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

Abstract

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 in vitro enzymatic digestion study. NOTA-Exendin 4 and NOTA-MVK-Exendin 4 were labeled with 64 Cu for in vivo dose-dependent micro-positron emission tomography (PET) studies. Groups 1 and 2 were injected with 0.2 and 0.8 nmol of 64 Cu-NOTA-Exendin 4, respectively. Groups 3−6 were injected with 0.2, 0.8, 1.0, and 1.4 nmol of 64 Cu-NOTA-MVK-Exendin 4, respectively. Groups 7 and 8 were co-injected with 0.2 nmol of 64 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 in vitro 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 64 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 in vitro and in vivo. 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.

Enzymological Characterization of 64Cu-Labeled Neprilysin Substrates and Their Application for Modulating the Renal Clearance of Targeted Radiopharmaceuticals

“…Although recent studies were focused on optimizing the linker sequences toward an enhanced enzymatic cleavage rate, 39,40 no quantitative data on the distinct substrate potential toward NEP or other brush border membrane enzymes are reported, which would lay the basis for a rationalized use of this strategy. Furthermore, the concept of cleavable linkers was predominantly applied to antibody derivatives and large peptides, such as exendin-4, 39,41,42 but only few studies are reported on adapting this strategy to small molecules or peptides.…”

“…Although recent studies were focused on optimizing the linker sequences toward an enhanced enzymatic cleavage rate, , no quantitative data on the distinct substrate potential toward NEP or other brush border membrane enzymes are reported, which would lay the basis for a rationalized use of this strategy. Furthermore, the concept of cleavable linkers was predominantly applied to antibody derivatives and large peptides, such as exendin-4, ,, but only few studies are reported on adapting this strategy to small molecules or peptides. , Herein, we synthesized a series of 64 Cu-labeled tri- and tetrapeptides and quantified their suitability to act as neprilysin substrates (NES) in a radiometric assay using recombinant NEP, which led to the exploration of the structural features that drive the cleavage rate.…”

A Systematic Investigation into the Influence of Net Charge on the Biological Distribution of Radiometalated Peptides Using [68Ga]Ga-DOTA-TATE Derivatives

“…Another approach is insertion of a cleavable linker (such as glycyl-lysine “GK” or methionine-valine-lysine “MVK” sequences) between the peptide/protein and the radiolabeled chelate moiety that is recognized and cleaved by enzymes on the lumen of renal tubules. ,− Consequently, the radionuclide-bearing portion of the molecule would be emancipated from the peptide/protein and excreted from the kidneys to the bladder without reabsorption. These enzyme-activated linkers are very sensitive to the molecular structure around the recognition sequence, and it is not trivial to perform detailed enzyme kinetics to validate the activity and selectivity of a new derivative. − Further, it is common for nontargeted enzymes in the blood or other healthy tissues to have some activity toward many of these cleavable linkers, making their application to new radiopharmaceuticals nontrivial.…”