WO2019205255A1 - Utilisation de polyméthylacryloyl-éthanolamine greffée à un disaccharide d'héparine sulfatée - Google Patents

Utilisation de polyméthylacryloyl-éthanolamine greffée à un disaccharide d'héparine sulfatée Download PDF

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Publication number
WO2019205255A1
WO2019205255A1 PCT/CN2018/091470 CN2018091470W WO2019205255A1 WO 2019205255 A1 WO2019205255 A1 WO 2019205255A1 CN 2018091470 W CN2018091470 W CN 2018091470W WO 2019205255 A1 WO2019205255 A1 WO 2019205255A1
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WIPO (PCT)
Prior art keywords
heparin
grafted
disaccharide
polymethacryloylethanolamine
sulfated
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PCT/CN2018/091470
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English (en)
Chinese (zh)
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陈敬华
蔡智
闫昳姝
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江南大学
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Publication of WO2019205255A1 publication Critical patent/WO2019205255A1/fr

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    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/795—Polymers containing sulfur
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/10—Esters
    • C08F120/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment

Definitions

  • the invention belongs to the field of biomedical materials, and particularly relates to the use of sulfated heparin disaccharide grafted polymethacryloylethanolamine.
  • Heparin which is often found in mast cells, is also present in tissues such as the lung, blood vessel wall, and intestinal mucosa, and is widely used as an anticoagulant drug in the clinic because of its various affinities. Heparin is best known for its role in blood coagulation, but it also exerts other effects such as anti-inflammatory, anti-angiogenic, and anti-tumor effects in biologically active functions. Among them, the anti-tumor effect of heparin has been paid close attention, and many studies have proved that heparin can inhibit the invasion and metastasis of tumor cells. However, natural heparin is a structurally heterogeneous mixture, which makes it difficult to define biological activity standards, and studies on structure-activity relationships and mechanisms of action are very difficult.
  • the strong anticoagulant activity of natural heparin may cause toxic side effects such as hemorrhage and thrombocytopenia. This limits the application of natural heparin.
  • the only source of natural heparin is animal tissue, which carries the risk of causing viral contamination and adverse reactions. Heparin may also be decomposed by heparinase and other enzymes in the body during treatment, resulting in loss of biological activity.
  • the anticoagulant activity of heparin is mainly derived from the specific pentose sequence contained in it.
  • the sequence structure can bind to antithrombin (AT-III), activate antithrombin, and is widely used due to the anticoagulant activity of heparin.
  • Heparin causes hemorrhage and induces thrombocytopenia; in addition, the researchers found that heparin's antitumor activity is not directly related to its anticoagulant capacity, but due to hydrogen bond formation accompanied by heparin sugar ring, and heparin sulfate negative charge
  • Chinese Patent Application No. CN2012103286497 discloses a method for controlling the production of low molecular weight heparin, which utilizes two or more heparinases of heparinase I, II, III to degrade heparin to produce low molecular weight or ultra low molecular weight heparin. Due to the specificity and polydispersity of heparin and its low molecular weight heparin structure, the risk of side effects is high.
  • a sulfated heparin disaccharide grafted polymethacryloylethanolamine the natural heparin is completely degraded by using excess heparinase I, heparinase II and heparinase III, and the natural disaccharide is separated and prepared.
  • the sugar is grafted onto a long chain of acrylate of a specific molecular weight and sulfated to obtain a heparin-like macromolecule having good chemical stability, low anticoagulant, excellent biocompatibility, and antitumor property.
  • the preparation method of the above sulfated heparin disaccharide grafted polymethacryloylethanolamine comprises the following steps:
  • the enzyme activity ratio of heparinase I, heparinase II and heparinase III in the step (1) is 1:1:1.
  • the specific step of synthesizing the methacryloylethanolamine in the step (2) is: weigh the ethanolamine hydrochloride and the hydroquinone, and add the methacryloyl chloride at 70 to 80 ° C. Reaction 2h.
  • the specific step of the step (2) synthesis of polymethacrylolethanolamine is: weighing propylene glycol ethanolamine, azobisisobutyronitrile (AIBN) at a molar ratio of 50 to 200:1:0.2. And 4-cyanovaleric acid dithiobenzoic acid (CTA), dissolved in N,N-dimethylformamide (DMF), reacted under nitrogen protection at 70-80 ° C for 24 h, wherein AIBN is used as an initiator. CTA acts as a polymeric chain transfer agent.
  • AIBN azobisisobutyronitrile
  • the mass ratio of heparin disaccharide to PAMA is 10-20:4-5, and the mass ratio of heparin disaccharide, EDC and NHS is 1:1.25-3:0.2. ⁇ 1.5.
  • the pH is adjusted to 9.5 to 10.5, and the reagent used is sodium carbonate.
  • the polymethyl methacrylate ethanol of a specific molecular weight is a component having a monodisperse molecular weight.
  • the tumor is melanoma.
  • the macromolecule of the present invention is heparin disaccharide degraded by heparinase, and is linked with an amino polyacrylate.
  • One end of the reactive group is a carboxyl group, and one end of the reactive group is an amino group, which is linked by an amide bond to form an anti-metastatic compound. Active heparin-containing sugar macromolecules.
  • the inhibitory effect of sulfated heparin disaccharide grafted polymethacryloylethanolamine on tumor cell migration and transmembrane ability is shown to be stronger than heparin, and the anticoagulant activity is determined in vitro. It shows that it basically eliminates anticoagulant activity and has great potential for development.
