A Novel Tumor Imaging Agent, Tc-99m and Fluorescence Labeled Gastrin-Releasing Peptide Analogues

Statement of the Problem: A variety of imaging probes have been developed for different molecular targets. The gastrin-releasing peptide (GRP) analogues (GRP, GRP18-27 and GRP21-27) were investigated vigorously as a targeting peptide for tumor, however, the cancer targeting abilities of these GRP analogues had not been compared head-to-head. Direct comparison of characteristics of these analogues will provide a useful information for the design of molecular imaging agent associated with GRP and an insight within the mechanism of tumor targeting using GRP analogues. Approach: In the present study, we developed Tc-99m and fluorescence (carboxytetramethylrhodamine, TAMRA) labeled GRP analogues containing three different peptides (TAMRA-GHEG-ECG-GRP, TAMRA-GHEG-ECG-GRP18-27 and TAMRA-GHEG-ECG-GRP21-27) to target the tumor cells and compared the tumor targeting abilities using in vitro and in vivo experiments. Peptides were synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of peptides with Tc-99m was done using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging study was performed in murine models with PC-3 tumors. The average counts per pixel within the ROIs were measured and target-to-non-target ratios were calculated. Results: After radiolabeling procedures with Tc-99m, Tc-99m labeled peptides were prepared in high yield $(>96\%)$. Tc-99m TAMRA-GHEG-ECG-GRP $(\mathbf{Kd}=\mathbf{7.9}\pm \mathbf{2.7}\ \mathbf{nM})$ showed highest binding affinity for PC-3 tumor cells and Tc-99m TAMRA-GHEG-ECG-GRP18-27 $(\mathbf{Kd}=\mathbf{12.6}\pm \mathbf{3.6}\ \mathbf{nM})$ showed relatively low binding affinity. Confocal microscopy images of PC-3 cells incubated with TAMRA-GHEG-ECG-GRP and TAMRA-GHEG-ECG-GRP21-27 showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-GRP21-27 in tumors. Conclusion: We developed three Tc-99m and TAMRA labeled GRP analogues as a molecular imaging agent for targeting tumor. In vitro studies demonstrated substantial binding affinity and cellular uptake of TAMRA-GHEG-ECG-GRP and TAMRA-GHEG-ECG-GRP21-27. In contrast, in vivo gamma imaging study revealed that only Tc-99m TAMRA-GHEG-ECG-GRP21-27 was significantly accumulated in the tumor tissue. Taken together, the present study suggest that 7-mer peptide, GRP21-27 is the best surrogate as a targeting ligand for tumor imaging.