Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177

Abstract

Targeting of cholecystokinin-2 receptor (CCK2R) expressing tumors using radiolabeled minigastrin (MG) analogs is hampered by rapid digestion of the linear peptide in vivo. In this study, a new MG analog stabilized against enzymatic degradation was investigated in preclinical studies to characterize the metabolites formed in vivo. The new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 comprising site-specific amino acid substitutions in position 2, 6 and 8 and different possible metabolites thereof were synthesized. The receptor interaction of the peptide and selected metabolites was evaluated in a CCK2R-expressing cell line. The enzymatic stability of the 177Lu-labeled peptide analog was evaluated in vitro in different media as well as in BALB/c mice up to 1 h after injection and the metabolites were identified based on radio-HPLC analysis. The new radiopeptide showed a highly increased stability in vivo with >56% intact radiopeptide in the blood of BALB/c mice 1 h after injection. High CCK2R affinity and cell uptake was confirmed only for the intact peptide, whereas enzymatic cleavage within the receptor specific C-terminal amino acid sequence resulted in complete loss of affinity and cell uptake. A favorable biodistribution profile was observed in BALB/c mice with low background activity, preferential renal excretion and prolonged uptake in CCK2R-expressing tissues. The novel stabilized MG analog shows high potential for diagnostic and therapeutic use. The radiometabolites characterized give new insights into the enzymatic degradation in vivo.

Investigation of Fluorine-18 Labelled Peptides for Binding to Cholecystokinin-2 Receptors with High Affinity

“…Therefore, truncated analogues of CP04 1 have also been investigated, including the peptide MG11 2 that lacks all N-terminal glutamic acid residues. This shortened peptide displayed significantly reduced renal uptake and retention, spurring additional refinements of the core MG11 2 structure to further reduce kidney uptake and enhance tumor targeting (Erba et al 2018;Grob et al 2020;Hörmann et al 2020;Klingler et al 2019;Maximilian et al 2020;Uprimny et al 2020).…”

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting

“…Among three different peptide derivatives with proline substitution studied, DOTA-MGS8 radiolabeled with indium-111 and lutetium-177 showed the most promising advantages in terms of enhanced tumor uptake with concomitant low kidney uptake [ 16 ]. A stabilizing effect against enzymatic degradation in vivo could be observed, as demonstrated by the absence of the metabolites with cleavage at position DGlu-Pro, Pro-Tyr and Tyr-Gly in the blood and urine of mice intravenously injected with [ 177 Lu]Lu-DOTA-MGS8 [ 29 ]. Thus, a considerable reduction of the enzymatic degradation compared to [ 111 In]In-DOTA-MG11 could be achieved [ 11 , 24 ].…”

A New Turn in Peptide-Based Imaging Agents: Foldamers Afford Improved Theranostics Targeting Cholecystokinin-2 Receptor-Positive Cancer

“…44 A common site of initial metabolic breakdown in CP04based peptides has been identified at Asp 12 −Phe 13 . 53,56 Journal of Medicinal Chemistry Therefore, we prepared one last peptide in this study with Nmethylated Nal 13 to investigate if that has any influence on the in vivo characteristics. N-methylation of Phe 13 has been shown to reduce binding affinity toward CCK-2R.…”