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 andtherapy. However, neutral endopeptidase (NEP)-based enzymatic hydrolysis to release and excrete the radioactive fragments hasbeen proven to be an effective and promising way to reduce renal accumulation. Despite the improvement, the effect is still far frombeing satisfactory. To further reduce kidney uptake, we studied the relationship between the enzymatic reaction rate and thesubstrate concentration and came up with a combined probe strategy. Model compounds Boc-MVK-Dde and Boc-MFK-Dde wereused for anin vitroenzymatic digestion study. NOTA-Exendin 4 and NOTA-MVK-Exendin 4 were labeled with64Cu forin vivodose-dependent micro-positron emission tomography (PET) studies. Groups 1 and 2 were injected with 0.2 and 0.8 nmol of64Cu-NOTA-Exendin 4, respectively. Groups 3-6 were injected with 0.2, 0.8, 1.0, and 1.4 nmol of64Cu-NOTA-MVK-Exendin 4,respectively. Groups 7 and 8 were co-injected with 0.2 nmol of64Cu-NOTA-MVK-Exendin 4 and NOTA-MVK-PEG5K (1.3 and 2.6nmol). The radioactivity uptakes were determined and compared within and among the groups. Thein vitrocleavage study for bothBoc-MVK-Dde and Boc-MFK-Dde indicated that within a certain concentration range, the enzyme digestion rate increased withincreasing substrate concentration. The microPET images showed that the renal clearance could be accelerated significantly byincreasing the injection dose of64Cu-NOTA-MVK-Exendin 4, with the kidney uptakes being 60.98, 43.01, and 16.10 % ID/g at 1 hfor groups 3, 4 and 5, respectively. Unfortunately, the tumor uptakes were also significantly inhibited as the injected dose of thetracer increased. However, with the co-injection of NOTA-MVK-PEG5K, the renal accumulation was significantly decreased withouthampering the tumor uptake. As a result, the tumor-to-kidney ratios were significantly improved, which were 1.93, 3.47, 1.74, and3.38 times that of group 3 at 1, 4, 24, and 48 h, respectively. The enzymatic reaction rate of NEP is dependent on the concentrationof the substrates bothin vitroandin vivo. The combined probe strategy developed in this study can dramatically reduce the renalaccumulation of a peptide radioligand without affecting the tumor uptake, which shows great potential in peptide-based radiotheranostics