Synthesis and preclinical characterization of [64Cu]NODAGA-MAL-exendin-4 with a Nε-maleoyl-L-lysyl-glycine linkage.

Introduction: Renal localization of high radioactivity levels during targeted imaging compromises tissue visualization in the kidney region and limits diagnostic accuracy. Radioiodinated antibody fragments with a renal enzyme-cleavable N(ε)-maleoyl-L-lysyl-glycine (MAL) linkage demonstrated low renal radioactivity levels in mice, from early postinjection times. This study tested the hypothesis whether a (64)Cu-labeled NODAGA-exendin-4 peptide with a MAL linkage ([(64)Cu]NODAGA-MAL-exendin-4) could decrease kidney radioactivity levels in rats, compared to a [(64)Cu]NODAGA-exendin-4 reference, without impairing the radioactivity levels in the target tissue.

Methods: NODAGA-MAL-exendin-4 was synthesized in a two-phase approach using solid support to prepare maleoyl-derivatized NODAGA followed by Michael addition to cysteine-derivatized exendin-4 in solution. Radiolabeling was performed in buffered aqua with [(64)Cu]CuCl2, which was produced via the (64)Ni(p,n)(64)Cu nuclear reaction. The in vitro and in vivo stability, lipophilicity, and distribution kinetics in major rat organs for [(64)Cu]NODAGA-MAL-exendin-4 were studied and compared to [(64)Cu]NODAGA-exendin-4. Labeling of pancreatic islets was assessed using autoradiography.

Results: NODAGA-MAL-exendin-4 was synthesized, with an overall yield of 9%, and radiolabeled with (64)Cu with high specific radioactivity. Serum incubation studies showed high stability for [(64)Cu]NODAGA-MAL-exendin-4. Similar tissue distribution kinetics was observed for [(64)Cu]NODAGA-MAL-exendin-4 and [(64)Cu]NODAGA-exendin-4, with high kidney radioactivity levels.

Conclusions: The incorporated MAL linkage in [(64)Cu]NODAGA-MAL-exendin-4 was unable to reduce kidney radioactivity levels, compared to [(64)Cu]NODAGA-exendin-4. The applicability of metabolizable linkages in the design of kidney-saving exendin-4 analogs requires further investigation.