WO2019205255A1 - 硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途 - Google Patents

硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途 Download PDF

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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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heparin
grafted
disaccharide
polymethacryloylethanolamine
sulfated
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陈敬华
蔡智
闫昳姝
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Jiangnan University
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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

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  • 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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Abstract

本发明属于生物医用材料领域，具体公开了一种硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途，所述硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺抗肿瘤活性强，低抗凝血活性的特点；本发明所用的硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺为肝素制备治疗抗肿瘤药物的研究提供一个新的方向。

Description

硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途 技术领域
本发明属于生物医用材料领域，具体涉及硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途。
背景技术
肝素，常常存在于肥大细胞中，也存在于肺、血管壁、肠粘膜等组织中，由于其各种亲和力在临床中被广泛用作抗凝药物。肝素最为人所熟知的是它在血液凝固过程中的作用，但在是生物活性功能中也能发挥其他作用比如抗炎、抗血管生成、及抗肿瘤作用。其中，肝素的抗肿瘤作用已经备受关注，许多研究已经证明了肝素能够抑制肿瘤细胞的侵袭和转移。然而天然肝素是在结构上不均一的混合物，导致生物活性标准难以界定，构效关系和作用机理的研究非常困难。而天然肝素的强抗凝血活性可能会引发出血和血小板减少症等毒副作用。这限制了天然肝素的应用。此外，天然肝素的唯一来源是动物组织，具有可能带来病毒污染和不良反应的风险。而肝素在治疗中还可能会被肝素酶和其他酶在体内分解，导致失去生物活性。
肝素的抗凝血活性主要来自其包含的特定戊糖序列，该序列结构能够与抗凝血酶(AT-III)结合，活化抗凝血酶，由于肝素具有的抗凝血活性，所以大量使用肝素会引起出血和诱导血小板减少等作用；此外，研究人员发现，肝素的抗肿瘤活性与其抗凝血能力并没有直接关联，而是由于肝素糖环伴随的氢键形成，和肝素硫酸根负电荷的作用，使肝素能够结合肿瘤转移过程中起重要作用的蛋白质的结果。因此，低抗凝血肝素的开发引起了很大关注，尤其在抑制肿瘤生长和转移方面。
申请号CN2012103286497的中国专利申请公开了一种控制生产低分子量肝素的方法，利用肝素酶I、II、III中两种以上的肝素酶来降解肝素从而生产低分子量或超低分子量肝素，然而，由于肝素及其低分子量肝素结构的特异性和多分散性，导致副作用的风险很高。
最近几年，许多研究者还尝试使用化学法合成结构明确的肝素二糖和肝素衍生物以减弱抗凝血活性，然而，这种方法伴随着生产的高成本和合成的困难，硫 酸化程度的降低也减弱了肝素的抗肿瘤活性。因此去除具有抗凝能力的五糖序列，将确定组分的肝素二糖接枝在丙烯酸酯长链上并加以硫酸化，就有可能降低结构异质性，消除抗凝血活性，并提高其抗肿瘤活性，合成刷状具有抗肿瘤作用的肝素类似物大分子。
发明内容
