基于LAT1的靶向药物的抗肿瘤活性及其药代动力学的研究

发布时间：2018-10-21 18:29

【摘要】：L-型氨基酸转运体1(L-type amino acid transporter 1,LAT1)是一个位于细胞膜上由507个氨基酸组成的跨膜转运蛋白,由重链和轻链通过二硫键连接后折叠环绕形成的13个跨膜区构成。不仅对大中性L-型氨基酸高效转运功能,还能协助转运一些与氨基酸具有相似结构的药物分子。本研究基于LAT1底物结构特点,用不同的氨基酸对DOX进行了结构修饰,并通过高效液相色谱HPLC、质谱MS和核磁共振氢谱(NMR)对合成产物进行了结构学验证,建立了体外筛选模型对合成产物进行了初步的筛选,最终确定了药物活性分子天冬阿霉素(Asp-DOX)。荧光显微镜观察结果和HPLC分析结果显示,Asp-DOX在肿瘤细胞的吸收量较原形药(DOX)显著提高了3倍多。不仅如此,新化合物Asp-DOX的外排率明显降低,这说明由P-gp蛋白引起的耐药性也降低。Asp-DOX药代动力学研究结果显示,Asp-DOX血液半衰期t1/2较DOX明显延长,药物浓度-时间曲线下面积(AUC)也较原形药DOX增加了8倍多。本研究还建立了HepG2、HCT116和H1299人移植瘤裸鼠模型,对Asp-DOX组织分布情况进行了研究,结果显示,Asp-DOX在HepG2和HCT116肿瘤组织中的含量都分别比原型药DOX提高了2.2倍和6.1倍多,在正常组织中则正好相反。这表明,Asp-DOX对LAT1过表达的肿瘤细胞及肿瘤组织的靶向性显著性提高,而在正常组织中尤其在心、肺和肾脏中的分布大大降低。体内药效学研究表明,Asp-DOX对LAT1阳性肿瘤(HepG2)的肿瘤组织的生长抑制作用较原形药显著性增强,相对肿瘤生长抑制率高达70%,且无明显的不良状况发生。以上结果表明,新化合物较原型药药代动力学参数明显改善,靶向抗肿瘤活性也显著性提高。LAT1为靶向抗肿瘤药物和肿瘤诊断试剂的设计提供了一个有效的靶点。本研进一步验证了LAT1的转运机制假说:1)LAT1蛋白分子上存在三个作用位点:氨基结合位点、羧基结合位点和疏水结合位点,这三个结合位点分别于底物分子上的氨基、羧基和疏水侧链相结合;2)底物分子必须呈中性;3)LAT1对底物的结合和跨膜转运是一种较为严格的识别过程,其驱动力由离子亲和力和疏水作用力提供。
[Abstract]:L- type amino acid transporter 1 (L-type amino acid transporter 1 + LAT1) is a 507 amino acid transmembrane transporter located on the cell membrane. It consists of 13 transmembrane regions formed by heavy chain and light chain linked by disulfide bond and folded around the membrane. It can not only transport large neutral L- amino acids, but also facilitate the transport of some drug molecules with similar structure to amino acids. In this study, DOX was modified with different amino acids based on the structural characteristics of LAT1 substrates, and the structure of the synthesized products was confirmed by HPLC, / MS and nuclear magnetic resonance (NMR) (NMR). An in vitro screening model was established for the screening of the synthetic products, and the drug active molecule adriamycin (Asp-DOX) was determined. The results of fluorescence microscope and HPLC analysis showed that the absorption of Asp-DOX in tumor cells was more than three times higher than that of (DOX). Moreover, the efflux rate of the new compound Asp-DOX was significantly decreased, which indicated that the drug resistance induced by P-gp protein was also decreased. The results of Asp-DOX pharmacokinetic study showed that the half life t 1 / 2 of Asp-DOX blood was significantly longer than that of DOX. The area (AUC) under the drug concentration-time curve also increased by more than 8 times compared with the original drug DOX. HepG2,HCT116 and H1299 human xenograft tumor models were established in this study. The distribution of Asp-DOX tissue was studied. The results showed that the contents of Asp-DOX in HepG2 and HCT116 tumor tissues were increased by 2.2 and 6.1 times, respectively, compared with the original drug DOX. In normal tissues, the opposite is true. The results showed that Asp-DOX significantly increased the targeting of LAT1 overexpressed tumor cells and tumor tissues, but decreased the distribution in normal tissues, especially in heart, lung and kidney. In vivo pharmacodynamics study showed that the inhibitory effect of Asp-DOX on the growth of LAT1 positive tumor (HepG2) was significantly higher than that of the original drug, and the inhibition rate of tumor growth was as high as 70%, and there was no obvious adverse situation. The results showed that the pharmacokinetic parameters of the new compounds were significantly improved than those of the original ones, and the targeted antitumor activity was also significantly improved. LAT1 provided an effective target for the design of targeted antitumor drugs and tumor diagnostic reagents. This study further verifies the hypothesis of LAT1 transport mechanism: 1) there are three action sites on LAT1 protein: amino binding site, carboxyl binding site and hydrophobic binding site. The combination of carboxyl group and hydrophobic side chain; (2) the substrate molecule must be neutral; (3) the binding and transmembrane transport of substrate by LAT1 is a strict recognition process, and the driving force is provided by ionic affinity and hydrophobic force.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R965
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本文编号：2285979