Abstract
1194
Objectives The presence of the gastrin-releasing peptide receptor (GRPR) has been documented in many human cancers including prostate cancer (Pca), breast cancer and lung cancer. Bombesin (BBN) peptide, an amphibian homologue of mammalian GRP, has demonstrated the ability to bind with high affinity and specificity to GRPR. Although a variety of BBN peptides and their analogs, including both agonists and antagonists, have been widely investigated as GRPR targeted imaging probes for cancer imaging in animal models and patients, a PET probe with better performance is still highly desired for clinical applications. In this work, we aim to develop a GRPR targeted PET probe with high clinical translation ability. A novel BBN analogs which contains an 8-amino octanoic acid spacer (named as ZY1) was thus designed and conjugated with the metal chelator NODAGA. The resulting NODAGA-ZY1 was radiolabeled with 68Ga and evaluated for PET imaging of PCa.
Methods Synthesis of the ZY1 was done using standard Fmoc solid-phase peptide chemistry, and the NODAGA conjugation was performed using standard coupling reaction. The NODAGA-ZY1 was radiolabeled with 68Ga in 0.1N sodium acetate (pH 5.5) at 55 oC within 15 min. For in-vitro characterization, receptor binding assay was performed to measure the binding affinity of the peptide using 125I-Tyr4-BBN as a radioligand. The competitive uptake and blocking studies in PC-3 cells and serum stability were further accomplished. To characterize the tumor targeting properties, in vivo static PET/CT-imaging at multiple time points (0.5, 1, and 2 h) and ex-vivo biodistribution to specific organs collected after sacrifice at 2 h post-injection were performed for 68Ga-NODAGA-ZY1 and a comparison probe 68Ga-NODAGA-RM1 in PC-3 tumor-bearing nude mice (n=4 per group). Blocking study with large excess of BBN peptides was also accomplished.
Results 68Ga-labeling gave high yield (>98 %) with high purity (>98 %) determined by HPLC. The binding affinity of NODAGA-ZY1 was measured to be 1.30±0.35 nM. The radiolabeled probe was stable after 2 hour serum incubation and showed specific uptake in PC-3 cells. Static PET/CT-imaging demonstrated that tumor was clearly imaged with high tumor to background contrast at 0.5, 1 and 2 h post-injection of 68Ga-NODAGA-ZY1. Quantification analysis of PET images revealed that tumor uptake of the probe was 4.64±0.40%, 5.30±0.41 and 5.80±0.42 %ID/g at 0.5, 1 and 2 h post-injection of the probe, respectively. In comparison, for contol group injected with 68Ga-NODAGA-RM1, much lower tumor PET signals were observed at all-time points. The tumor to background ratio (T/N) ratios for both probes were significantly higher than those of the blocking groups. Importantly, the T/N was 17.66±2.47 at 2 h time point for 68Ga-NODAGA-ZY1 , which was nearly two times of the control group with the T/N value of 9.42±1.39. Ex-vivo biodistribution performed at 2 h post-injection also verified the relative low accumulation in the liver (4.21±0.42 %ID/g) and kidney (3.41±0.46 %ID/g), which suggested that the clearance routes of 68Ga-NODAGA-ZY1 were through hepatobiliary and renal systems. Furthermore, much lower tumor uptake of 68Ga-NODAGA-ZY1 was obtained (0.77±0.08 %ID/g at 2 h) in the presence of the blocking peptide, indicating the specific targeting properties of this probe.
Conclusions A novel GRPR targeted 68Ga-NODAGA-ZY1 has been facile prepared. In vitro assay and in vivo studies highlight that 68Ga-NODAGA-ZY1 exhibits high stability, binding affinity, specificity, and excellent in vivo tumor imaging properties. It represents a highly promising probe for the PET imaging of PCa for clinical applications.