Site-specific stabilization of minigastrin analogs against enzymatic degradation for enhanced cholecystokinin-2 receptor targeting

Abstract

Minigastrin (MG) analogs show high affinity to the cholecystokinin-2 receptor (CCK2R) and have therefore been intensively studied to find a suitable analog for imaging and treatment of CCK2R-expressing tumors. The clinical translation of the radioligands developed thus far has been hampered by high kidney uptake or low enzymatic stability. In this study, we aimed to develop new MG analogs with improved targeting properties stabilized against degradation through site-specific amino acid modifications.Method: Based on the lead structure of a truncated MG analog, four new MG derivatives with substitutions in the C-terminal part of the peptide (Trp-Met-Asp-Phe-NH2) were synthesized and derivatized with DOTA at the N-terminus for radiolabeling with trivalent radiometals. The in vitro properties of the new analogs were characterized by analyzing the lipophilicity, the protein binding, and the stability of the Indium-111 (111In)-labeled analogs in different media. Two different cell lines, AR42J cells physiologically expressing the rat CCK2R and A431 cells transfected with human CCK2R (A431-CCK2R), were used to study the receptor affinity and cell uptake. For the two most promising MG analogs, metabolic studies in normal BALB/c mice were carried out as well as biodistribution and imaging studies in tumor xenografted athymic BALB/c nude mice.Results: Two out of four synthesized peptide analogs (DOTA-MGS1 and DOTA-MGS4) showed retained receptor affinity and cell uptake when radiolabeled with 111In. These two peptide analogs, however, showed a different stability against enzymatic degradation in vitro and in vivo. When injected to normal BALB/c mice, for 111In-DOTA-MGS1 at 10 min post injection (p.i.) no intact radiopeptide was found in the blood, whereas for 111In-DOTA-MGS4 more than 75% was still intact. 111In-DOTA-MGS4 showed a clear increase in injected activity per gram tissue (IA/g) for A431-CCK2R xenografts (10.40±2.21% IA/g 4 h p.i.) when compared to 111In-DOTA-MGS1 (1.23±0.15% IA/g 4 h p.i.). The tumor uptake of 111In-DOTA-MGS4 was also combined with a low uptake in stomach and kidney leading to high-contrast NanoSPECT/CT images.Conclusion: Of the four new MG analogs developed, the best results in terms of enzymatic stability and increased tumor targeting were obtained with 111In-DOTA-MGS4 showing two substitutions with N-methylated amino acids. 111In-DOTA-MGS4 was also superior to other MG analogs reported thus far and seems therefore an extremely promising targeting molecule for theranostic use with alternative radiometals.

Resveratrol attenuates cerebral ischaemia reperfusion injury via modulating mitochondrial dynamics homeostasis and activating AMPK‐Mfn1 pathway

“…Transfection with siRNA was used to inhibit Mfn1 expression in resveratrol‐treated N2a cells. Briefly, siRNA against Mfn1 (Yangzhou Ruibo Biotech Co., Ltd.) was used to infect cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol . The negative control group was transfected with negative control siRNA.…”

“…Briefly, siRNA against Mfn1 (Yangzhou Ruibo Biotech Co., Ltd.) was used to infect cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. 41,42 The negative control group was transfected with negative control siRNA. Transfection was performed for approximately 48 hours.…”

DOTA-MGS5, a New Cholecystokinin-2 Receptor-Targeting Peptide Analog with an Optimized Targeting Profile for Theranostic Use

“…DOTA-MGS5 (DOTA-D-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 ) was synthesized and analyzed following a previously described protocol (14). The chemical structure is displayed in Figure 1.…”

“…We have recently described a new stabilization strategy based on site-specific amino acid modifications with unnatural aromatic amino acids and N-methylated amino acids preventing the metabolization of the C-terminal receptor-specific sequence. When applied to the truncated minigastrin analog DOTA-MG11 missing the penta-Glu motif (7), these modifications led to compounds with high CCK2R affinity, improved bioavailability, limited kidney retention, and highly increased tumor uptake (14).…”

Radiolabelled Peptides for Positron Emission Tomography and Endoradiotherapy in Oncology

“…Another relatively easy strategy for stability improvement towards endopeptidases is based on the use of d-amino acids or unnatural amino acids such as naphthylalanine, phenylglycine, norleucine, and cyclohexylalanine as found, e.g., by Murza et al [9] or Klingler et al [10]. In many cases, a combination of N/C-terminal modification with stabilisation via d-amino acids or unnatural amino acids is used as described for Tyr 3 -octreotide or different RGD-derivatives.…”