von Guggenberg E, Behe M, Behr T M, Saurer M, Seppi T, Decristoforo C
Department of Nuclear Medicine, Leopold-Franzens-University of Innsbruck, Austria.
Bioconjug Chem. 2004 Jul-Aug;15(4):864-71. doi: 10.1021/bc0300807.
Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.
胃泌素/CCK - 2受体在多种肿瘤中过度表达，如甲状腺髓样癌（MTC）和小细胞肺癌（SCLC）。最近，[D - Glu1] - 小胃泌素（MG）已用131I、111In和90Y进行放射性标记，并在患者中进行了评估。本研究描述了使用两种不同的标记方法用99mTc对MG进行标记和评估：使用HYNIC作为双功能偶联剂，以及使用（Nα - His）Ac作为99mTc（CO3）标记的三齿配体。使用Tricine和EDDA作为HYNIC - MG的共配体以及[99mTc（OH2）3（CO）3]+作为（Nα - His）Ac - MG的共配体，在高比活度下进行标记。通过反相HPLC分析在PBS、血清、组氨酸和半胱氨酸溶液以及大鼠肝脏和肾脏匀浆中进行稳定性实验。使用CCK - 2受体阳性的AR42J大鼠胰腺肿瘤细胞进行受体结合和内化实验。通过注射99mTc标记的肽，在有或没有共同注射50μg人小胃泌素I（MGh）的情况下，对携带AR42J肿瘤的裸鼠进行生物分布实验。HYNIC - MG和（Nα - His）Ac - MG可以在高比活度（>1 Ci/μmol）下进行放射性标记。对于HYNIC - MG，使用Tricine和EDDA作为共配体可实现高标记产率（>95%）。所有99mTc标记的偶联物的稳定性实验表明，标记物在PBS和血清中以及在组氨酸和半胱氨酸的挑战下具有高稳定性。在肾脏匀浆中孵育导致所有偶联物快速降解，在37℃下60分钟后完整肽<10%，放射性标记的偶联物之间没有显著差异；在肝脏匀浆中观察到降解速率略低。蛋白质结合差异很大，99mTc - EDDA/HYNIC - MG的水平最低。未标记的偶联物在AR42J膜上与[125I - Tyr12] - 胃泌素I的竞争实验表明，所研究的所有偶联物对CCK - 2受体具有高亲和力。所有放射性标记的偶联物的内化行为都非常迅速，顺序为99mTc - （Nα - His）Ac - MG>99mTc - EDDA/HYNIC - MG>99mTc - Tricine/HYNIC - MG。在荷瘤裸鼠中，观察到99mTc - EDDA/HYNIC - MG的肿瘤摄取最高（8.1%ID/g），其次是99mTc - Tricine/HYNIC - MG（2.2%ID/g）和99mTc - （Nα - His）Ac - MG（1.2%ID/g），这与肾脏摄取相关（分别为101.0%ID/g、53.8%ID/g、1.8%ID/g）。在这一系列化合物中，99mTc - EDDA/HYNIC - MG除肾脏外具有非常高的肿瘤/器官比，似乎是靶向CCK - 2受体最有前景的药物。尽管在受体结合、内化和体外稳定性方面具有良好的特性，但99mTc - （Nα - His）Ac - MG在体内显示出低肿瘤摄取。
