量是沉淀2～3倍的去离子水溶解，使用0.22μm的膜过滤，收集透过液并放置在-80℃低温冰箱冻成坚实的冰块，然后送入冻干机(冷阱温度为-50℃)冻干，然后用研钵或者小型粉碎机粉碎成粉末。将得到的粉末20g溶于0.6L去离子水，在其中添加同等体积的0.2M高碘酸钠溶液(现配)，300rpm、4℃避光反应22小时。加入80mL乙二醇中和过量高碘酸钠，再添加28g硼氢化钠于4℃反应16小时。用HCl调节pH至7.0。经0.22μm滤膜抽滤，收集过滤样品。再利用透析袋除盐或者通过Millipore超滤装置加1K滤膜进行超滤浓缩和除盐，直至滤出液经0.1M AgNO 3检验无颜色变化即认为除盐完成。样品于-80℃冷冻后置于冻干机中冻干，然后用研钵或小型粉碎机粉碎成粉末保存，得到去抗凝肝素(命名为NAHep-1)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为2IU/mg，抗IIa为15.8IU/mg。并利用上述方法测定了该肝素衍生物的分子量及其分布，结果如下表4或5所示。利用上述方法测定了该肝素衍生物的寡糖分布，其结果如表7所示。
对比例6
将精品肝素溶于反应缓冲液，每隔0.5～1h向该溶液添加按照ZL200410038098.6方法制备的肝素酶I，每次添加20IU肝素酶I，使用光程差为1cm的石英比色皿和紫外分光光度计监测溶液231nm的光吸收A231(使用pH7.4 的缓冲液对仪器进行校准调零，为了检测结果的准确性，当紫外分光光度计读数A231大于0.6时，将溶液稀释一定倍数，使读数在0.2～0.6来测定)。当检测到A231达到98时，使反应结束。结束的方法为在100℃沸水浴中对反应溶液中的酶灭活5～10min，然后取出反应体系冷却至室温，向反应溶液中添加6倍体积的无水乙醇，在室温下搅拌10min，然后在室温下以4000r/min的速度离心15min，收集沉淀，加入质量是沉淀2～3倍的去离子水溶解，使用0.22μm的膜过滤，收集透过液并放置在-80℃低温冰箱冻成坚实的冰块，然后送入冻干机(冷阱温度为-50℃)冻干，然后用研钵或者小型粉碎机粉碎成粉末。将得到的粉末20g溶于0.6L去离子水，在其中添加同等体积的0.2M高碘酸钠溶液(现配)，300rpm、4℃避光反应22小时。加入80mL乙二醇中和过量高碘酸钠，再添加28g硼氢化钠于4℃反应16小时。用HCl调节pH至7.0。经0.22μm滤膜抽滤，收集过滤样品。再利用透析袋除盐或者通过Millipore超滤装置加1K滤膜进行超滤浓缩和除盐，直至滤出液经0.1M AgNO 3检验无颜色变化即认为除盐完成。样品于-80℃冷冻后置于冻干机中冻干，然后用研钵或小型粉碎机粉碎成粉末保存，得到去抗凝肝素(命名为NAHep-2)。利用上述方法检测得到的去抗凝肝素的抗凝活性，结果显示抗Xa为1.8IU/mg，抗IIa为8.8IU/mg。并利用上述方法测定了该肝素衍生物的分子量及其分布，结果如下表4或5所示。利用上述方法测定了该肝素衍生物的寡糖分布，其结果如表7所示。
对比例7
从河北常山生化购买未分级肝素，该肝素的重均分子量Mw为17223(命名为HP)。利用上述方法检测抗凝活性，结果显示抗Xa为187.3IU/mg，抗IIa为197.2IU/mg。并利用上述方法测定了其分子量及其分布，结果如下表7所示。
对比例8
