Nuclear Medicine Department, Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India.
Chem Biol Drug Des. 2014 Jan;83(1):58-70. doi: 10.1111/cbdd.12166. Epub 2013 Nov 1.
During the past decade, several peptides containing Arg-Gly-Asp sequence have been conjugated with different chelating agents for labeling with various radionuclides for the diagnosis of tumor development. In this study, we report the synthesis of two tetrapeptides (Asp-Gly-Arg-His and Asp-Gly-Arg-Cys) and one hexapeptide [Asp-Gly-Arg-D-Tyr-Lys-His] by changing the amino acid sequence of the Arg-Gly-Asp motif. Peptide synthesis was initiated from aspartic acid. Aspartic acid placed at C-terminal end of the peptide chain can be conjugated with different drug molecules facilitating their transport to the site of action. The peptides were synthesized in excellent yield and labeled using freshly prepared (99m) Tc(CO)3 (H2 O)3 intermediate. A complexation yield of over 97% was achieved under mild conditions even at low ligand concentrations of 10(-2) m. Radiolabeled peptides were characterized by HPLC and were found to be substantially stable in saline, in His solution as well as in rat serum and tissue (kidney, liver) homogenates. Internalization studies using Ehrlich ascites carcinoma cell line showed rapid and significant internalization (30-35% at 30 min of incubation attaining maximum value of about 40-60% after 2-4 h incubation). A good percentage of quick internalization was also observed in αv β3 -receptor-positive B16F10 mouse melanoma cell line (14-16% after 30 min of incubation and 25-30% after 2-4 h incubation). Imaging and biodistribution studies were performed in Swiss albino mice bearing Ehrlich ascites tumor in right thigh. Radiolabeled peptides exhibited fast blood clearance and rapid elimination through the urinary systems. (99m) Tc(CO)3 -tetra-Pep2 exhibited remarkable localization at tumor site (1.15%, 1.17%, and 1.37% ID/g at 2, 4, and 6 h p.i., respectively) which could be due to slow clearance of the radiolabeled peptide from blood in comparison with the other two radiolabeled peptides. However, (99m) Tc(CO)3 -hexa-Pep exhibited the highest tumor to muscle and tumor to blood ratios among the three. The preliminary results with these amino acid-based peptides are encouraging enough to carry out further experiments for targeting tumor.
在过去的十年中，已经有几种含有精氨酸-甘氨酸-天冬氨酸序列的肽与不同的螯合剂结合，用于标记各种放射性核素来诊断肿瘤的发展。在这项研究中，我们报告了两种四肽（天冬氨酸-甘氨酸-精氨酸-组氨酸和天冬氨酸-甘氨酸-精氨酸-半胱氨酸）和一种六肽[天冬氨酸-甘氨酸-精氨酸-D-酪氨酸-赖氨酸-组氨酸]的合成，方法是改变精氨酸-甘氨酸-天冬氨酸基序中的氨基酸序列。肽的合成从天冬氨酸开始。位于肽链 C 末端的天冬氨酸可以与不同的药物分子结合，促进它们向作用部位运输。这些肽以优异的产率合成，并使用新制备的(99m)Tc(CO)3(H2O)3中间体进行标记。即使在配体浓度低至 10(-2)m 的情况下，在温和条件下也能获得超过 97%的络合产率。放射性标记的肽通过 HPLC 进行了表征，并在盐水中、His 溶液中以及在大鼠血清和组织（肾脏、肝脏）匀浆中均表现出相当稳定。使用艾氏腹水癌细胞系进行的内化研究表明，内化速度快且显著（孵育 30 分钟时为 30-35%，孵育 2-4 小时后达到约 40-60%的最大值）。在αvβ3-受体阳性 B16F10 小鼠黑色素瘤细胞系中也观察到了相当大的快速内化百分比（孵育 30 分钟后为 14-16%，孵育 2-4 小时后为 25-30%）。在右大腿患有艾氏腹水瘤的瑞士白化病小鼠中进行了成像和生物分布研究。放射性标记的肽表现出快速的血液清除和通过泌尿系统的快速消除。(99m)Tc(CO)3-四肽 2 在肿瘤部位表现出显著的定位（分别在 2、4 和 6 hpi 时为 1.15%、1.17%和 1.37%ID/g），这可能是由于与另外两种放射性标记的肽相比，放射性标记的肽从血液中的清除速度较慢。然而，(99m)Tc(CO)3-六肽表现出三种中最高的肿瘤与肌肉和肿瘤与血液的比值。这些基于氨基酸的肽的初步结果令人鼓舞，足以进行进一步的肿瘤靶向实验。
