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基于八臂PEG聚集和阿拉伯半乳聚糖-PEG修饰的长效葡激酶及其药学性质研究
祁芳冰
Subtype博士
Thesis Advisor胡涛
2019-07-01
Degree Grantor中国科学院大学
Degree Discipline生物化工
Keyword葡激酶,聚乙二醇,阿拉伯半乳聚糖,药代动力学,生物活性
Abstract

葡激酶(SAK)由于具有良好的溶解纤维蛋白的活性,可用于冠状动脉血栓和急性心肌梗塞的治疗。然而,SAK在体内的循环半衰期很短,需要重复给药才能维持其生理作用,这将给患者带来极大的痛苦;此外,SAK具有较强的免疫原性、稳定性较差,这些都限制了SAK的临床应用。聚乙二醇(PEG)修饰和多糖(PS)修饰可以延长SAK的循环半衰期,并降低其免疫原性,但同时会降低SAK的生物活性。为了解决以上问题,本论文选择去掉N-末端前10位氨基酸残基,且C-末端引入Gly-Gly-Cys的重组SAK作为研究对象,以降低野生型SAK的免疫原性,且能实现远离SAK活性区域的定点修饰。本论文设计采用8-arm PEG定点修饰SAK,探索使SAK多聚化的修饰方法对SAK药代动力学和生物活性等性质的影响。另外,采用低分子量的PEG为连接桥,介导阿拉伯半乳聚糖(AG)修饰SAK,探索该修饰方法对SAK免疫原性、药代动力学和生物活性等性质的影响。 采用8-arm PEG-MAL 10 kDa定点修饰SAK的C-末端巯基,并用两种不同的线性PEG修饰剂mPEG-MAL 10 kDa和MAL-PEG-MAL 10 kDa定点修饰SAK作对照,制备并纯化了3种SAK的PEG修饰产物。结果表明,3种PEG修饰方法均不会改变SAK的二级结构,但是会对SAK的三级结构造成轻微的影响。SAK经PEG修饰后,其水化动力学体积均明显增大,热稳定性显著提高,体内循环半衰期得以延长,其他药代动力学参数也有显著的改善。其中,SAKp-PEG具有最大的水化动力学体积,表现出了最佳的药代动力学性质。PEG修饰会屏蔽SAK的活性区域,导致3种PEG修饰产物的体外生物活性有所降低。其中,SAKp-PEG能保持高达90%以上的生物活性。PEG修饰也会对SAK的抗原表位产生屏蔽作用,导致SAK免疫原性降低,但其降低的程度会随着SAK的二聚化或多聚化导致的分子量增大而被抵消。与SAK相比,SAKp-PEG的免疫原性仅稍微增强。此外,SAKp-PEG对小鼠的心、肝、肾均无明显的毒性。因此,通过采用8-arm PEG使蛋白质药物多聚化,能大幅延长蛋白质药物的半衰期,并保留了较高的生物活性。 采用两种不同分子量的异型双功能低分子量PEG分别连接AG和SAK,得到SAK-P2K-AG和SAK-P5K-AG,设计SAK-AG、SAK-P2K和SAK-P5K三个对照组,制备并纯化了的5种SAK的修饰产物。研究结果表明,AG和PEG修饰均不会改变SAK的二级结构。与PEG相比,AG会对SAK产生更强的屏蔽作用,导致SAK-AG在5种修饰产物中荧光强度最弱、生物活性最低、免疫原性显著降低。而在SAK和AG之间引入PEG连接桥,能够降低AG对SAK的空间屏蔽作用,导致SAK-P2K-AG和SAK-P5K-AG比SAK-AG的荧光强度有显著增强、生物活性大幅提高、免疫原性降低。AG和PEG修饰能协同降低SAK的免疫原性,且不会引发抗AG和抗PEG的抗体产生。SAK经AG和PEG修饰后,其水化动力学体积均明显增大,导致体内循环半衰期延长,其他药代动力学参数也有显著的改善。其中,SAK-P5K-AG的水化动力学体积最大,药代动力学性质改善最为显著。这表明AG和更高分子量的PEG修饰可以协同增大SAK的水化动力学体积,并改善药代动力学性质。此外,AG和PEG修饰均不会对小鼠的心、肝、肾产生明显的毒性。该研究的修饰策略为研制新型长效蛋白质药物提供了一种有效的方法。 ;Staphylokinase (SAK) is useful for the treatment of coronary thrombosis and acute myocardial infarction due to its high fibrinolytic activity. However, the serum half-life of SAK is very short and requires repeated administration to maintain its physiological effects, which renders great pain to patients. In addition, SAK suffers from strong immunogenicity and poor stability. These problems limit the clinical application of SAK. Polyethylene glycol (PEG) modification and polysaccharide (PS) modification can prolong the serum half-life of SAK and reduce its immunogenicity. But these modifications can reduce the bioactivity of SAK. In order to solve the above problems, a recombinant SAK was used, which was connected with Gly-Gly-Cys at its C-terminus and lacked the first 10 amino acids of native SAK. The immunogenicity of the recombinant SAK was lower than native SAK and it can be modified at the site far from its bioactive domain. Eight-arm PEG was used to modify SAK, and the effects of polymerization on pharmacokinetics and bioactivity of SAK were investigated. In addition, PEG with low molecular weight was used as a linker to mediate the AG-modified SAK, and the effects of modification on immunogenicity, pharmacokinetics and bioactivity of SAK were investigated. Eight-arm PEG-MAL 10 kDa was used for site-directed conjugation with the C-terminal of SAK. Two linear PEGs (m-PEG-MAL 10 kDa and MAL-PEG-MAL 10 