本文選題：依諾肝素鈉 + 硫酸魚精蛋白�。� 參考：《山東大學(xué)》2014年碩士論文

【摘要】：肝素作為經(jīng)典的抗凝藥物有其獨(dú)特的優(yōu)勢,如抗凝作用強(qiáng)大,起效快,而且已經(jīng)積累了豐富的臨床經(jīng)驗(yàn)。但是,肝素也有一些缺點(diǎn),例如需要靜脈給藥,生物半衰期短,藥代動力學(xué)特征難以預(yù)測,用藥個體差異大,有出血危險等。 低分子肝素與肝素相比組成更均一,有更高的抗FⅩa/抗FⅡa比值,抗血栓活性與肝素相當(dāng),生物利用度高,體內(nèi)半衰期長,出血危險小,可以皮下注射給藥,且藥代動力學(xué)特征可以預(yù)測,所以低分子肝素現(xiàn)在在臨床使用非常廣泛,尤其是依諾肝素鈉(Enoxaparin sodium)。依諾肝素鈉是肝素經(jīng)過堿性β-消除降解法制備的低分子肝素,擁有最多的臨床適應(yīng)癥,是所有低分子肝素產(chǎn)品中最熱銷的一種。雖然包括依諾肝素鈉在內(nèi)的所有低分子肝素出血危險與肝素相比大幅降低,但是,一旦出血,不能像肝素那樣有效地被硫酸魚精蛋白所中和。所以針對這一問題,本研究通過親和層析技術(shù)對依諾肝素鈉進(jìn)行分級分離,獲得了三個組分,對比研究其理化性質(zhì)、生物活性、被硫酸魚精蛋白中和程度、抗血栓活性及體內(nèi)藥代特性,為獲得有效且更安全的低分子肝素奠定基礎(chǔ)。 本研究以瓊脂糖凝膠為載體,魚精蛋白為配體,將魚精蛋白偶聯(lián)到瓊脂糖凝膠上制備親和層析介質(zhì),填裝了親和層析柱；利用該親和柱對依諾肝素鈉進(jìn)行分級分離,得到與魚精蛋白親和程度不同的組分；然后以依諾肝素鈉為對照進(jìn)行IR、NMR、糖鏈組成(chain mapping)、雙糖組成、分子量分布等理化性質(zhì)的研究；通過羊血漿法和生色底物法測定體外抗凝活性；采用生色底物法測定了所得組分的抗凝活性被魚精蛋白中和的程度；采用靜脈血栓模型研究了其抗血栓活性,以APTT、PT、小鼠尾部出血凝固為指標(biāo)考察了其抗凝活性；研究了其大鼠皮下注射給藥后藥代動力學(xué)。本課題主要取得了如下研究成果： 1.以瓊脂糖凝膠CNBr活化的Sepharose4B為載體,以硫酸魚精蛋白為配體制備了親和層析介質(zhì),并填裝了親和柱。 2.利用所制備的硫酸魚精蛋白親和層析柱,對依諾肝素鈉原料藥(EP)進(jìn)行了分級分離,分別用含Omol/L NaCl、0.5mol/L NaCl、1.0mol/L NaCl三種洗脫液(均以0.05mol/L Tris-HCl為溶劑)依次洗脫獲得了三個組分,采用超濾離心法多次反復(fù)除鹽后,凍干后得到三種粉末,分別是與硫酸魚精蛋白低強(qiáng)度、中強(qiáng)度以及高強(qiáng)度結(jié)合的組分,簡稱為L-EP, M-EP和H-EP,收率分別為56.8%、27.6%,2.6%。由于H-EP的組分量太少,收率過低,所以未對這一組分進(jìn)行研究。 3.對所得組分進(jìn)行了UV, IR,1H-NMR測定,采用CTA-SAX HPLC測定了糖鏈組成,徹底酶解后采用SAX-HPLC測定了雙糖組成,采用GPC-HPLC和多角度激光散射儀測定了其相對和絕對分子量,還測定了組分的S042-/COO"。結(jié)果表明與魚精蛋白的結(jié)合強(qiáng)弱程度與組分的分子量,電荷密度有關(guān),分子量越大,電荷密度越大的組分,與魚精蛋白的結(jié)合越強(qiáng)。 4.對所得組分進(jìn)行了體外抗凝活性研究。采用羊血漿法測了其抗凝效價,采用生色底物法測定了抗FXa、抗FIIa活性。結(jié)果表明,EP、M-EP、L-EP的抗凝效價分別為25.2IU/mg、30.3IU/mg、7.2IU/mg。EP、M-EP、L-EP的抗FXa效價分別為112IU/mg、114IU/mg.96IU/mg,其抗FIIa效價分別為32.8IU/mg.38.0IU/mg、28.2IU/mg。 5.采用生色底物法測定了所得組分被硫酸魚精蛋白中和的程度。結(jié)果表明,魚精蛋白加入量為樣品量的2倍時,中和程度達(dá)到飽和,此時,EP、M-L-EP的抗FXa含量被中和的百分率分別為55.4%、91%、47.9%,抗FIIa含量被中和的百分率分別為100%、100%、0%,可見M-EP的的抗凝活性,無論抗FXa和抗FIIa活性,均可較徹底地被中和,而組分L-EP的抗凝活性被中和效率最低。綜合抗凝活性說明,M-EP保留了EP的抗凝活性,而且大大提高了PS的中和程度,用藥安全性更高。 6.采用是靜脈血栓模型和經(jīng)典的Wessler模型,以EP為對照,將M-EP和L-EP通過大鼠皮下注射給藥,采用了APTT, PT,靜脈血栓濕重,Wessler評分以及大鼠尾靜脈凝血時間五個指標(biāo)來比較M-EP和L-EP的抗凝,抗血栓能力。結(jié)果表明血栓濕重的大小為NSL-EPEPM-EP,四組之間都有顯著性差異(P0.05),說明抗栓效果M-EPEPL-EPNS。各組APTT, PT,尾靜脈凝固時間數(shù)值的大小為M-EPEPL-EPNS,說明抗凝效果M-EPEPL-EPNS。 7.采用生色底物法測定了大鼠皮下注射各組分的藥代動力學(xué)。以EP為對照,將M-EP和L-EP通過大鼠皮下注射給藥,測定了不同時間點(diǎn)血漿抗FⅩa和FⅡa活性,分別繪制了抗FⅩa活性隨時間的變化曲線和抗FⅡa活性隨時間的變化曲線。結(jié)果表明,曲線的最高點(diǎn)均在2h出現(xiàn),抗FⅩa活性-時間曲線中EP、M-EP、L-EP三組Cmax分別為1.04IU/ml、1.069IU/ml、0.957IU/ml, AUC分別為4.589IU*h/mL、6.598IU*h/mL.6.314IU*h/mL;抗FⅡa-時間曲線中,EP、M-EP、L-EP三組Cmax分別為0.304IU/ml、0.356IU/ml、0.282IU/ml,AUC分別為1.987IU*h/mL、2.199IU*h/mL、1.86IU*h/ml。結(jié)果表明三種樣品大鼠體內(nèi)藥代動力學(xué)特征一致。 總之,本研究所得的與硫酸魚精蛋白較強(qiáng)結(jié)合的組分M-EP具有電荷密度更大,分子量分布更均一,其抗血栓和抗凝活性優(yōu)于EP,但較后者更安全。再者,本研究所建立的硫酸魚精蛋白親和層析法也適用于分級分離其他低分子肝素產(chǎn)品,以獲得更安全的新一代低分子肝素產(chǎn)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 .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R973.2

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本文編號：1910276