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.

Keywords: cholecystokinin-2 receptor; lutetium-177; minigastrin; molecular imaging; targeted radiotherapy.

Figure 1

Amino acid sequence and chemical structure of 1.

Figure 2

Radio-HPLC chromatograms of [¹⁷⁷Lu]Lu-1 and its ¹⁷⁷Lu-labeled metabolites M1–M8 (* indicating radiolabeled with lutetium-177).

Figure 3

Stability of (a) [¹⁷⁷Lu]Lu-1 after incubation in human serum as well as in rat liver and rat kidney homogenates, and of (b) selected radiolabeled metabolites M1–M4 in human serum, as analyzed up to 24 h after incubation.

Figure 4

Cell uptake of (a) [¹⁷⁷Lu]Lu-1 in A431-CCK2R and A431-mock cells and (b) ¹⁷⁷Lu-labeled M1–M4 in A431-CCK2R cells, after 1 h and 2 h incubation.

Figure 5

Competitive binding curves against [¹²⁵I][3-iodo-Tyr¹²,Leu¹⁵]gastrin-I for (a) non-labeled 1 in comparison with pentagastrin, as well as (b) non-labeled M1–M4.

Figure 6

Intact [¹⁷⁷Lu]Lu-1 detectable in blood and urine, as well as liver and kidney homogenates obtained from BALB/c mice, as analyzed up to 60 min p.i.

Figure 7

Uptake values in selected tissues as obtained from metabolic biodistribution studies with [¹⁷⁷Lu]Lu-1 in BALB/c mice at 10, 30 and 60 min p.i.: (a) background activity in blood and muscle, (b) excretory organs kidney and liver as well as (c) CCK2R-expressing pancreas and stomach. Values are expressed as % IA/g.

Figure 8

Radiochromatograms obtained from (a) blood, (c) liver, (d) kidneys and (b) urine of BALB/c mice injected with [¹⁷⁷Lu]Lu-1 for the time point of 60 min p.i.; radiochromatograms of ¹⁷⁷Lu-labeled peptides 1 and M1–M8 are shown for comparison (* indicating radiolabeled with lutetium-177).