从药店购买美沙拉秦缓释颗粒(5-Amino Salicylic Acid，5-ASA)。适应症为：溃疡性结肠炎，用于溃疡性结肠炎的急性发作，防止复发；克罗恩病，用于频繁发病的克罗恩病病人，预防急性发作。
对比例9
将精品肝素溶于反应缓冲液，每隔0.5～1h向该溶液添加按照ZL200410038098.6方法制备的肝素酶I，每次添加20IU肝素酶I，使用光程差为1cm的石英比色皿和紫外分光光度计监测溶液231nm的光吸收A231(使用pH7.4的缓冲液对仪器进行校准调零，为了检测结果的准确性，当紫外分光光度计读数A231大于0.6时，将溶液稀释一定倍数，使读数在0.2～0.6来测定)。当检测到A231达到45时，使反应结束。结束的方法为在100℃沸水浴中对反应溶液中的酶灭活5～10min，然后取出反应体系冷却至室温，向反应溶液中添加6倍体积的无水乙醇，在室温下搅拌10min，然后在室温下以4000r/min的速度离心15min，收集沉淀，加入质量是沉淀2～3倍的去离子水溶解，使用0.22μm的膜过滤，收集透过液并放置在-80℃低温冰箱冻成坚实的冰块，然后送入冻干机(冷阱温度为-50℃)冻干，然后用研钵或者小型粉碎机粉碎成粉末，得到低分子肝素(命名为I-45)。利用上述方法检测得到的低分子肝素的抗凝活性，结果显示抗Xa为99IU/mg，抗IIa为43.3IU/mg。并利用上述方法测定了该低分子肝素的分子量及其分布，结果如下表4或5所示。利用上述方法测定了该低分子肝素的寡糖分布，其结果如表7所示。
对比例10
将精品肝素溶于反应缓冲液，每隔0.5～1h向该溶液添加按照ZL200410038098.6方法制备的肝素酶I，每次添加20IU肝素酶I，使用光程差为1cm的石英比色皿和紫外分光光度计监测溶液231nm的光吸收A231(使用pH7.4的缓冲液对仪器进行校准调零，为了检测结果的准确性，当紫外分光光度计读数A231大于0.6时，将溶液稀释一定倍数，使读数在0.2～0.6来测定)。当检测到A231达到98时，使反应结束。结束的方法为在100℃沸水浴中对反应溶液中的酶灭活5～10min，然后取出反应体系冷却至室温，向反应溶液中添加6倍体积的无水乙醇，在室温下搅拌10min，然后在室温下以4000r/min的速度离心15min，收集沉淀，加入质量是沉淀2～3倍的去离子水溶解，使用0.22μm的膜过滤，收集透过液并放置在-80℃低温冰箱冻成坚实的冰块，然后送入冻干机(冷阱温度为-50℃)冻干，然后用研钵或者小型粉碎机粉碎成粉末，得到低分子肝素(命名为I-98)。利用上述方法检测得到的低分子肝素的抗凝活性，结果显示抗Xa为42.4IU/mg，抗IIa为21.4IU/mg。并利用上述方法测定了该低分子肝素的分子量及其 分布，结果如下表4或5所示。
表4实施例和对比例中的各样品的分子量分布
Figure PCTCN2018075359-appb-000004
表5实施例和对比例中的各样品的分子量分布
Figure PCTCN2018075359-appb-000005
表6实施例和比较例中各样品的抗Xa和抗IIa结果
实施例 样品 抗Xa(IU/mg) 抗IIa(IU/mg)
实施例7 NAHP 5.8 5.8
实施例8 LNAHP 3.2 4.0
实施例9 ULNAHP 3.7 5.2
对比例5 NAHep-1 2 15.8
对比例6 NAHep-2 1.8 8.8
对比例7 HP 187.3 197.2
