定点聚乙二醇化和可逆聚乙二醇化的策略研究
文献类型：学位论文
| 作者 | 赵永江 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-05-30 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 马光辉 |
| 关键词 | 定点PEG化 PEG丙醛 分支型PEG醛 PEG硫代磺酸酯 可逆PEG化 PEG前体药物 N-Mannich碱 |
| 其他题名 | Study on Strategies of Site Specific PEGylation and Reversible PEGylation |
| 学位专业 | 生物化工 |
| 中文摘要 | 聚乙二醇化(PEG化，PEGylation)是一种重要的先进给药方式，然而传统的PEG化方式在改善药物药代动力学性质的同时，不可避免地带来药效学活性的下降，因此在PEG化过程中对药物的这两方面性质进行平衡非常重要。本论文针对PEG化过程中平衡药物药代动力学性质和药效学性质的策略进行了两方面研究：蛋白质药物的定点修饰策略和小分子抗癌药物的可逆修饰策略。论文的第一部分针对天然蛋白质用于定点修饰的两个重要位点N端氨基和自由巯基，对N端特异性修饰剂和巯基特异性修饰剂进行了研究；第二部分以蒽环类抗肿瘤药物为模型药物，发展了一种新的可逆PEG化策略。主要工作包括： 1．PEG丙醛是一种重要的蛋白质N端定点修饰剂，但是其传统的合成方法Williamson缩合法由于存在副反应，合成收率较低。本论文对Williamson缩合法的过程进行了动力学分析，依此为基础对反应过程进行了系统优化，优化实验的结果证实了动力学分析的合理性，并给出了获得定量转化PEG丙醛的合成工艺。使用所合成的PEG丙醛对类细胞集落刺激因子(G-CSF)和干扰素(IFN)进行了修饰，可达到较高的定点修饰效果。 2．以本课题组成熟发展的分支型PEG羧酸为基础，合成了分支型PEG丙醛。分支型PEG丙醛既具有PEG醛类修饰剂的特点，对被修饰的蛋白质的N端具有一定选择性，并保持所修饰氨基的正电荷；同时具有分支型的空间结构，能够提供比直链PEG更大的空间位阻。使用分支型PEG醛修饰剂对G-CSF进行了修饰，电泳分析确证了分支型PEG丙醛对蛋白质的定点修饰效果。 3．设计并合成了一类新的巯基特异性mPEG修饰试剂——mPEG硫代磺酸酯。以硫代甲磺酸钠或硫代苯磺酸钠对PEG卤代物进行S-烷基化反应，得到了两种mPEG磺酸酯——mPEG硫代甲磺酸酯(mPEG-MTS)和mPEG硫代苯磺酸酯(mPEG-PTS)。使用两种修饰剂对木瓜蛋白酶和G-CSF进行了修饰，修饰结果证明PEG硫代磺酸酯对半胱氨酸残基选择性修饰。细胞增殖实验表明巯基定点修饰的PEG5k-G-CSF可以保留59%的天然蛋白质活性。最后考察了PEG硫代磺酸酯对表面巯基进行修饰的能力，红外和ζ电位分析确证了PEG的偶联和水合层的形成。上述实验证明PEG硫代磺酸酯可以在温和条件下选择性地与巯基反应，反应速度快，几乎定量转化，这些性质对修饰药用蛋白质具有极大的优势。 4．以阿霉素和柔红霉素两种蒽环类抗肿瘤药物为模型药物，设计并合成了以水杨酰胺N-Mannich碱为可逆联结的新型PEG前体药物。其合成历程为选取5-醛基-水杨酰胺及其2位保护的衍生物作为间臂，通过N-Mannich反应偶联药物，进而与PEG酰肼偶联后形成所设计的PEG前体药物。体外释放实验表明水杨酰胺2位羟基的保护可以调节大分子前体药物的释放行为，这一概念允许构建稳定、靶向的大分子前体药物，使母体药物以可控制的方式从大分子载体中释放。新合成的PEG前体药物在细胞增殖实验中表现出与母体药物相当的细胞毒性(IC50值)，对癌细胞仍然具有较强的抑制能力。选取2位保护的mPEG20k前体药物进行体内评价，结果表明2位保护的mPEG20k前体药物可以有效地延长药物的循环半衰期。最后使用荷S-180瘤的小鼠对2位保护的mPEG20k前体药物进行了体内抗癌活性评价，结果表明在相同剂量下前体药物比母体药物具有较好的抑癌活性，同时也表现出较低的毒性。本论文将N-Mannich碱前体药物形式引入到PEG前体药物领域中，构建了一种药物释放可控，并具有较高抑癌活性的大分子前体药物平台，拓展了PEG前体药物的策略库。 |
| 英文摘要 | PEGylation is one of the most important advanced drug delivery systems recently developed. Nevertheless, traditional PEGylation manner lower the activity of drug after modification while improving its pharmacokinetic property. Therefore, it is essential to develop PEGylation strategy which can balance the pharmacokinetic and pharmacodynamic properties of the PEGylated drug. Site specific PEGylation for protein drug and reversible PEGylation for small drug have been two promising strategies tackling the problem of activity lost of PEGylated drug. In the first part of this dissertation, site specific PEGylation strategies for N-terminal and free cysteine residue, the most important sites in native protein for specific modification, were studied. A novel methodology for constructing PEG prodrug of anthracycline anticancer drugs was developed in the second part. Main work of this dissertation included: 1. mPEG propionaldehyde is an important polymer derivative used for N-terminal modification of protein, which is usually synthesized by Williamson reaction of 3-chloropropionaldehde diethyl acetal with PEG alkoxide followed by deprotection of the aldehyde group. However, the side reaction of Williamson reaction has been a severe drawback leading to low yield. A kinetic model was established to depict the competition of Williamson reaction and its side reaction. Based on kinetic analysis, experiments had been performed to systemically investigate the influence of process parameters on yield of PEG aldehyde. Experimental results confirmed the rationale of kinetic model and achieved quantitative yield of mPEG propionaldehyde under optimum conditions. The newly synthesized PEG propionaldehyde was used to modify granulocyte colony-stimulating factor (G-CSF) and Interferon (IFN). SDS-PAGE verified the N-terminal modification of proteins. 