基于PEPT1和氨基肽酶N的3-溴丙酮酸胰腺癌靶向前药的研究

Cancer was the first threat to the people in the world. Though the chemotherapy has benefited many cancer patients, it brought a lot of side effects to people. The membrane transporters and some aminopeptidases expressed in the tumor tissue have become promising targets for the drug delivery. In the present study, some peptidomimetic prodrugs of 3-Bromopyruvate (3-BrPA) targeted to peptide transporter 1 (PEPT1) and aminopeptidase N (APN) highly expressed in the pancreatic cancer were synthesized by attaching amino acid or dipeptide to the carboxyl group. The prodrugs were evaluated by the affinity to PEPT1 and the hydrolysis rate by APN, and the transport by PEPT1 and the activation by APN in pancreatic cancer cell was also studied. To help the rational design of successful prodrugs, the quantitative structure-affinity/hydrolysis rate relationships were constructed by analyzing the physiochemical and structural parameter of the prodrug. The pharmacokinetics, the tissue distribution and the pharmacodynamics of the candidate prodrug was studied in rat to delineate the hydrolysis, and the target efficiency. The preliminary result showed the present study was feasible.

癌症已经成为人类健康的最大威胁，目前临床上使用的很多化疗药物，虽然挽救了很多患者的生命，但是也带来了很多副作用。肿瘤细胞上高度表达的膜转运蛋白和氨基肽酶，利用它们为靶点来提高药物的肿瘤靶向是非常有前途的。本课题以高度表达在胰腺癌细胞上的寡肽转运蛋白（PEPT1）和氨基肽酶N(APN)为靶点,选择3-溴丙酮酸（3-BrPA）为模型药物，利用计算机辅助药物设计，在3-BrPA的羧基位引入氨基酸或者二肽，从与PEPT1亲和性和APN激活前药的速率两方面对3-BrPA拟肽前药进行筛选;在胰腺癌细胞上考察前药的转运和激活与药效的关系；结合药物理化和结构参数，建立定量结构-亲和性/降解速率关系，指导前药的合理设计。选择适宜的前药在小鼠体内进行药动学、组织分布和药效学研究，考察体内的活化、分布行为，并筛选出最佳候选前药。前期结果表明本项目可行性良好。

以胰腺癌细胞上特异表达的的寡肽转运蛋白1（Oligopeptide transporter 1, PEPT1）和氨基肽酶N (Aminopeptidase N, APN)为靶点，以3-溴丙酮酸（3-Bromopyruvate, 3-BrPA）为模型药物，对3-BrPA的羧基进行结构改造形成拟肽类衍生物，以提高母药在肿瘤中的分布并实现前药在肿瘤细胞内的快速激活，以达到3-BrPA的靶向治疗效果。. 首先合成8个3-BrPA前药。通过质谱、高效液相、核磁氢谱和碳谱分析，与目标化合物的分子量和结构一致，纯度均在95%以上。.利用Caco-2细胞研究表明，8个3-BrPA前药的膜渗透率比3-BrPA都有不同程度的提高；竞争性抑制实验表明，8个3-BrPA前药都是PEPT1的底物，可以被PEPT1特异性识别并主动转运进至细胞内部。. 3-BrPA前药在PEPT1表达阳性肿瘤细胞上（PEPT1+，AsPC-1）抑制活性明显高于PEPT1阴性肿瘤细胞（PEPT1-，SW480、Hela、A549）， 因此前药对高表达PEPT1的AsPC-1细胞表现出良好的细胞毒选择性和PEPT1靶向性；利用流式细胞仪开展的前药诱导凋亡实验表明，3-BrPA前药对PEPT1+/PEPT1-不同肿瘤细胞表现出诱导凋亡的选择性，进一步验证了前药具有很好的PEPT1靶向性。.稳定性研究表明，前药在血浆中能够相对稳定的存在，为前药以完整形式到达肿瘤细胞提供了可能性；3-BrPA前药能够被APN特异性水解，表明前药在吸收进入肿瘤细胞后能被激活成母药。. 建立了衍生化法测定生物样本中3-BrPA浓度的HPLC-MS/MS法。在荷AsPC-1 细胞的裸鼠模型上，药动学和组织分布研究表明，候选前药3-BrPA-Leu-Val-OH给药后，3-BrPA在肿瘤中的浓度显著高于母药给药后。在荷AsPC-1 细胞的裸鼠模型上，前药的抑瘤率也显著高于母药 (67.4%vs 43.6%)。