对比例9 I-45 99 43.3
对比例10 I-98 42.4 21.4
表7实施例和比较例中的各样品的寡糖含量
Figure PCTCN2018075359-appb-000006
实验例4硫酸葡聚糖钠诱导小鼠溃疡性结肠炎的试验数据
2.5-3％硫酸葡聚糖钠盐(DSS，Mw:36,000-50,000,MP biomedicals,LLC)溶于C57BL/6J品系小鼠的饮用水中，同时设置DSS造模组、健康对照组以及药物治疗组。健康对照组不做任何处理，DSS造模组持续自由饮用含2.5-3％的DSS饮用水直至实验结束，药物治疗组持续自由饮用含2.5-3％的DSS饮用水直至实验结束，并且采用实施例7、8、9得到的去抗凝肝素，以及对比例7和8的样品用生理盐水溶解，从出现症状起给予灌胃给药，给药剂量为30mg/kg/天，持续至实验结束。
小鼠体重变化如图6所示，在诱导第三天开始出现体重下降、稀便和脓血便等症状。从出现症状开始给予不同药物治疗，其中(超)低分子去抗凝肝素治疗组能显著减缓DSS诱导的体重降低。第七天结束实验，处死小鼠后眼眶取血，取脾脏称重，从盲肠端到肛门处剪取全段结直肠拍照测量长度，留近端1/3-1/2冻存，另外部分剪开，PBS洗净并卷好置于石蜡包埋用塑料夹中，浸没入4％多聚甲醛中固定。
结直肠组织病理切片HE染色如图7所示，结果表明经过DSS诱导之后结肠上皮糜烂，腺体结构严重破坏，黏膜和黏膜下炎性细胞浸润增多。在给予去抗凝 肝素治疗后，结肠上皮得到一定程度保护，腺体结构相对完整。其中，给予(超)低分子去抗凝肝素治疗效果最优。
结肠病理组织切片：
(一)常规石蜡切片的制备：
(1)取材与固定：切取组织时应使用锋利的刀、剪，将结肠组织用剪刀剖开，用灭菌的PBS溶液洗净，截取约1.5-2.0cm远端结肠并从肛门侧向近端卷好放入蜡块包埋夹。结直肠组织块用10％福尔马林溶液固定24～48h。
(2)包埋：先经梯度乙醇脱水后用二甲苯透明，然后入溶融的石蜡中浸透每次30min，共3次；再包埋。
(3)切片：包埋好的石蜡块即可进行切片；切片的厚度为5μm左右。
(二)苏木精-伊红(hematoxylin and eosin,HE)染色方法：
(1)脱蜡：主要用二甲苯脱蜡。
(2)梯度乙醇水化。
(3)自来水冲洗。
(4)苏木精染色：水化后的切片放入苏木精染液中浸5～20min，染细胞核。自来水冲洗3～5min。
(5)1％盐酸乙醇分化5～30s。自来水冲洗1～3min。
(6)弱碱性水溶液返蓝30s～1min。自来水充分冲洗5～10分钟。
(7)伊红染色：充分水化后的切片直接入伊红染色液中，染细胞质5～15min左右。
(8)梯度乙醇脱水。
(9)二甲苯透明。
(10)中性树胶封片。显微镜下观察组织结构的变化。
结直肠HE染色组织学变化。NC组(正常组)：结肠上皮组织完整，结构清晰，上皮细胞排列整齐，腺体完整；DSS造模组：结肠粘膜上皮细胞萎缩、坏死、脱落，腺体异常，腺体杯状细胞消失，炎症细胞广泛浸润，基底膜断裂或消失，腺上皮与粘膜肌层间隙增大，粘膜下层毛细血管增生，并扩张出血；HP治疗组：结肠粘膜上皮细胞萎缩、坏死、脱落，腺体不完整，可见炎症细胞浸润，基底膜增厚，未见断裂，腺上皮与粘膜肌层间隙增大，粘膜下层毛细血管增生，并扩张出血；NAHP治疗组：结肠粘膜上皮见部分脱落，腺体轻度增生，少数腺体结构 不完整，可见少量炎症细胞浸润，粘膜下层未见异常；ULNAHP治疗组和LNAHP治疗组：结肠上皮组织完整，结构清晰，上皮细胞排列整齐，腺体完整，部分腺体增生，粘膜下层未见异常；5-ASA(美沙拉秦)治疗组(阳性对照治疗组)：结肠粘膜上皮细胞坏死、脱落，腺体异常，排列不整齐，杯状细胞增生或坏死，炎症细胞浸润，病变未累及粘膜下层。
