基于依诺肝素钠的新一代抗血栓药物的研究

发布时间：2018-05-19 13:27

本文选题：依诺肝素钠 + 硫酸鱼精蛋白　；参考：《山东大学》2014年硕士论文

【摘要】：肝素作为经典的抗凝药物有其独特的优势,如抗凝作用强大,起效快,而且已经积累了丰富的临床经验。但是,肝素也有一些缺点,例如需要静脉给药,生物半衰期短,药代动力学特征难以预测,用药个体差异大,有出血危险等。低分子肝素与肝素相比组成更均一,有更高的抗FⅩa/抗FⅡa比值,抗血栓活性与肝素相当,生物利用度高,体内半衰期长,出血危险小,可以皮下注射给药,且药代动力学特征可以预测,所以低分子肝素现在在临床使用非常广泛,尤其是依诺肝素钠(Enoxaparin sodium)。依诺肝素钠是肝素经过碱性β-消除降解法制备的低分子肝素,拥有最多的临床适应症,是所有低分子肝素产品中最热销的一种。虽然包括依诺肝素钠在内的所有低分子肝素出血危险与肝素相比大幅降低,但是,一旦出血,不能像肝素那样有效地被硫酸鱼精蛋白所中和。所以针对这一问题,本研究通过亲和层析技术对依诺肝素钠进行分级分离,获得了三个组分,对比研究其理化性质、生物活性、被硫酸鱼精蛋白中和程度、抗血栓活性及体内药代特性,为获得有效且更安全的低分子肝素奠定基础。本研究以琼脂糖凝胶为载体,鱼精蛋白为配体,将鱼精蛋白偶联到琼脂糖凝胶上制备亲和层析介质,填装了亲和层析柱；利用该亲和柱对依诺肝素钠进行分级分离,得到与鱼精蛋白亲和程度不同的组分；然后以依诺肝素钠为对照进行IR、NMR、糖链组成(chain mapping)、双糖组成、分子量分布等理化性质的研究；通过羊血浆法和生色底物法测定体外抗凝活性；采用生色底物法测定了所得组分的抗凝活性被鱼精蛋白中和的程度；采用静脉血栓模型研究了其抗血栓活性,以APTT、PT、小鼠尾部出血凝固为指标考察了其抗凝活性；研究了其大鼠皮下注射给药后药代动力学。本课题主要取得了如下研究成果： 1.以琼脂糖凝胶CNBr活化的Sepharose4B为载体,以硫酸鱼精蛋白为配体制备了亲和层析介质,并填装了亲和柱。 2.利用所制备的硫酸鱼精蛋白亲和层析柱,对依诺肝素钠原料药(EP)进行了分级分离,分别用含Omol/L NaCl、0.5mol/L NaCl、1.0mol/L NaCl三种洗脱液(均以0.05mol/L Tris-HCl为溶剂)依次洗脱获得了三个组分,采用超滤离心法多次反复除盐后,冻干后得到三种粉末,分别是与硫酸鱼精蛋白低强度、中强度以及高强度结合的组分,简称为L-EP, M-EP和H-EP,收率分别为56.8%、27.6%,2.6%。由于H-EP的组分量太少,收率过低,所以未对这一组分进行研究。 3.对所得组分进行了UV, IR,1H-NMR测定,采用CTA-SAX HPLC测定了糖链组成,彻底酶解后采用SAX-HPLC测定了双糖组成,采用GPC-HPLC和多角度激光散射仪测定了其相对和绝对分子量,还测定了组分的S042-/COO"。结果表明与鱼精蛋白的结合强弱程度与组分的分子量,电荷密度有关,分子量越大,电荷密度越大的组分,与鱼精蛋白的结合越强。 4.对所得组分进行了体外抗凝活性研究。采用羊血浆法测了其抗凝效价,采用生色底物法测定了抗FXa、抗FIIa活性。结果表明,EP、M-EP、L-EP的抗凝效价分别为25.2IU/mg、30.3IU/mg、7.2IU/mg。