kDa) were used for conjugation with SAK, which acted as the control groups. Three PEGylated SAKs were prepared and purified. The results showed that conjugation with the three PEGs did not alter the secondary structure of SAK, but slightly perturb the tertiary structure of SAK. After conjugation with PEG, the hydrodynamic volume and thermal stability of SAK increased significantly. This prolonged the in vivo half-life and improved other pharmacokinetic parameters. SAKp-PEG exhibited the largest hydrodynamic volume and the best pharmacokinetic properties due to polymerization of SAK. The large hydration shell of PEG around SAK could sterically shield the bioactive domain of SAK, thereby decreasing the bioactivity of SAK. SAKp-PEG could maintain up to 90% bioactivity of SAK. PEG could also shield the epitope of SAK, thereby decreasing the immunogenicity of SAK. However, the reduction of immunogenicity was offset by an increase in molecular weight due to polymerization of SAK. Compared to SAK, SAKp-PEG exhibited slightly enhanced immunogenicity. In addition, SAKp-PEG showed no apparent toxicity to the cardiac, liver and renal functions of mice. Eight-arm PEG was used to conjugate with a protein drug, leading to greatly prolonged half-life and high bioactivity of the protein drug. Two heterobifunctional PEGs with low molecular weights were used as the spacer arm to conjugate AG with SAK. SAK-P2K-AG and SAK-P5K-AG were thus prepared. SAK-AG, SAK-P2K and SAK-P5K were prepared as the control groups. The results showed that conjugation with AG and PEG did not alter the secondary structure of SAK. Compared with PEG, AG exerted a stronger shielding effect on SAK, resulting in the lowest fluorescence intensity, the lowest bioactivity and the significantly low immunogenicity of SAK-AG in the five modified products. The PEG linker between SAK and AG could decrease the steric shielding effect of AG on SAK. Compared with SAK-AG, SAK-P2K-AG and SAK-P5K-AG both showed significantly increased fluorescence intensity, improved bioactivity and decreased immunogenicity. Conjugation with AG and PEG could synergistically decrease the immunogenicity of SAK without triggering the production of anti-AG and anti-PEG IgG. After conjugated with AG and PEG, the hydrodynamic volume of SAK increased significantly, leading to prolonged half-life in vivo and improved other pharmacokinetic parameters. SAK-P5K-AG exhibited the largest hydrodynamic volume and the best pharmacokinetic properties. Conjugation with AG and higher molecular weight PEG could synergistically increase the hydrodynamic volume of SAK and improve the pharmacokinetic properties. In addition, conjugation with AG-PEG showed no apprant toxicity to the heart, liver and renal functions of mice. The modification strategy of this study could be effective for the development of new long-acting protein drugs. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/40664
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
祁芳冰. 基于八臂PEG聚集和阿拉伯半乳聚糖-PEG修饰的长效葡激酶及其药学性质研究[D]. 中国科学院大学,2019.
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