Evaluation of fluorine-labeled gastrin-releasing peptide receptor (GRPR) agonists and antagonists by LC/MS

An LC/MS method was used to evaluate 2-fluoropropionyl (FP) and 4-fluorobenzoyl (FB) modified bombsin peptides: GRPR agonist [Aca-QWAVGHLM-NH2] and antagonist [fQWAVGHL-NHEt], and their hydrophilic linker modified counterparts with the attachment of GGGRDN sequence. This study developed strategies to evaluate the in vitro receptor mediated cell uptake and metabolic profile of the various GRPR agonists and antagonists. We identified the metabolites produced by rat hepatocytes and quantitatively analyzed the uptake and internalization of the ligands in PC-3 human prostate cancer cells. The major metabolites of both GRPR agonists and antagonists were the result of peptide bond hydrolysis between WA and AV. The agonists also formed a unique metabolite resulting from hydrolysis of the C-terminal amide. The antagonists showed significantly higher stability against metabolism compared to the agonists in rat hepatocytes. The directly modified agonists (FP-BBN and FB-BBN) had higher internalization with similar cell binding compared to the unmodified agonist (BBN), whereas the hydrophilic linker modified agonists (G-BBN and FG-BBN) had much lower total cell uptake. The labeled antagonists (FP-NBBN, FB-NBBN, G-NBBN and FP-G-NBBN) displayed lower internalization. The optimal imaging agent will depend on the interplay of ligand metabolism, cellular uptake, and internalization in vivo.