2. A branched PEG aldehyde was developed based on a lab-developed branched PEG acid, which combined the site specific manner of aldehyde and branched structure. The newly synthesized branched PEG aldehyde was used to modify G-CSF and the N-terminal PEGylated product was verified by SDS-PAGE. 3. A novel kind of thiol-selective mPEG derivatives, mPEG thiosulfonate was developed. Methoxy PEG methanethiosulfonate (mPEG-MTS) and mPEG phenylthiosulfonate (mPEG-PTS) were synthesized by one-step S-alkylation of mPEG-halide with the corresponding sodium thiosulfonate. The thiol selectivity of both mPEG-MTS and mPEG-PTS were confirmed by modification the cysteine residue of papain and G-CSF. The PEGylated G-CSF (5k) with thiol-selective manner preserved 59% biological activity compared with native G-CSF. Furthermore, mPEG-MTS was used to modify BSA nanoparticles coated with sulfhydryl groups. FT-IR spectroscopy and ζ-potential measurement demonstrated mPEG thiosulfonate can be effectively immobilized on the nanoparticle surface to form hydrophilic and neutral corona. These experimental results indicated that the reaction of PEG thiosulfonate with thiol groups proceeded extremely rapidly under mild conditions with high selectivity and quantitative conversion. These characteristics render PEG thiolsulfonate suitable for modification of pharmaceutical protein. 4. A novel methodology for constructing PEG prodrug of anthracycline anticancer drugs: daunorubicin (DNR) and doxorubicin (DXR) was developed. 5-formyl-salicylamide and its O-acyloxymethylated derivative were used as amide moiety to synthesize N-Mannich bases of anthracycline anticancer drug. PEG prodrugs were constructed afterward by introducing PEG carrier through the formyl group. In vitro hydrolysis evaluation revealed the release behavior of newly synthesized PEG prodrugs could be adjusted by the status of 2-hydroxy group of salicylamide. The O-blocked conception of salicylamide N-Mannich base allowed for the formation of macromolecular prodrug with tunable drug release behavior. Newly synthesized PEG prodrugs also demonstrated superior cytotoxicity in antiproliferative assay. O-blocked PEG20k was chosen for further in vivo test and presented longer circulating life in pharmacokinetic experiment. This high molecular prodrug was also found to be more efficacious against S-180 xenografted tumor and lower toxic than equivalent amount of doxorubicin. The current work has now introduced the usefulness of N-Mannich base prodrug strategy to macromolecular prodrug area, and we believe these findings will extend the PEG prodrug methodology. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 165 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1197]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 赵永江. 定点聚乙二醇化和可逆聚乙二醇化的策略研究[D]. 过程工程研究所. 中国科学院过程工程研究所. 2009. |
入库方式： OAI收割
来源：过程工程研究所
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