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 (K_i = 23.4-32.3 nM), while [Lys(Ga-DOTA)⁴, Har⁶]BVD15 (P05067) showed poor binding affinity (K_i > 1000 nM). In addition, CCZ01055 exhibited low binding affinity (K_i > 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.