In vitro and in vivo evaluation of ⁶⁴Cu-labeled SarAr-bombesin analogs in gastrin-releasing peptide receptor-expressing prostate cancer

Bombesin is a 14-amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radio-labeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7-14) for radiolabeling with ⁶⁴Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[ 6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin (7-14), radiolabel the conjugate with ⁶⁴Cu, and evaluate in vitro and in vivo. Methods: SarAr was synthesized as previously published and conjugated to bombesin(7-14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7-14) to yield the resulting SarAr-SA-Aoc-bombesin(7- 14) and SarAr-SA-Aoc-GSG-bombesin(7-14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and ¹²⁵I-Tyr⁴-bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with ⁶⁴Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies. Results: The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSGbombesin(7-14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with ⁶⁴Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for ⁶⁴Cu-SarAr-SA-Aoc-bombesin(7-14) and ⁶⁴Cu-SarAr-SA-Aoc- GSG-bombesin(7-14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported ⁶⁴Cu-labeled bombesin analogs. The ⁶⁴Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) had more rapid blood clearance and lower tumor and normal-tissue uptake than ⁶⁴Cu-SarAr-SA-Aoc-bombesin(7-14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for ⁶⁴Cu-SarAr-SA-Aoc- bombesin(7-14) and ⁶⁴Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h). Conclusion: These studies demonstrate that ⁶⁴Cu-SarAr-SA-Aoc-bombesin(7-14) and ⁶⁴Cu-SarAr-SA-Aoc- GSG-bombesin(7-14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.