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

Abstract

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 <b>1</b>) or NODAGA (in <b>2</b>) chelators via a 6-aminohexanoic acid linker. The Cu-<b>1</b>/<b>2</b> metalated peptides were synthesized by reacting <b>1</b>/<b>2</b> with CuCl₂ and were characterized by LC-ESI-MS and HR-ESI-MS. Cu-<b>1</b>/<b>2</b> 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-<b>1</b>/<b>2</b> were prepared in quantitative radiochemical yield (by radio-HPLC), and their identities were confirmed by coelution with their Cu-<b>1</b>/<b>2</b> standards via comparative HPLC studies. Lipophilicity was measured in 1-octanol/PBS (pH 7.4), and the negative log <i>D</i>_7.4 values (≤-1) confirmed the anticipated hydrophilic character for [⁶⁴Cu]Cu-<b>1</b>/<b>2</b>. Both tracers demonstrated excellent <i>in vitro</i> 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-<b>1</b>, 3.92 ± 1.03 for [⁶⁴Cu]Cu-<b>2</b>) 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-<b>1</b>/<b>2</b> tracers show promise for further development as diagnostic PET imaging agents of GRPR-expressing tumors.