针对现有问题的不足，本发明的目的是提供硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途。本发明研究结果表明，硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺能够有效提高抗转移活性，降低抗凝血活性，从而为肝素制备治疗抗肿瘤药物提供支持。
本发明解决其技术问题采用的技术方案是：
一种硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺，通过联用过量肝素酶I、肝素酶II、肝素酶III，将天然肝素完全降解，分离制备得到天然二糖，将二糖接枝在特定分子量的丙烯酸酯长链上，加以硫酸化，得到具有良好化学稳定性，低抗凝血，优异生物相容性、抗肿瘤性能的类肝素大分子。
上述硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的制备方法，包括如下步骤：
(1)肝素二糖的制备与分离：首先加入过量肝素酶I、肝素酶II和肝素酶III将天然肝素完全酶解；然后利用G25、强阴离子色谱交换柱、G10凝胶脱盐得肝素二糖；
(2)聚甲基丙烯乙醇胺(PAMA)的合成：以乙醇胺盐酸盐、甲基丙烯酰氯为原料，合成甲基丙烯乙醇胺单体，再利用RAFT反应制备特定分子量的聚甲基丙烯乙醇胺；特定分子量的聚甲基丙烯乙醇胺通过RAFT反应实现，反应中通过控制添加引发剂、链转移剂添加量，同时控制反应条件实现分子量可控；
(3)接枝反应：称取肝素二糖、1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)和N-羟基丁二酰亚胺(NHS)，溶于pH 5.4～5.6的MES缓冲液，调节控制pH在7.5～8.5，再加入聚甲基丙烯乙醇胺，室温下反应8～12h，反应液离心除沉淀后，透析，冻干，得肝素二糖接枝聚甲基丙烯乙醇胺(GPHD)；
(4)硫酸化反应：称取肝素二糖接枝聚甲基丙烯酰乙醇胺、三氧化硫吡啶溶于超纯水中，调节pH至碱性，50～80℃反应24h，再用盐酸溶液中和，透析， 冻干，即得。
作为本申请的优选技术方案，所述步骤(1)中肝素酶I、肝素酶II和肝素酶III的酶活添加比例为1∶1∶1。
作为本申请的优选技术方案，所述步骤(2)中甲基丙烯酰乙醇胺的合成具体步骤为：称取乙醇胺盐酸盐与对苯二酚混合，70～80℃下加入甲基丙烯酰氯，反应2h。
作为本申请的优选技术方案，所述步骤(2)聚甲基丙烯乙醇胺的合成具体步骤为：按摩尔比50～200∶1∶0.2称取甲基丙烯乙醇胺、偶氮二异丁腈(AIBN)和4-氰基戊酸二硫代苯甲酸(CTA)，并溶于N，N-二甲基甲酰胺(DMF)，70～80℃氮气保护下反应24h，其中，AIBN作为引发剂，CTA作为聚合链转移剂。
作为本申请的优选技术方案，所述步骤(3)中，肝素二糖与PAMA的质量比10～20∶4～5，肝素二糖、EDC和NHS的质量比为1∶1.25～3∶0.2～1.5。
作为本申请的优选技术方案，所述步骤(4)中，调节pH为9.5～10.5，所用试剂为碳酸钠。
作为本申请的优选技术方案，所述步骤(2)中，特定分子量的聚甲基丙烯乙醇胺为分子量单分散的组分。
上述硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺在制备治疗抗肿瘤药物中的用途。
作为本申请的优选技术方案，所述的肿瘤是黑色素瘤。
本发明所述的大分子是由肝素酶降解的肝素二糖，与氨基聚丙烯酸酯连接，一端反应基团是羧基，一端反应基团是氨基，通过形成酰胺键相连，合成的具有抗转移活性的类肝素含糖大分子。
本发明实施例中，硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺对肿瘤细胞迁移、穿膜能力的抑制作用，与肝素相比都显示出更强的抑制作用，且体外抗凝活性测定显示，其基本消除了抗凝血活性，非常具有发展潜力。
其具体合成路线如下：
Figure PCTCN2018091470-appb-000001
本发明通过联用肝素酶I、肝素酶II和肝素酶III，将天然肝素完全降解，经G25凝胶初步过滤分离，强阴离子色谱交换柱分离制备得到天然二糖，G10凝胶脱盐后，将二糖接枝在特定分子量的聚甲基丙烯乙醇胺长链上，加以硫酸化，得到具有良好化学稳定性，低抗凝血，优异生物相容性、抗肿瘤性能的类肝素大分子。
本发明提供的硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺以及制备方法，与现有技术相比，具有以下优点：
(1)本发明首先从天然肝素酶解获得二糖原料，免去了化学法合成肝素糖单元的繁琐，同时也消除了肝素抗凝血活性中心的数量，极大地降低了抗凝血活性；
(2)本发明的肝素二糖是天然二糖，具有安全性；高分子和肝素的选择均具有高度的生物安全性做前提；