EP、M-EP、L-EP的抗FXa效价分别为112IU/mg、114IU/mg.96IU/mg,其抗FIIa效价分别为32.8IU/mg.38.0IU/mg、28.2IU/mg。 5.采用生色底物法测定了所得组分被硫酸鱼精蛋白中和的程度。结果表明,鱼精蛋白加入量为样品量的2倍时,中和程度达到饱和,此时,EP、M-L-EP的抗FXa含量被中和的百分率分别为55.4%、91%、47.9%,抗FIIa含量被中和的百分率分别为100%、100%、0%,可见M-EP的的抗凝活性,无论抗FXa和抗FIIa活性,均可较彻底地被中和,而组分L-EP的抗凝活性被中和效率最低。综合抗凝活性说明,M-EP保留了EP的抗凝活性,而且大大提高了PS的中和程度,用药安全性更高。 6.采用是静脉血栓模型和经典的Wessler模型,以EP为对照,将M-EP和L-EP通过大鼠皮下注射给药,采用了APTT, PT,静脉血栓湿重,Wessler评分以及大鼠尾静脉凝血时间五个指标来比较M-EP和L-EP的抗凝,抗血栓能力。结果表明血栓湿重的大小为NSL-EPEPM-EP,四组之间都有显著性差异(P0.05),说明抗栓效果M-EPEPL-EPNS。各组APTT, PT,尾静脉凝固时间数值的大小为M-EPEPL-EPNS,说明抗凝效果M-EPEPL-EPNS。 7.采用生色底物法测定了大鼠皮下注射各组分的药代动力学。以EP为对照,将M-EP和L-EP通过大鼠皮下注射给药,测定了不同时间点血浆抗FⅩa和FⅡa活性,分别绘制了抗FⅩa活性随时间的变化曲线和抗FⅡa活性随时间的变化曲线。结果表明,曲线的最高点均在2h出现,抗FⅩa活性-时间曲线中EP、M-EP、L-EP三组Cmax分别为1.04IU/ml、1.069IU/ml、0.957IU/ml, AUC分别为4.589IU*h/mL、6.598IU*h/mL.6.314IU*h/mL;抗FⅡa-时间曲线中,EP、M-EP、L-EP三组Cmax分别为0.304IU/ml、0.356IU/ml、0.282IU/ml,AUC分别为1.987IU*h/mL、2.199IU*h/mL、1.86IU*h/ml。结果表明三种样品大鼠体内药代动力学特征一致。总之,本研究所得的与硫酸鱼精蛋白较强结合的组分M-EP具有电荷密度更大,分子量分布更均一,其抗血栓和抗凝活性优于EP,但较后者更安全。再者,本研究所建立的硫酸鱼精蛋白亲和层析法也适用于分级分离其他低分子肝素产品,以获得更安全的新一代低分子肝素产品。
[Abstract]:Heparin as a classical anticoagulant has its unique advantages , such as strong anticoagulant effect , rapid onset of action , and abundant clinical experience . However , heparin also has some disadvantages , such as the need for intravenous administration , short half - life of the biological half - life , unpredictable pharmacokinetic characteristics , large individual differences in medication , risk of bleeding , etc .