表8组织损伤评分标准
Figure PCTCN2018075359-appb-000007
按照上表8中的评分标准对图7中的照片进行组织学评分，结果如图8所示。可以看出，NAHP治疗组的评分明显低于DSS造模组，而ULNAHP治疗组和LNAHP治疗组的效果更优于NAHP治疗组，评分均显著优于5-ASA治疗组。
溃疡性结肠炎诱导成功后，小鼠肠道肌肉痉挛收缩，导致全结直肠长度缩短。连续7天给予小鼠3％DSS或同时给予药物治疗后，颈椎脱臼法处死动物，取全结直肠，测量长度。各组全段结肠长度如图9所示，除未分级肝素外，其余肝素类药物都能一定程度缓解结肠缩短。其中，NAHP治疗组，酶法降解的ULNAHP治疗组与LNAHP治疗组较5-ASA治疗组效果更佳。酶解低分子和超低分子肝素治疗组效果优于NAHP治疗组。
实验例5肝素衍生物缓解硫酸葡聚糖钠诱导的人正常结肠上皮细胞NCM460细胞凋亡的试验数据。
细胞凋亡在UC的发生发展中起到重要作用。在炎症性肠病病理条件下，上皮细胞沿隐窝绒毛轴增殖一分化一凋亡的正常顺序可能被破坏。UC炎症活动区域的粘膜上皮细胞凋亡速率明显增加，可能是UC上皮屏障功能的破坏的另一大重要原因。细胞凋亡早期改变发生在细胞膜表面，这些细胞膜表面的改变之一是磷脂酰丝氨酸(PS)从细胞膜内转移到细胞膜外，使PS暴露在细胞膜外表面。Annexin V作为一种Ca 2+依赖的磷脂结合蛋白，对PS有高度的亲和性。因此，该 蛋白可充当一敏感的探针检测暴露在细胞膜表面的PS。PS转移到细胞膜外不是凋亡所独特的，也可发生在细胞坏死中。两种细胞死亡方式间的差别是在凋亡的初始阶段细胞膜是完好的，而细胞坏死在其早期阶段细胞膜的完整性就破坏了。因此结合碘化丙啶(PI)染细胞核可以区分凋亡细胞和坏死细胞。
在本实验中应用3％硫酸葡聚糖钠盐(DSS)诱导人正常结肠上皮细胞NCM460(购自ATCC(Rockefeller,MD,USA))凋亡，来模拟DSS诱导的溃疡性结肠炎小鼠肠上皮细胞凋亡过程。在给予细胞2mg/ml的不同去抗凝肝素衍生物处理48小时后，通过FITC-Annexin/PI染色并进行流式细胞仪分选检测NCM460细胞凋亡情况，探讨和评估各肝素衍生物缓解结肠上皮细胞凋亡效果。如图10所示，(超)低分子去抗凝肝素衍生物治疗组(ULNAHP组和LNAHP组)中的肠上皮细胞凋亡水平明显低于DSS诱导凋亡组，有效证明了ULNAHP和LNAHP对DSS诱导的细胞凋亡作用的缓解效果，其保护效果优于5-ASA组和NAHP组。
实验例6肝素衍生物缓解硫酸葡聚糖钠诱导的人正常结肠上皮细胞NCM460细胞膜通透性异常的试验数据。
