Truncated NPY-based NPY(Y1)R-specific radiopeptides: Improved in vivo PET tumor imaging by application of peptidase inhibitors

The neuropeptide Y receptor subtype 1 (NPY(Y 1 )R) exhibits high expression rates on human breast cancer and is therefore an important target structure for the sensitive and specific visualization and characterization of the disease by Positron Emission Tomography (PET). However, imaging of this receptor type has been of limited success so far due to the low stability of peptide-based NPY-derived NPY(Y 1 )R-specific radiotracer candidates due to in vivo degradation. Given the challenges in stabilizing these agents, our study sought to explore whether the stability of NPY(Y 1 )R-specific radiopeptides could be enhanced. We aimed to achieve this by either modifying the peptide structure with various molecular scaffolds or by applying peptidase inhibitors. This evaluation aimed to identify the optimal approach for achieving effective NPY(Y 1 )R-specific imaging in the following. To validate our approach, we systematically investigated four new truncated 68 Ga-labeled analogs of NPY and the reference compound [ 68 Ga]Ga-[Lys 4 (N ε -DOTA)]BVD15, bearing different molecular scaffolds such as a DOTA or NODA-GA chelator, a 4-APipAc linker, a Bip unit, and an N -terminal Lys(lauryl) group. The four new radiotracers as well as the reference compound were obtained in high chemical and radiochemical yields with molar activities of 33–39 GBq/μmol. The radiopeptides exhibited varying log D 7.4 values, ranging from −3.37 ± 0.09 to +0.35 ± 0.11, and showed different levels of stability in human serum and liver microsomes, depending on their molecular structure. Subsequently, the influence of the peptidase inhibitors actinonin, phosphoramidon, captopril and E−64 on the in vitro stability of the radiotracers was investigated. In these studies, only actinonin demonstrated a positive effect on the stability of all radiopeptides. In contrast, phosphoramidon yielded variable results, and neither captopril nor E−64 showed a significant stabilizing effect. Consequently, the effect of actinonin administration on the in vivo PET/CT imaging results of the most promising ligand [ 68 Ga]Ga-[Lys 4 (N ε -NODA-GA)]BVD15 ([ 68 Ga]Ga- 2 ) was investigated in a T47D tumor-bearing xenograft mouse model, followed by ex vivo biodistribution studies. In these experiments, the administration of 250 μg actinonin resulted in a significantly increased uptake of [ 68 Ga]Ga- 2 in the T47D tumor (5.9 ± 1.0 % ID/g (with actinonin) instead of 3.1 ± 0.9 % ID/g (without actinonin) at 2h p.i.), and an increase in tumor-to-muscle ratios from 1.8 to 4.0 upon co-administration of the inhibitor. The results impressively demonstrate the positive influence of actinonin on the in vivo stability of the NPY(Y 1 )R-specific radiopeptide [ 68 Ga]Ga- 2 , resulting in an increased tumor accumulation and improved tumor-to-background ratios. These findings thus provide important incentive for further advancement of NPY(Y 1 )R-specific tumor imaging using PET. • Peptidic NPY(Y 1 )R-specific radioligands usually exhibit a limited in vivo stability. • Structural modifications of the peptide do not sufficiently solve the problem. • Peptidase inhibitors can decelerate radiopeptide degradation. • Actinonin is a suitable inhibitor for stabilizing NPY(Y 1 )R-specific radiopeptides. • Actinonin demonstrated to decelerate radiopeptide degradation in vitro and in vivo .