Compared with heparin , the low molecular weight heparin is more uniform and has higher anti - F Xa / anti - F鈪 ratio , the antithrombotic activity is comparable with heparin , the bioavailability is high , the in vivo half - life is long , the bleeding risk is small , the drug can be injected subcutaneously , and the pharmacokinetic characteristics can be predicted , so the low molecular weight heparin is now very widely used in clinical use , in particular to enothersodium . It is the most popular among all the low molecular weight heparin products . Although the risk of hemorrhage is greatly reduced compared with heparin , it can not be effectively neutralized by heparan sulfate . However , once the hemorrhage is not as effective as heparin , it has been obtained three components . The study also studies its physical and chemical properties , biological activity , neutralization degree , antithrombotic activity and pharmacokinetics in vivo , thus laying the foundation for the effective and safer low molecular weight heparin .

In this study , an affinity chromatography column was prepared by coupling the sperm protein to an agarose gel using an agarose gel as a carrier , and then coupling it to an agarose gel to prepare an affinity chromatography medium ;
the affinity column is utilized to carry out fractional separation on the enohepatic sodium to obtain the components with different affinity to the fish refined protein ;
IR , NMR , sugar chain mapping , disaccharide composition and molecular weight distribution were studied .
The anti - coagulation activity in vitro was determined by goat plasma method and chromogenic substrate method .
The anticoagulant activity of the obtained components was determined by chromogenic substrate method .
Antithrombotic activity was studied by using venous thrombosis model , and its anticoagulant activity was investigated by means of PT , PT and coagulation of tail bleeding in mice .
The pharmacokinetics of subcutaneous injection after subcutaneous injection in rats was studied . The main results were as follows :

1 . Sepharose 4B activated by agarose gel CNBr was used as carrier , affinity chromatography media was prepared by using sulfuric acid as ligand , and affinity column was filled in .

2 . Three kinds of components were obtained by using the prepared sulfuric acid fish - concentrate affinity chromatography column . The three components were successively eluted with 0 . 05mol / L NaCl , 0.5 mol / L NaCl and 1.0 mol / L NaCl ( all with 0.05 mol / L Tris - HCl as solvent ) . The yield of the three components was 56.8 % , 27 . 6 % and 2.6 % respectively .

3 . The components were determined by UV , IR and 1H - NMR . The composition of the sugar chain was determined by CTA and HPLC . The relative and absolute molecular weights of the components were determined by GPC - HPLC and multi - angle laser scattering instrument . The results showed that the binding intensity of the components was related to the molecular weight and charge density of the components . The greater the molecular weight , the greater the charge density , the stronger the binding of the protein .

The anti - FXa titer of EP , M - EP and L - EP was 25 . 2IU / mg , 30 . 3 IU / mg , 7.2 IU / mg . EP , M - EP and L - EP were 112IU / mg , 114IU / mg . 96IU / mg , respectively .

The results showed that the anti - coagulation activity of EP , M - L - EP was 55.4 % , 91 % , 47.9 % , and the percentage of anti - FXa was 100 % , 100 % , 47.9 % , respectively .

6 . The anticoagulant and antithrombotic effects of M - EP and L - EP were compared by subcutaneous injection of M - EP and L - EP by subcutaneous injection of M - EP and L - EP by subcutaneous injection of EP and L - EP by subcutaneous injection of EP and L - EP . The results showed that the size of thrombus wet weight was NSL - EPEPM - EP , and there was a significant difference between the four groups ( P0.05 ) . The results showed that the magnitude of the anticoagulant effect was M - EPEPL - EPNS . The results showed that the anticoagulant effect was M - EPEPL - EPNS .

7 . The pharmacokinetics of various components of subcutaneous injection in rats was determined by chromogenic substrate . The changes of plasma anti - F X a and F鈪 activity were measured by subcutaneous injection of EP and L - EP in rats . The results showed that the highest concentrations of EP , M - EP and L - EP were 1.04IU / ml , 1.069IU / ml , 0.282IU / ml , 1.86IU / ml , 2.199IU / ml and 1.86IU * h / ml respectively . The results showed that the pharmacokinetic characteristics of the three groups were consistent .

In conclusion , the components M - EP with stronger binding to the concentrate of sulfuric acid in the Institute have higher charge density , higher molecular weight distribution , better antithrombotic and anticoagulant activity than EP , but the latter is safer . Furthermore , the method of affinity chromatography of sulfuric acid expressed by the Institute is also applicable to the fractionation of other low molecular weight heparin products to obtain a safer new generation of low molecular weight heparin products .
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R973.2

【参考文献】