(3)本发明将肝素二糖接枝在PAMA上，形成“糖簇效应”，产品也呈现低毒性和可忽略的抗凝血活性；
(4)本发明用硫酸化提高提高了产物活性位点的电荷密度，提高了抗转移活性；(5)制备方法较为简单，具有原料低廉、反应条件温和易控等优点。
附图说明
图1为由肝素二糖的强阴离子交换色谱图；
图2为特定分子量聚甲基丙烯乙醇胺的GPC分子量测定图；
图3为采用划痕实验检测肝素和硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺对B16黑色素瘤细胞迁移的抑制情况；其中，Control代表无药物添加的空白对照组，Heparin代表肝素，SGPHD代表硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺；图为0h与24h后药物存在时细胞划痕愈合情况的对比；
图4为采用划痕实验检测肝素和硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺 对B16黑色素瘤细胞穿膜的抑制情况；其中，Control代表无药物添加的空白对照组，Heparin代表肝素，SGPHD代表硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺。
具体实施方式
以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的，均视为可以通过商业购买的常规产品。
实施例1：
1.酶法降解制备肝素二糖
将6g肝素溶解于120mL包含5mM CaCl，20mM NaCl的pH 7.4Tris缓冲液中，加入肝素酶I、肝素酶II、肝素酶III各10IU，置于37℃摇床150转/分钟震荡反应24h。反应结束后，反应液被煮沸3min使酶失活，5000r/min离心15min，取上清液冻干，得到肝素寡糖，本实施例通过提高温度使酶失活。
2.G25凝胶初步过滤分离
Sephadex G-25经处理，装成1.2×100cm的玻璃层析柱。将冻干寡糖使用超纯水配置成40mg/mL溶液，上样1mL，使用1mL/min超纯水流速洗脱，使用紫外检测器监测232nm吸收波长曲线，分管收集各峰并冻干。
3.强阴离子交换色谱检测二糖组分
采用HPLC法，色谱条件为：ProPac SAX-10色谱柱，以20mM-1.5M pH 3.5的NaCl溶液为流动相梯度洗脱，流速0.5mL/min，柱温25℃。分别上样分管收集的肝素二糖样品20μL，监测洗脱曲线，确定包含二糖组分，如图1所示，1-8峰分别为肝素结构中包含的八种二糖。
4.G10凝胶脱盐
将检测到的肝素二糖组分40℃旋转蒸发浓缩后，使用1.2×100cm Sephadex G10柱脱盐，流动相为超纯水，流速为1mL/min。脱盐二糖再次旋转蒸发浓缩后，冻干待用。
5.甲基丙烯酰乙醇胺单体的合成
将1g干燥的乙醇胺盐酸盐与30mg对苯二酚混合，置于25mL圆底烧瓶中，80℃加热搅拌状态下，滴入2mL甲基丙烯酰氯，70～80℃冷凝回流搅拌反应2h；向溶化的产物加入3mL四氢呋喃溶解，使用100mL乙醚8000r/min沉淀离 心；沉淀用乙醚洗三次，真空干燥得到甲基丙烯酰乙醇胺单体。
6.特定分子量聚甲基丙烯乙醇胺的合成
将662mg甲基丙烯乙醇胺、4.24mg AIBN、0.821 mg4-氰基戊酸二硫代苯甲酸溶于5mL无水DMF，加入聚合管。聚合管使用液氮冷却，抽真空后解冻，置换高纯氮气，重复三次。聚合管70-80℃油浴搅拌反应24h后，使用100mL乙醇沉淀，再使用乙醇洗三次，将沉淀出的聚合物真空干燥。聚合物分子量使用GPC测定(图2)，结果显示聚合物为分子量单分散的组分。
7.肝素二糖接枝聚甲基丙烯酰乙醇胺
取200mg混合肝素二糖、47mg NHS与257mg EDC溶于50mL pH 5.5MES缓冲液，室温下搅拌30min。使用三乙胺调节pH到8，再加入50mg聚甲基丙烯乙醇胺，室温搅拌反应8h。反应液离心除去沉淀，使用截留分子量3000透析袋透析，冻干得到产物肝素二糖接枝聚甲基丙烯酰乙醇胺。
8.肝素二糖接枝聚甲基丙烯酰乙醇胺的硫酸化
取二糖接枝聚甲基丙烯酰乙醇胺50mg，三氧化硫吡啶75mg，溶于5mL超纯水中，使用碳酸钠调节pH为9.5～10.5，反应液在50～80℃氮气氛下搅拌反应24h后使用0.1M盐酸溶液中和，使用截留分子量3000透析袋透析，冻干得到产物硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺。
性能测试
1.硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺体外对肿瘤细胞迁移能力的抑制作用
