Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.
Bioconjug Chem. 2010 May 19;21(5):911-20. doi: 10.1021/bc9004602.
To increase the therapeutic index of chemotherapeutic drugs, prodrugs have been investigated as anticancer agents, as they may present fewer cytotoxic side effects than conventional cytotoxic drugs, while therapeutic efficacy is maintained or even increased. Extracellular beta-glucuronidase (beta-GUS) in the tumors has been investigated as a target enzyme for prodrug therapy, as it can convert nontoxic prodrugs into cytostatic drugs. To optimize beta-GUS-based prodrug therapies, PET imaging could be a useful tool by providing information regarding the localization and quantification of beta-GUS. Here, we describe our first PET tracer for extracellular beta-GUS, [(18)F]-FEAnGA, which consists of a 2-[(18)F]fluoroethylamine ([(18)F]-FEA) group bound to a glucuronic acid via a self-immolative nitrophenyl spacer. [(18)F]-FEAnGA was synthesized by alkylation of its imidazole carbamate precursor with [(18)F]-FEA, followed by deprotection of the sugar moiety with NaOH in 10-20% overall radiochemical yield. [(18)F]-FEAnGA is about 10-fold more hydrophilic than the cleavage product [(18)F]-FEA, and it is stable in PBS and rat plasma for at least 3 h. In the presence of either Escherichia coli beta-GUS or bovine liver beta-GUS, in vitro cleavage of [(18)F]-FEAnGA with complete release of [(18)F]-FEA was observed within 30 min. C6 glioma cells incubated with the tracer and Escherichia coli beta-GUS or bovine liver beta-GUS showed a 4- and 1.5-fold higher uptake of radioactivity, respectively, as compared to control C6 cells without beta-GUS. Incubation of CT26 murine colon adenocarcinoma cells or the genetically engineered CT26mbetaGUS cells, which expressed membrane-anchored GUS on the outer cell membrane, with the tracer, resulted in a 3-fold higher uptake into GUS-expressing cells as compared to control cells. In a preliminary microPET study in mice bearing both CT26 and CT26mbetaGUS tumors, [(18)F]-FEAnGA exhibited a 2-fold higher retention of radioactivity in the tumor expressing beta-GUS than in the control tumor. [(18)F]-FEA did not show any difference in tracer uptake between tumors. These results suggest that [(18)F]-FEAnGA may be a suitable PET tracer for evaluation of beta-GUS activity, since it is specifically cleaved by beta-GUS and the released [(18)F]-FEA remains attached to targeted cells.
为了提高化疗药物的治疗指数，人们研究了前药作为抗癌药物，因为它们可能比传统的细胞毒性药物具有更少的细胞毒性副作用，同时保持或甚至增加治疗效果。肿瘤中的细胞外β-葡糖苷酸酶（β-GUS）已被研究作为前药治疗的靶酶，因为它可以将无毒的前药转化为细胞抑制药物。为了优化基于β-GUS 的前药治疗，正电子发射断层扫描（PET）成像可能是一种有用的工具，可提供有关β-GUS 定位和定量的信息。在这里，我们描述了我们的第一个用于细胞外β-GUS 的 PET 示踪剂，[(18)F]-FEAnGA，它由通过自毁硝苯基间隔基连接到葡萄糖醛酸的 2-[(18)F]氟乙基胺（[(18)F]-FEA）基团组成。[(18)F]-FEAnGA 通过用 [(18)F]-FEA 烷基化其咪唑碳酸酯前体，然后用 NaOH 在 10-20%的总放射化学产率下脱保护糖部分来合成。[(18)F]-FEAnGA 比裂解产物 [(18)F]-FEA 亲水性高约 10 倍，并且在 PBS 和大鼠血浆中至少稳定 3 小时。在存在大肠杆菌β-GUS 或牛肝β-GUS 的情况下，观察到在 30 分钟内完全释放 [(18)F]-FEA 对 [(18)F]-FEAnGA 的体外裂解。与没有β-GUS 的对照 C6 细胞相比，用示踪剂孵育的 C6 神经胶质瘤细胞和大肠杆菌β-GUS 或牛肝β-GUS 分别显示放射性摄取增加了 4 倍和 1.5 倍。用示踪剂孵育 CT26 小鼠结肠腺癌细胞或表达在细胞膜外的膜锚定 GUS 的基因工程 CT26mbetaGUS 细胞导致 GUS 表达细胞的摄取增加了 3 倍，而对照细胞则没有。在携带 CT26 和 CT26mbetaGUS 肿瘤的小鼠的初步 microPET 研究中，[(18)F]-FEAnGA 在表达β-GUS 的肿瘤中比在对照肿瘤中保留了 2 倍的放射性。[(18)F]-FEA 在肿瘤摄取示踪剂方面没有显示出任何差异。这些结果表明，[(18)F]-FEAnGA 可能是评估β-GUS 活性的合适的 PET 示踪剂，因为它被β-GUS 特异性切割，释放的[(18)F]-FEA 仍附着在靶向细胞上。
