Preclinical Evaluation of Novel ⁶⁴Cu-Labeled Gastrin-Releasing Peptide Receptor Bioconjugates for PET Imaging of Prostate Cancer

We report herein the preclinical evaluation of new [⁶⁴Cu]­Cu-gastrin-releasing peptide receptor (GRPR)-targeting tracers, employing the potent peptide antagonist _DPhe-Gln-Trp-Ala-VaI-Gly-His-Sta-Leu-NH₂ conjugated to NOTA (in 1) or NODAGA (in 2) chelators via a 6-aminohexanoic acid linker. The Cu-1/2 metalated peptides were synthesized by reacting 1/2 with CuCl₂ and were characterized by LC-ESI-MS and HR-ESI-MS. Cu-1/2 exhibited high GRPR-binding affinities with IC₅₀ values <3 nM, as measured in a competition assay using the GRPR-expressing human PC-3 prostate cancer cell line and [¹²⁵I]­I-Tyr⁴-BBN as the competing ligand. Tracers [⁶⁴Cu]­Cu-1/2 were prepared in quantitative radiochemical yield (by radio-HPLC), and their identities were confirmed by coelution with their Cu-1/2 standards via comparative HPLC studies. Lipophilicity was measured in 1-octanol/PBS (pH 7.4), and the negative log D_7.4 values (≤−1) confirmed the anticipated hydrophilic character for [⁶⁴Cu]­Cu-1/2. Both tracers demonstrated excellent in vitro stability, with ≥98% remaining intact through 24 h at physiological conditions (PBS, pH 7.4, 37 °C). Biodistribution in PC-3 tumor-bearing mice demonstrated good tumor uptake (%ID/g at 4 h: 4.34 ± 0.71 for [⁶⁴Cu]­Cu-1, 3.92 ± 1.03 for [⁶⁴Cu]­Cu-2) and rapid renal clearance (≥87% ID at 4 h). Tumor uptake was receptor-mediated, as verified by parallel GRPR-blocking studies. Small-animal PET/CT imaging studies validated the biodistribution data. These preclinical data support that the [⁶⁴Cu]­Cu-1/2 tracers show promise for further development as diagnostic PET imaging agents of GRPR-expressing tumors.