将易转移的B16黑色素瘤细胞接种在24孔板上，密度为5×10 4/孔。待孔中细胞密度大于90％后，使用200μL枪头于每孔细胞层上平行画出划痕。使用PBS清洗两次，更换为无血清培养基，同时分组加入肝素(512mg/L)，不同浓度的硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺(128mg/L、256mg/L和512mg/L)，继续培养24h，观察并拍照划痕愈合情况。
结果如图3显示，没有加药处理的B16黑色素瘤细胞层划痕经过24h后基本愈合，说明其原本的迁移能力很强。而加入肝素和硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺均对这种愈合有明显的抑制作用，而且硫酸化肝素二糖接枝聚甲基 丙烯酰乙醇胺对B16黑色素瘤细胞迁移的抑制能力更强。
2.硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺体外对肿瘤细胞穿膜能力的抑制作用
于24孔板各孔分别加入700μL含有不同浓度的肝素和硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺(128mg/L、256mg/L和512mg/L)的培养基，装置Transwell小室。向每个小室上层接种100μL密度为1×10 6/mL的B16黑色素瘤细胞悬浮液，继续培养24h后，使用棉签擦去小室上层的B16黑色素瘤细胞，并使用0.1％结晶紫溶液对穿过小室膜的B16黑色素瘤细胞染色，PBS清洗残余染料三次后，观查并拍照细胞穿膜情况。
结果如图4显示，肝素和硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺均对这种穿膜运动有明显的抑制作用，且硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺显示出了更强的抑制能力。
3.硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺体外抗凝活性测定
硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺体外抗凝活性使用欧洲药典7.0版本的底物显色法测定。6.05g/L Tris溶液被盐酸调节pH至8.4作为稀释液。160μL不同浓度的药物与过量的抗凝血酶AT-III以及20μL人血清在37℃水浴60s后，加入40μL 2mM凝血因子Xa(S-2765)或IIa(S-2238)显色底物，10-30s时间段测定405nm处吸收。使用肝素标准品(220U/mg)按上述方法测定不同浓度肝素的抗Xa或IIa活性，并绘制标准曲线，根据标准曲线，计算硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的抗Xa或II a活性。
表1硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺(SGPHD)及作为对照组的肝素、肝素二糖体外抗凝活性
Figure PCTCN2018091470-appb-000002
由表1可以看出，硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺具有与肝素二糖相当的抗凝血能力，比肝素低50倍以上，因此，当用作抗肿瘤药物时，可忽略SGPHD的出血风险；肝素的抗凝血活性由单链糖蛋白抗凝血酶III介导，该活性主要来源于NA/NS混合区域中特异性的可与抗凝血酶特异性结合的五糖序列，由于SGPHD的肝素二糖不含有上述五糖结构，所以SGPHD理论上可以降低其抗凝血活性。
由以上的性能测试数据可以看出，SGPHD对抑制B16细胞迁移、侵入和粘附与肝素相比显示了更强的作用，同时还消除了肝素抗凝血活性中心的数量，极大地降低了抗凝血活性，这些结果对抗肿瘤药物的进一步合理设计与应用产生重要影响。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (2)

  1. 硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺在制备治疗抗肿瘤药物中的用途。
  2. 权利要求1所述的硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺在制备治疗抗肿瘤药物中的用途，所述的肿瘤是黑色素瘤。
PCT/CN2018/091470 2018-04-28 2018-06-15 硫酸化肝素二糖接枝聚甲基丙烯酰乙醇胺的用途 Ceased WO2019205255A1 (zh)

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