Combined Probe Strategy to Increase the Enzymatic Digestion Rate and Accelerate the Renal Radioactivity Clearance of Peptide Radiotracers.

High and sustained renal radioactivity accumulation is a major challenge in peptide-based radionuclide imaging and therapy. However, neutral endopeptidase (NEP)-based enzymatic hydrolysis to release and excrete the radioactive fragments has been proven to be an effective and promising way to reduce renal accumulation. Despite the improvement, the effect is still far from being satisfactory. To further reduce kidney uptake, we studied the relationship between the enzymatic reaction rate and the substrate concentration and came up with a combined probe strategy. Model compounds Boc-MVK-Dde and Boc-MFK-Dde were used for an enzymatic digestion study. NOTA-Exendin 4 and NOTA-MVK-Exendin 4 were labeled with Cu for dose-dependent micro-positron emission tomography (PET) studies. Groups 1 and 2 were injected with 0.2 and 0.8 nmol of Cu-NOTA-Exendin 4, respectively. Groups 3-6 were injected with 0.2, 0.8, 1.0, and 1.4 nmol of Cu-NOTA-MVK-Exendin 4, respectively. Groups 7 and 8 were co-injected with 0.2 nmol of Cu-NOTA-MVK-Exendin 4 and NOTA-MVK-PEG5K (1.3 and 2.6 nmol). The radioactivity uptakes were determined and compared within and among the groups. The cleavage study for both Boc-MVK-Dde and Boc-MFK-Dde indicated that within a certain concentration range, the enzyme digestion rate increased with increasing substrate concentration. The microPET images showed that the renal clearance could be accelerated significantly by increasing the injection dose of Cu-NOTA-MVK-Exendin 4, with the kidney uptakes being 60.98, 43.01, and 16.10 % ID/g at 1 h for groups 3, 4 and 5, respectively. Unfortunately, the tumor uptakes were also significantly inhibited as the injected dose of the tracer increased. However, with the co-injection of NOTA-MVK-PEG5K, the renal accumulation was significantly decreased without hampering the tumor uptake. As a result, the tumor-to-kidney ratios were significantly improved, which were 1.93, 3.47, 1.74, and 3.38 times that of group 3 at 1, 4, 24, and 48 h, respectively. The enzymatic reaction rate of NEP is dependent on the concentration of the substrates both and . The combined probe strategy developed in this study can dramatically reduce the renal accumulation of a peptide radioligand without affecting the tumor uptake, which shows great potential in peptide-based radiotheranostics.