【作者】 章丽辉；

【导师】 徐宇虹；

【作者基本信息】 上海交通大学 ， 药剂学， 2012， 博士

【摘要】 口服给药方便、经济，病患的依从性好，一直是最理想的给药途径，但口服生物利用度不高往往是造成许多药物无法口服给药的重要原因。在提高口服生物利用度的众多策略中，靶向转运体，尤其是靶向寡肽转运蛋白（PEPT1）的载体前药研究，一直是国内外近十年来的研究热点。作为膜蛋白的PEPT1，3D晶体结构至今未破解，基于靶标PEPT1结构的理性化设计难以实现，因此，目前研究思路一般集中在母体化合物的羟基位引入氨基酸修饰，形成氨基酸酯而做的PEPT1底物改造，同一母体化合物的二肽修饰效果优于单个氨基酸修饰。一般认为，肽键、羧基末端、氨基末端等是修饰后前药化合物被PEPT1识别和转运的必要结构特征。而在母体化合物的非羟基位修饰，却罕见文献报道。然而在后续研究中证实，除氨基末端以外，肽键和羧基末端并非PEPT1识别的必要结构，因此，PEPT1的底物模型特征还难有定论。我们通过对文献17个头孢菌素类化合物在Caco-2细胞单层膜的透过系数的数据分析，认为末端氨基也不是PEPT1底物识别和转运所必不可少的特征结构。因此，基于PEPT1底物的可能设计趋向以及我们对底物特征结构的分析和推测，在本论文研究中，我们尝试在母体化合物齐多夫定的非羟基位引入二肽修饰，同时舍弃氨基末端，设计一个具有新型结构特征的小型化合物库，并通过PEPT1亲和性筛选研究和体内外转运和吸收研究，来证实PEPT1对该结构特征的化合物的识别和转运。本论文结合点击化学（Click Chemistry）与固相合成技术，建立简便、高效的合成方法，完成了包含61个化合物库的合成，并对全库化合物进行了收率、HPLC、MS以及1H NMR的表征、30%化合物的高分辨质谱表征，以及15%化合物的全谱表征，建立了一个具有新型结构的二肽修饰化合物库。并且，通过寡肽转运蛋白PEPT1已知底物头孢氨苄的竞争性抑制实验，完成了化合物库的PEPT1亲和性研究，部分二肽修饰化合物对头孢氨苄的细胞摄取的抑制率达到80以上；进一步地，通过代表性化合物在PEPT1高表达的Hela细胞模型上的体外细胞摄取实验，以及在体大鼠肠灌流实验研究，证实PEPT1参与并介导了该类化合物的细胞摄取和小肠转运过程。本论文研究的主要结果：发现了多个对PEPT1具有很高亲和性的二肽序列，并未见文献报道；首次证实具有该新型结构的化合物能够被PEPT1识别和转运；通过本论文筛选获得的高亲和性二肽序列修饰，能够显著提高其母体化合物的小肠吸收。因此，本论文的研究结果对扩大靶向PEPT1载体前药的母体化合物的选择范围、二肽修饰的位点和键合方式具有重要意义，从而进一步拓展PEPT1在靶向载体前药策略中的应用。更多还原

【Abstract】 Oral administration is best accepted for its safty, convenience andless cost as well. But poorly oral bioavaibility is a critical obstacle in oralstrategies. Great efforts were involved in improving oral bioavaibility,among which, target carrier prodrug, especially targeting to PEPT1, was ahotspot in recent years.As a membrane protein, PEPT1is very hard to be purified,crystallized and homology modeling also. Since that, investigation onsubstrates structure based on in vitro or in vivo models was still the mostimportant part in carrier prodrug strategy targeted to PEPT1. Researchworks were mainly focusing on designing amino acid ester prodrugs ofparent compound via hydroxyl group, and dipeptide mono ester prodrugswere reported to have higher affinity for PEPT1than its amino acid monoester prodrug. But non hydroxyl group modification was hardly studied. Additionally, including peptide bond, α amino group and carboxyl groupwere considered as necessary structures for PEPT1recognition andtransport. But in the latter studies, except for α amino group, peptidebond and carboxyl group were proved to be not necessary. Therefore,substrate specificity of PEPT1is not fully understood.On analyzing the Peffof17cephalosporins on Caco-2monolayer, wefound that α amino group could be unnecessary for PEPT1recognitionand transport yet. According to the possible new trends in prodrug designtargeted to PEPT1and the hypothesis of α amino group to be unessential,in this study, a dipeptide modifided library was designed and synthesizedbased on click chemistry and solid phase peptide synthesis. Modificationon non hydroxyl group and non α amino group were two key structuralfeatures of the library and which was well characterized. Isolated purity,MS and~1H NMR were employed into the characterization of the wholelibrary, and30%compounds were confirmed with HR MS, additionally,15%compounds were fully characterized.Competitive inhibition assay was designed to evaluate the affinity toPEPT1of the library operated on transfected Hela cell model. SomeAZT-dipeptide conjugates can compete off over80%cell uptake ofcephalexin. In the further, cell uptake in vitro was conducted on Hela cellmodel and single-pass intestinal perfusion in vivo was operated on ratsrespectively. In this study, we found some dipeptide sequences with very highaffinity to PEPT1which were not reported before and could be used inprodrug strategy. Furthermore, results in vitro and in vivo affirmed thatPEPT1was involved in the uptake and transport of compound candidateswith two key structures, which was also not reported before. Moreover,our results also showed that the intestinal absorption of compoundcandidates with our selected dipeptide sequences was improvedsignificantly compared with parent structure.Findings in this study could enlarge parent drug selection andsignificantly expand the application of PEPT1in target carrier prodrugstrategy.更多还原