基于LAT1的靶向藥物的抗腫瘤活性及其藥代動力學的研究
發(fā)布時間：2018-10-21 18:29
【摘要】：L-型氨基酸轉(zhuǎn)運體1(L-type amino acid transporter 1,LAT1)是一個位于細胞膜上由507個氨基酸組成的跨膜轉(zhuǎn)運蛋白,由重鏈和輕鏈通過二硫鍵連接后折疊環(huán)繞形成的13個跨膜區(qū)構(gòu)成。不僅對大中性L-型氨基酸高效轉(zhuǎn)運功能,還能協(xié)助轉(zhuǎn)運一些與氨基酸具有相似結(jié)構(gòu)的藥物分子。本研究基于LAT1底物結(jié)構(gòu)特點,用不同的氨基酸對DOX進行了結(jié)構(gòu)修飾,并通過高效液相色譜HPLC、質(zhì)譜MS和核磁共振氫譜(NMR)對合成產(chǎn)物進行了結(jié)構(gòu)學驗證,建立了體外篩選模型對合成產(chǎn)物進行了初步的篩選,最終確定了藥物活性分子天冬阿霉素(Asp-DOX)。熒光顯微鏡觀察結(jié)果和HPLC分析結(jié)果顯示,Asp-DOX在腫瘤細胞的吸收量較原形藥(DOX)顯著提高了3倍多。不僅如此,新化合物Asp-DOX的外排率明顯降低,這說明由P-gp蛋白引起的耐藥性也降低。Asp-DOX藥代動力學研究結(jié)果顯示,Asp-DOX血液半衰期t1/2較DOX明顯延長,藥物濃度-時間曲線下面積(AUC)也較原形藥DOX增加了8倍多。本研究還建立了HepG2、HCT116和H1299人移植瘤裸鼠模型,對Asp-DOX組織分布情況進行了研究,結(jié)果顯示,Asp-DOX在HepG2和HCT116腫瘤組織中的含量都分別比原型藥DOX提高了2.2倍和6.1倍多,在正常組織中則正好相反。這表明,Asp-DOX對LAT1過表達的腫瘤細胞及腫瘤組織的靶向性顯著性提高,而在正常組織中尤其在心、肺和腎臟中的分布大大降低。體內(nèi)藥效學研究表明,Asp-DOX對LAT1陽性腫瘤(HepG2)的腫瘤組織的生長抑制作用較原形藥顯著性增強,相對腫瘤生長抑制率高達70%,且無明顯的不良狀況發(fā)生。以上結(jié)果表明,新化合物較原型藥藥代動力學參數(shù)明顯改善,靶向抗腫瘤活性也顯著性提高。LAT1為靶向抗腫瘤藥物和腫瘤診斷試劑的設(shè)計提供了一個有效的靶點。本研進一步驗證了LAT1的轉(zhuǎn)運機制假說:1)LAT1蛋白分子上存在三個作用位點:氨基結(jié)合位點、羧基結(jié)合位點和疏水結(jié)合位點,這三個結(jié)合位點分別于底物分子上的氨基、羧基和疏水側(cè)鏈相結(jié)合;2)底物分子必須呈中性;3)LAT1對底物的結(jié)合和跨膜轉(zhuǎn)運是一種較為嚴格的識別過程,其驅(qū)動力由離子親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.
【學位授予單位】：天津大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R965
，
本文編號：2285979
[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.
【學位授予單位】：天津大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R965
，
本文編號：2285979
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