新型预活化巯基化聚合物促进蛋白多肽药物跨膜转运机理研究

The oral bioavailability of proteins and peptides is strongly limited by an insufficient uptake from the mucosa. In order to overcome the absorption barrier, permeation enhancers, enzyme inhibitors or other auxiliary agents are used in oral drug delivery systems. State of the art most of the agents are in low molecular mass. They are easily absorbed through the gastrointestinal mucosa into the circulation and systemic toxic side-effects of these auxiliary agents can consequently not excluded. In contrast, macromolecular permeation enhancers such as poly(acrylic acid) derivatives, chitosan derivatives and thiolated polymers are too big in size to be taken up into the systemic circulation. In addition, they remain at the site where drug absorption shall take place for a comparatively longer time period..In recent years, thiomers have gained considerable attention. Thiomers are a group of polymers bearing thiol substructures. Due to the immobilization of thiol groups on already well-established polymers such as polyacrylates or chitosans, features such as mucoadhesive, in-situ gelling, efflux pump inhibiting and permeation enhancing properties are strongly improved. However, there are also shortcomings of thiomers, in particular regarding storage stability. Thiomers in solutions and semisolid formulations are subject to thiol oxidation at pH above 5, unless sealed under inert conditions..To overcome this drawback, novel thiomers - designated preactivated thiomers- are synthesised in this project. The concept for these novel thiomers is based on the reaction scheme for covalent chromatography of resins such as thiopropyl sepharose? 6B. The thiolated resin being activated by a mercaptopyridin group is stable towards oxidation and can react with solutes containing thiol groups under mild conditions to form mixed disulfides. In analogy the reaction can also take place when such polymers come into contact with the mucosa where thiol-rich substructures are available..Following this strategy novel preactivated thiomers will be prepared. Poly(acrylic acid) and chitosan are chosen as backbones and they are preactivated with 6,6'-dithiodinicotinic acid. It is anticipated at preactivated thiomers are stable at a wide range pH and they also have the same characteristics as thiomers: bioadhension, perrmeation enhancement, enzyme activity inhibition, efflux pump inhibition, etc. Furthermore, because the introduction of mercaptopyridin group into the molecule, it is also anticipated at preactivated thiomers are more active than thiomers. .The goal of this study is to synthesis a series of preactivated thiomers, evaluate their function on absorption enhancement and elucidate their absorption enhancement mechanisms on proteins and peptides systemly.

生物技术快速发展使蛋白多肽药物大量涌现，而此类药物具有分子量大、体内不稳定、半衰期短和不易通过生物膜等特点，其非注射给药特别是口服给药面临巨大挑战，是长期悬而未决的科学难题。多功能高分子聚合物在促进蛋白多肽吸收方面具有明显优势。本项目以阴离子型和阳离子型聚合物为模板合成一系列结构新颖的预活化巯基化聚合物，重点探索其生物黏附、吸收促进、稳定化(pH,酶抑制)、抗耐药、原位凝胶、控释等方面的作用规律，系统阐明其促进蛋白多肽药物跨膜转运的作用机理，具有重要的理论意义。与传统聚合物相比，本研究选择安全性好的活化剂制备结构全新的巯基化聚合物，使之在广泛pH范围内具有更好的稳定性，并具有更强的生物黏附、促渗透、酶抑制、抗耐药、原位凝胶、控释等功能，具有明显的创新性。本团队在蛋白多肽和纳米载体的跨膜转运方面有长期的工作经历，在巯基化聚合物的研究方面有较好的工作基础，科研力量较强，完成任务的可行性较强。

生物技术快速发展使蛋白多肽药物大量涌现，而此类药物具有分子量大、体内不稳定、半衰期短和不易通过生物膜等特点，其非注射给药特别是口服给药面临巨大挑战，是长期悬而未决的科学难题。多功能高分子聚合物在促进蛋白多肽吸收方面具有明显优势。本项目首先以阴离子型聚合物聚丙烯酸和阳离子型聚合物壳聚糖为模板合成一系列结构新颖的预活化巯基化聚合物，并对其结构进行了表征。然后对其促进生物大分子药物吸收的功能和分子机制、抗耐药的功能和机制进行了深入研究。聚合物功能研究结果表明，与传统巯基化聚合物相比，预活化巯基化聚合物在广泛pH 范围内具有更好的稳定性；在离体小肠，Caco-2、MDCK细胞单层上均表现出良好的促渗透效果和逆转P-糖蛋白多药耐药的功能。机制研究结果表明：（1）聚合物打开细胞间紧密连接的分子机制为：巯基化聚合物通过与细胞表面富含半胱氨酸的受体（EGFR，IGFR）相互作用，引发受体的激活，进而引起其下游相关蛋白Src激酶的磷酸化。Src激酶的激活引起紧密连接蛋白Claudin-4的迁移，由细胞间隙向细胞胞浆内分布，从而导致紧密连接的打开。这种机理在一定程度上填补了该领域的空白。（2）其抑制P-糖蛋白外排功能的作用机制为：这种抑制作用与细胞膜流动性的改变有关，与P-gp ATP酶活性的降低有关，而材料对Caco-2细胞内ATP水平以及P-gp蛋白表达水平无显著的影响。这些研究结果为预活化巯基化聚合物在药剂学领域的应用提供了扎实的理论基础，具有重要的学术价值。. 发表SCI论文3篇，中文核心期刊1篇以及硕士学位论文2篇。另外2017年将有硕士学位论文1篇，有一些核心数据还在整理中，尚未发表。