68Ga-NOTA-exendin-4

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 ⁶⁴Cu 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 ⁶⁴Cu-NOTA-Exendin 4, respectively. Groups 3-6 were injected with 0.2, 0.8, 1.0, and 1.4 nmol of ⁶⁴Cu-NOTA-MVK-Exendin 4, respectively. Groups 7 and 8 were co-injected with 0.2 nmol of ⁶⁴Cu-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 ⁶⁴Cu-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.

Noninvasive imaging methods for the diagnosis and risk stratification of pheochromocytomas (PHEOs) remain a great clinical challenge. The glucagon-like peptide-1 receptor (GLP-1R) has been validated to be overexpressed in PHEOs and therefore may be a reliable target for PHEOs. In this study, we firstly synthesized a novel radiotracer 68Ga-NOTA-MAL-Cys39-exendin-4 that specifically targets GLP-1R and evaluated the performance of GLP-1R PET for the diagnosis and risk stratification of PHEOs. Cys39-exendin-4 was conjugated to NOTA-MAL and then radiolabeled with 68Ga. The reaction was completed within 20 min with a yield of 91.6 ± 2.8%. In vitro cell uptake studies validated its high specificity. PET images showed promising tumor visualization with high uptake (1.88 ± 0.10 %ID/g for PC-12 poorly differentiated tumors and 1.09 ± 0.003 %ID/g for PC-12 highly differentiated tumors at 30min after injection). There was a significant difference in the uptake of 68Ga-NOTA-MAL-Cys39-exendin-4 between PC-12 poorly and highly differentiated tumors (p < 0.001), but no significant difference could be observed by 18F-FDG PET. Biodistribution results confirmed the findings of GLP-1R PET and demonstrated that 131I-MIBG couldn't be used for the risk stratification of PHEOs. Immunohistochemistry (IHC) staining revealed differences in GLP-1R expression between PC-12 poorly and highly differentiated tumor tissues. These results demonstrated that 68Ga-NOTA-MAL-Cys39-exendin-4 can specifically target GLP-1R with favorable pharmacokinetic properties. GLP-1R PET can be used for PHEOs detection and has potential for the risk stratification of PHEOs.