Molecular imaging investigations of a 67Ga/64Cu labeled bivalent ligand, [RGD-Glu-(DO3A)-6-Ahx-RM2], targeting GRPR/α v β 3 biomarkers: a comparative study

Abstract In the present study, we report the metallation, characterization, in vitro and in vivo studies comparing 67 Ga/ 64 Cu-radiolabeled bivalent peptide ligands as targeting probes with the capability of targeting the α v β 3 integrin or the gastrin releasing peptide receptor (GRPR) for preclinical and clinical use in single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of prostate tumors. The ligand precursor, [RGD-Glu-6-Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 ), was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA), purified by reversed-phase high-performance liquid chromatography (RP-HPLC), characterized via electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with 67 Ga or 64 Cu. The in vitro investigations of the binding affinities of the natural-metallated ligands for the GRPR or the α v β 3 integrin were conducted via competitive displacement binding assays in human prostate PC-3 and glioblastoma U87-MG cell lines. Following stability investigations via RP-HPLC, the in vivo evaluations of the Ga 67 /Cu 64 -radiolabeled ligands were performed in CF-1 mice and SCID mice bearing PC-3 tumors. The in vitro studies of the natural-metallated ligands showed high binding affinities for the GRPR (7.78 ± 2.42, 8.64 ± 2.16 nM; Ga, Cu respectively) and moderate binding affinity for the α v β 3 integrin receptor (307 ± 40.0, 308 ± 42.6 nM; Ga, Cu respectively). In vivo biodistribution studies displayed high tumor uptake (7.44 ± 1.09, 10.85 ±4.02% ID/g at 1 h post-intravenous injection; 67 Ga, 64 Cu respectively) and prolonged tumor retention (4.89 ± 1.11, 4.09 ±0.96% ID/g at 24 h post-intravenous injection; 67 Ga, 64 Cu respectively) in PC-3 tumor-bearing mice. Micro-single photon emission computed tomography (microSPECT) and micro-positron emission computed tomography (microPET) molecular imaging studies produced high-quality, high-contrast images in PC-3 tumor-bearing mice at 18 h post-intravenous injection. Both radiolabeled ligands show satisfactory tumor uptake and retention in PC-3 tumor-bearing mice. However, [RGD-Glu-( 67 Ga-DO3A)-6-Ahx-RM2] demonstrates superior pharmacokinetic profiles to [RGD-Glu-( 64 Cu-DO3A)-6-Ahx-RM2], presumably due to more favorable in vivo stability.