Cholecystokinin-2 Receptor Targeting with Novel C-terminally Stabilized HYNIC-Minigastrin Analogs Radiolabeled with Technetium-99m

Abstract

The high overexpression of cholecystokinin-2 receptors (CCK2R) in tumors, such as medullary thyroid carcinoma, allows for highly specific diagnostic and therapeutic targeting with radiolabeled peptide probes derived from natural ligands for the receptor. Based on the ideal imaging characteristics, high availability and low cost of technetium-99m (99mTc)-labeled radiopharmaceuticals we have developed two hydrazinonicotinic acid (HYNIC) conjugated minigastrin analogs allowing labeling at high specific activity. The CCK2R targeting peptide conjugates show specific amino acid substitutions in the C-terminal receptor-specific sequence with the aim to increase stability and tumor targeting. The CCK2R affinity and the cell uptake of the new radioligands were analyzed using A431 human epidermoid carcinoma cells stably transfected with human CCK2R and mock transfected cells. Metabolic studies in BALB/c mice revealed a high resistance against enzymatic degradation for both radioligands. Biodistribution studies in tumor-xenografted athymic BALB/c nude mice at 1 h and 4 h p.i. showed that the two 99mTc-labeled compounds showed varying uptake in receptor expressing organs, stomach and pancreas (1.3–10.4% IA/g), as well as kidneys, the main route of excretion (7.8–19.9% IA/g). The tumor uptake in A431-CCK2R xenografts was 24.75 ± 4.38% IA/g for [99mTc]Tc-HYNIC-MGS5 and 42.48 ± 6.99% IA/g for [99mTc]Tc-HYNIC-MGS11 at 4 h p.i., whereas the tumor-to-kidney ratio was comparable (2.6–3.3). On demand availability and potential application for radioguided surgery of a 99mTc-labeled minigastrin analog support the further evaluation of these highly promising new compounds.

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

“…In our recent studies we could introduce new modifications within the C-terminal sequence Trp-Met-Asp-Phe-NH 2 , known to be essential for CCK2R binding [ 27 , 28 , 29 ]. Most favorable properties were found for the new MG analog with the sequence DOTA-DGlu-Ala-Tyr-Gly-Trp-( N -Me)Nle-Asp-1Nal-NH 2 (DOTA-MGS5), in which methionine is replaced by N -methylated norleucine (( N -Me)Nle) and phenylalanine by 1-naphtylalanine (1Nal) [ 28 ].…”

“…With the aim of further improving the in vivo stability of DOTA-MGS5, we have explored additional modification of the peptide sequence. The amino acid proline (Pro) is a promising candidate for amino acid exchange and forms a tertiary amide bond which similarly to N -methylated peptide bonds and triazoles may improve the stability in vivo [ 27 , 30 ]. In this study, a preliminary preclinical characterization of the new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-( N -Me)Nle-Asp-1Nal-NH 2 ( 1 ) was carried out focusing on the enzymatic stability of the 177 Lu-labeled radiopeptide in vivo.…”

Stabilization Strategies for Linear Minigastrin Analogues: Further Improvements via the Inclusion of Proline into the Peptide Sequence

“… 12 , 25 As previously reported by our group, it is possible to hinder the metabolization of this vulnerable part of the peptide by introducing bulky unnatural aromatic amino acids as well as N-methylated amino acids. 26 − 28 Most interestingly, the introduction of such amino acids within the receptor-binding sequence resulted in novel MG analogues, which besides increased resistance against enzymatic degradation, also showed an enhanced receptor interaction, leading to increased cell uptake of the radiolabeled peptides in CCK2R-expressing cell lines. Comprehensive preclinical investigations evaluating the new modifications introduced within this part of the peptide sequence led to very promising results.…”

“…These improvements were independent of the radiometal-chelator approach used for radiolabeling. 27 , 28 Introduction of an additional N-methylated peptide bond between Asp in position 12 and 1-Nal in position 13 further increased the resistance against enzymatic degradation in vivo and the tumor uptake. However, also the kidney uptake was drastically increased, a drawback especially with regard to therapeutic applications.…”

Correction: Klingler, M., et al. Cholecystokinin-2 Receptor Targeting with Novel C-terminally Stabilized HYNIC-Minigastrin Analogs Radiolabeled with Technetium-99m. Pharmaceuticals 2019, 12, 13

“…In our paper [1], an error unfortunately occurred in Figure 1. We therefore would like to provide the readers with the corrected figure and apologize for this inconvenience.…”