Targeting the Neuropeptide Y1 Receptor for Cancer Imaging by Positron Emission Tomography Using Novel Truncated Peptides.

The neuropeptide Y1 receptor (Y1R) is overexpressed in many human cancers, particularly breast cancer. Due to stability issues, limited success has been achieved for Y1R imaging agents, including full length and truncated neuropeptide Y (NPY) analogues. The goal of this study was to evaluate the possibility of using radiolabeled truncated NPY analogues to visualize Y1R expression in a preclinical model of Y1R-positive tumor. Four truncated NPY analogues were synthesized based on the sequence of [Pro30, Tyr32, Leu34]NPY(28-36), also known as BVD15. We substituted Tyr5 and Arg6 with unnatural amino acids aiming to enhance plasma stability while maintaining good receptor binding affinity to Y1R. In addition, we substituted Leu4 to Lys4 in order to conjugate via an optional linker the DOTA chelator for 68Ga labeling. Receptor binding affinity and plasma stability of these compounds were evaluated. Positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies were performed using immune-compromised mice bearing HEK293T::WT and HEK293T::hY1R tumors. [Lys(Ga-DOTA)4, Bip5]BVD15 (CCZ01035), [Lys(Ahx-Ga-DOTA)4, Bip5]BVD15 (CCZ01053), and [Lys(Pip-Ga-DOTA)4, Bip5]BVD15 (CCZ01055) demonstrated good binding affinity to Y1R (Ki = 23.4-32.3 nM), while [Lys(Ga-DOTA)4, Har6]BVD15 (P05067) showed poor binding affinity (Ki > 1000 nM). In addition, CCZ01055 exhibited low binding affinity (Ki > 1000 nM) to Y2R and Y4R, demonstrating its selectivity to Y1R. The former three peptides showed improved in vitro plasma stability of 7-16% remaining intact after 1 h incubation. PET/CT imaging and biodistribution studies for 68Ga-labeled CCZ01053, CCZ01035, and CCZ01055 showed that radioactivity was mainly cleared by the renal pathway, and HEK293T::hY1R tumors were clearly visualized with minimal background activity with the latter two. Of these two tracers, [68Ga]CCZ01055 provided lower kidney accumulation and higher contrast, i.e., average uptake ratios of Y1R tumor to wild type tumor, blood, and muscle are 3.87 ± 0.83, 4.12 ± 1.14, and 17.6 ± 4.64, respectively. Furthermore, Y1R tumor uptake with [68Ga]CCZ01055 was significantly reduced with coinjection of 100 μg of peptide YY, confirming the specificity of tumor accumulation was receptor mediated. We successfully developed the first Y1R-targeting truncated NPY analogues for PET imaging in a preclinical model, and [68Ga]CCZ01055 is a critical template for designing improved imaging agents to detect Y1R expressing cancers.