  • the invention completely degrades natural heparin by using heparinase I, heparinase II and heparinase III, and is separated by G25 gel for preliminary filtration and separation, and a strong anion chromatography exchange column is used to prepare natural disaccharide, and G10 gel is desalted. Thereafter, the disaccharide is grafted onto a long chain of polymethyl propylene glycolamine of a specific molecular weight and sulfated to obtain a heparin-like macromolecule having good chemical stability, low anticoagulant, excellent biocompatibility, and antitumor property. .
  • the present invention firstly obtains a disaccharide raw material by enzymatic hydrolysis from natural heparin, thereby eliminating the cumbersome chemical synthesis of heparin sugar units, and also eliminating the number of heparin anticoagulant active centers, thereby greatly reducing anticoagulant activity. ;
  • the heparin disaccharide of the present invention is a natural disaccharide, which is safe; the selection of a polymer and heparin are highly prevalent on biosafety;
  • the present invention grafts heparin disaccharide on PAMA to form a "sugar cluster effect", and the product also exhibits low toxicity and negligible anticoagulant activity;
  • the use of the sulfation in the present invention increases the charge density of the active site of the product and improves the anti-metastatic activity; (5) the preparation method is simple, and has the advantages of low raw materials, mild reaction conditions and easy control.
  • Figure 1 is a strong anion exchange chromatogram of heparin disaccharide
  • Figure 3 shows the inhibition of migration of B16 melanoma cells by heparin and sulfated heparin disaccharide grafted polymethacryloylethanolamine using a scratch test; wherein Control represents a blank control group without drug addition, Heparin represents heparin, SGPHD Representative of sulfated heparin disaccharide grafted polymethacryloylethanolamine; the figure shows the comparison of cell scratch healing in the presence of drug after 0h and 24h;
  • Figure 4 shows the inhibition of B16 melanoma cell transmembrane by heparin and sulfated heparin disaccharide grafted polymethacryloylethanolamine using a scratch test; wherein Control represents a blank control group without drug addition, Heparin represents heparin, SGPHD stands for sulfated heparin disaccharide grafted polymethacryloylethanolamine.
  • heparin 6 g was dissolved in 120 mL of pH 7.4 Tris buffer containing 5 mM CaCl, 20 mM NaCl, and 10 IU of heparinase I, heparinase II and heparinase III were added, and shaken at 150 rpm for 37 rpm shaking at 37 ° C. 24h. After the reaction was completed, the reaction solution was boiled for 3 minutes to inactivate the enzyme, centrifuged at 5000 r/min for 15 min, and the supernatant was lyophilized to obtain heparin oligosaccharide. In this example, the enzyme was inactivated by increasing the temperature.
  • Sephadex G-25 was processed and packed into a 1.2 x 100 cm glass column.
  • the lyophilized oligosaccharide was configured into a 40 mg/mL solution using ultrapure water, loaded with 1 mL, and eluted with a flow rate of 1 mL/min ultrapure water.
  • the absorption wavelength curve of 232 nm was monitored using a UV detector, and the peaks were collected and lyophilized.
  • the HPLC method was carried out, and the chromatographic conditions were: ProPac SAX-10 column, eluted with a mobile phase gradient of 20 mM - 1.5 M NaCl 3.5 as a mobile phase, a flow rate of 0.5 mL/min, and a column temperature of 25 °C. 20 ⁇ L of the collected heparin disaccharide sample was separately sampled, and the elution curve was monitored to determine the inclusion of the disaccharide component. As shown in Fig. 1, the 1-8 peaks were respectively the eight disaccharides contained in the heparin structure.
  • the detected heparin disaccharide fraction was concentrated by rotary evaporation at 40 ° C, and then desalted using a 1.2 ⁇ 100 cm Sephadex G10 column, and the mobile phase was ultrapure water at a flow rate of 1 mL/min.
  • the desalted disaccharide was again concentrated by rotary evaporation and lyophilized for use.
  • the easily transferable B16 melanoma cells were seeded on a 24-well plate at a density of 5 x 10 4 /well. After the cell density in the well was greater than 90%, a scratch was drawn in parallel on the cell layer of each well using a 200 ⁇ L tip. Wash twice with PBS, replace with serum-free medium, and add heparin (512 mg / L) in different concentrations, different concentrations of sulfated heparin disaccharide grafted polymethacryloylethanolamine (128 mg / L, 256 mg / L and 512 mg / L), continue to culture for 24h, observe and take photos of the scratch healing.
  • the in vitro anticoagulant activity of sulfated heparin disaccharide grafted polymethacryloylethanolamine was determined using the substrate chromogenic method of the European Pharmacopoeia version 7.0.
  • the 6.05 g/L Tris solution was adjusted to pH 8.4 with hydrochloric acid as a diluent.
  • the absorption at 405 nm was measured during the -30 s period.
  • the anti-Xa or IIa activity of different concentrations of heparin was determined by the above method using heparin standard (220 U/mg), and a standard curve was drawn.
  • the anti-Xa of sulfated heparin disaccharide grafted polymethacryloylethanolamine was calculated according to the standard curve. II a activity.
  • the sulfated heparin disaccharide grafted polymethacryloylethanolamine has anticoagulant ability comparable to heparin disaccharide, which is 50 times lower than that of heparin. Therefore, when used as an antitumor drug, Neglecting the risk of bleeding from SGPHD; the anticoagulant activity of heparin is mediated by the single-chain glycoprotein antithrombin III, which is mainly derived from the specific binding of anti-thrombin in the NA/NS mixed region.
  • the sugar sequence since heparin disaccharide of SGPHD does not contain the above-mentioned pentasaccharide structure, SGPHD can theoretically reduce its anticoagulant activity.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Polymers & Plastics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