上皮细胞间紧密连接是维持粘膜上皮机械屏障和通透性的重要结构。ZO-1蛋白是细胞紧密连接蛋白的重要组成蛋白之一，不但参与维持和调节上皮屏障功能，还参与细胞物质转运和维持上皮极性等重要过程。在本实验中应用3％硫酸葡聚糖钠盐(DSS)诱导人正常结肠上皮细胞NCM460(购自ATCC(Rockefeller,MD,USA))的细胞膜通透性增大，来模拟DSS诱导的溃疡性结肠炎小鼠肠上皮细胞通透性增大的细胞破坏作用。在给予细胞2mg/ml的实施例7、8、9的去抗凝肝素衍生物、以及对比例5、6、7、9、10的物质处理48小时后，通过蛋白质印迹法检测NCM460细胞中ZO-1蛋白的表达情况，探讨和评估各肝素衍生物对通透性增大的结肠上皮细胞的保护作用。如图11所示，DSS诱导条件下，相比正常细胞(NC组)ZO-1水平明显降低，HP处理组无效，NAHP组能缓解ZO-1水平的降低，而ULNAHP和LNAHP去抗凝肝素衍生物处理组中ZO-1蛋白表达水平均明显高于DSS诱导组细胞ZO-1蛋白的表达水平，但通过先酶解后去抗凝修饰得到的NAHep-1(对比例5)和NAHep-2(对比例6)，以及仅酶解低分子肝素(对比例9、10)并不能起到缓解ZO-1降低的效果。该实验有效证明了先去抗凝修饰再酶解得到的去抗凝肝素衍生物对DSS诱导的细胞膜通透性异常的缓解效果。

Claims (20)

  1. 一种去抗凝肝素衍生物，其抗Xa因子小于等于70IU/mg，优选抗Xa因子小于等于60IU/mg，优选抗Xa因子小于等于50IU/mg，优选抗Xa因子小于等于40IU/mg，优选抗Xa因子小于等于30IU/mg，优选抗Xa因子小于等于20IU/mg，优选抗Xa因子小于等于10IU/mg，并且
    其抗IIa因子小于等于175IU/mg，优选抗IIa因子小于等于170IU/mg，优选抗IIa因子小于等于160IU/mg，优选抗IIa因子小于等于150IU/mg，优选抗IIa因子小于等于140IU/mg，优选抗IIa因子小于等于130IU/mg，优选抗IIa因子小于等于120IU/mg，优选抗IIa因子小于等于110IU/mg，优选抗IIa因子小于等于100IU/mg，优选抗IIa因子小于等于90IU/mg，优选抗IIa因子小于等于80IU/mg，优选抗IIa因子小于等于70IU/mg，优选抗IIa因子小于等于60IU/mg，优选抗IIa因子小于等于50IU/mg，优选抗IIa因子小于等于40IU/mg，优选抗IIa因子小于等于30IU/mg，优选抗IIa因子小于等于20IU/mg，优选抗IIa因子小于等于10IU/mg。
  2. 根据权利要求1所述的去抗凝肝素衍生物，其中，所述去抗凝肝素衍生物的重均分子量为8000以上。
  3. 根据权利要求1所述的去抗凝肝素，其中，所述去抗凝肝素衍生物的重均分子量为小于8000。
  4. 一种去抗凝肝素衍生物在用于制备治疗炎症性肠病，以及炎症性肠病相关并发症及发病机理相似的疾病的药物中的用途，其中，炎症性肠病相关并发症及发病机理相似的疾病包括但不限于肠易激综合征、关节炎和其他肠外并发症包括强直性脊柱炎、坏疽性脓皮病、结节性红斑、虹膜炎、葡萄膜炎、巩膜外层炎和原发性硬化性胆管炎。
  5. 根据权利要求4所述的用途，其中，所述去抗凝肝素衍生物的抗Xa因子小于等于70IU/mg，优选抗Xa因子小于等于60IU/mg，优选抗Xa因子小于等于50IU/mg，优选抗Xa因子小于等于40IU/mg，优选抗Xa因子小于等于30IU/mg，优选抗Xa因子小于等于20IU/mg，优选抗Xa因子小于等于10IU/mg，并且
