A High-Affinity ⁶⁴Cu-Labeled Ligand for PET Imaging of Hepsin: Design, Synthesis, and Characterization

Hepsin, a cell surface serine protease, is a potential biomarker for the detection of prostate cancer due to its high expression in prostate cancer but not in normal prostate. This study aimed to develop a radioligand for positron emission tomography (PET) imaging of hepsin. Six leucine-arginine (Leu-Arg) dipeptide derivatives (two diastereomers for each of three ligands) were synthesized and evaluated for their binding affinities and selectivity for hepsin. Based on the binding assay, a ^natCu-1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA)-conjugated ligand (3B) was selected for the development of a PET radioligand. [⁶⁴Cu]3B was synthesized by labeling the DOTA-conjugated compound 11B with [⁶⁴Cu]CuCl₂ at 80 ^°C for 20 min. The radioligand was evaluated for prostate cancer cell binding and PET imaging in a prostate tumor mouse model. The results demonstrated that [⁶⁴Cu]3B exhibited high binding to LNCaP cells, intermediate binding to 22Rv1 cells, and low binding to PC3 cells. PET studies of [⁶⁴Cu]3B in mice, implanted with 22Rv1 and PC3 cells on each flank, revealed that the radioligand uptake was high and persistent in the 22Rv1 tumors over time, whereas it was low in PC3 tumors. The results of this study suggest that [⁶⁴Cu]3B is a promising PET radioligand for hepsin imaging.