La présente invention appartient au domaine des matériaux biomédicaux. En particulier, l'invention concerne une utilisation de polyméthylacryloyl-éthanolamine greffée à un disaccharide d'héparine sulfatée. La polyméthylacryloyl-éthanolamine greffée à un disaccharide d'héparine sulfatée présente des propriétés de forte activité anti-tumorale et de faible activité anti-coagulante. La polyméthylacryloyl-éthanolamine greffée à un disaccharide d'héparine sulfatée utilisée dans la présente invention fournit une nouvelle direction pour des études sur la préparation d'un médicament anti-tumoral à base d'héparine.
PCT/CN2018/091470 2018-04-28 2018-06-15 Utilisation de polyméthylacryloyl-éthanolamine greffée à un disaccharide d'héparine sulfatée WO2019205255A1 (fr)

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CN201810399166.3A CN108498536B (zh) 2018-04-28 2018-04-28 硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途
CN201810399166.3 2018-04-28

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103705534A (zh) * 2013-12-30 2014-04-09 中国药科大学 一种天然活性物质构建的高分子复合药物的制备及其在抑制血管生成中的应用
CN108403704A (zh) * 2018-05-31 2018-08-17 江南大学 肝素二糖接枝硫酸化聚甲基丙烯酰乙醇胺的用途
CN108641018A (zh) * 2018-04-28 2018-10-12 江南大学 一种硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺及其制备方法

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CN102864191A (zh) * 2011-12-16 2013-01-09 深圳市海普瑞药业股份有限公司 肝素双糖混合物及其制备方法和应用
CN103087219B (zh) * 2011-12-30 2014-11-26 北京大学 一种树枝状肝素纳米材料修饰的生物型人造血管
CN105891343B (zh) * 2014-12-06 2019-01-08 烟台东诚药业集团股份有限公司 一种舒洛地特组分精细结构分析检测方法
CN104892807B (zh) * 2015-06-02 2017-05-24 江南大学 一种表面糖修饰聚合物胶束及其制备方法和应用
CN105504097B (zh) * 2015-12-30 2018-07-03 深圳市海普瑞药业集团股份有限公司 一种硫酸化肝素寡糖及其制备方法和应用
CN106932494A (zh) * 2015-12-30 2017-07-07 深圳市海普瑞药业股份有限公司 一种酶解-hplc法检测舒洛地特的方法
EP3398971A4 (fr) * 2015-12-30 2019-09-25 Shenzhen Hepalink Pharmaceutical Group Co., Ltd. Oligosaccharide d'héparine sulfaté et procédé de préparation et application de celui-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103705534A (zh) * 2013-12-30 2014-04-09 中国药科大学 一种天然活性物质构建的高分子复合药物的制备及其在抑制血管生成中的应用
CN108641018A (zh) * 2018-04-28 2018-10-12 江南大学 一种硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺及其制备方法
CN108403704A (zh) * 2018-05-31 2018-08-17 江南大学 肝素二糖接枝硫酸化聚甲基丙烯酰乙醇胺的用途

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