    其抗IIa因子小于等于175IU/mg，优选抗IIa因子小于等于170IU/mg，优选抗IIa因子小于等于160IU/mg，优选抗IIa因子小于等于150IU/mg，优选抗 IIa因子小于等于140IU/mg，优选抗IIa因子小于等于130IU/mg，优选抗IIa因子小于等于120IU/mg，优选抗IIa因子小于等于110IU/mg，优选抗IIa因子小于等于100IU/mg，优选抗IIa因子小于等于90IU/mg，优选抗IIa因子小于等于80IU/mg，优选抗IIa因子小于等于70IU/mg，优选抗IIa因子小于等于60IU/mg，优选抗IIa因子小于等于50IU/mg，优选抗IIa因子小于等于40IU/mg，优选抗IIa因子小于等于30IU/mg，优选抗IIa因子小于等于20IU/mg，优选抗IIa因子小于等于10IU/mg。
  6. 根据权利要求4或5所述的用途，其中，所述去抗凝肝素衍生物的重均分子量为8000以上。
  7. 根据权利要求4或5所述的用途，其中，所述去抗凝肝素衍生物的重均分子量为小于8000。
  8. 根据权利要求1所述的去抗凝肝素衍生物，其重均分子量为600～8000，优选重均分子量为1000～7800，进一步优选重均分子量为1500～7500，进一步优选重均分子量为2000～7000，其数均分子量为600～6000，进一步优选数均分子量为1200～5800，进一步优选数均分子量为1500～5500，进一步优选数均分子量为1800～5000。
  9. 根据权利要求1或权利要求8所述的去抗凝肝素衍生物，其2糖单元占全部糖成分的含量在40重量％以上，进一步优选在45重量％以上，进一步优选在50重量％以上，进一步优选在55重量％以上。
  10. 根据权利要求1、权利要求8或9中任一项所述的去抗凝肝素衍生物，其4糖单元占全部糖成分的含量为10～30重量％，进一步优选为15～25重量％。
  11. 根据权利要求1或权利要求8～10中任一项所述的去抗凝肝素衍生物，其6糖单元占全部糖成分的含量在15重量％以下，优选在12重量％以下，进一步优选在10重量％以下，进一步优选在8重量％以下。
  12. 根据权利要求1或权利要求8～11中任一项所述的去抗凝肝素衍生物，其重均分子量与数均分子量之比(重均分子量/数均分子量)在1.4以上，优选在1.45以上，进一步优选在1.50以上。
  13. 一种制备去抗凝肝素衍生物的方法，其包括：
    对原料肝素进行去抗凝处理，得到去抗凝处理的产物，以及
    随后利用酶对经去抗凝处理的产物进行酶解以获得去抗凝肝素衍生物。
  14. 根据权利要求13所述的方法，其中，所述酶为肝素酶。
  15. 根据权利要求14所述的方法，其中，所述肝素酶为肝素酶I。
  16. 根据权利要求13～15中任一项所述的方法，其中，所述对原料肝素进行去抗凝处理是利用高碘酸氧化法对原料肝素进行处理。
  17. 根据权利要求13～16中任一项所述的方法，其中，所述去抗凝肝素衍生物是(1)或(8)～(12)中任一项所述的去抗凝肝素衍生物。
  18. 根据权利要求4所述的用途，其中，所述去抗凝肝素衍生物是权利要求1或8～12中任一项所述的去抗凝肝素衍生物。
  19. 根据权利要求18所述的用途，其中，所述去抗凝肝素衍生物是利用权利要求13～17中任一项所述的方法制备的去抗凝肝素衍生物。
  20. 一种治疗炎症性肠病，以及炎症性肠病相关并发症及发病机理相似的疾病的方法，其包括：
    向有需要的受试者给药去抗凝肝素衍生物。
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