Improved ⁶⁸Ga-labelled truncated peptides targeting the neuropeptide Y1 receptor for cancer imaging by positron emission tomography

Objectives Many human cancers express the neuropeptide Y1 receptor (Y1R), notably breast cancers. Limited success has been achieved for Y1R imaging agents due to the short biological half-life of the peptide, and truncated neuropeptide Y (NPY) analogues have not been successfully used for Y1R imaging to date. The goal of this study was to evaluate the possibility of using radiolabeled modified truncated NPY analogues to visualize Y1R expression in a preclinical model of Y1R-positive tumor.


Methods Truncated NPY analogues were synthesized using solid phase peptide synthesis. These peptides were derived from the sequence [Pro³⁰, Tyr³², Leu³⁴]NPY(28-36), also known as BVD15. Tyr-5 and Arg-6 were substituted with unnatural amino acids in order to enhance plasma stability while maintain the receptor binding affinity to Y1R. In addition, we substituted Leu-4 to Lys-4 in order to conjugate a DOTA chelator for ⁶⁸Ga labeling, with or without a neutral or cationic linker. Four different peptides were evaluated. The receptor binding affinity and in vitro plasma stability of these compounds were evaluated. PET/CT imaging and biodistribution studies were performed using immune-compromised mice bearing HEK293T::WT and HEK293T::hY1R tumors.

Results [Lys(DOTA)⁴, Bip⁵]BVD15 (CCZ01035), [Lys(Ahx-DOTA)⁴, Bip⁵]BVD15 (CCZ01053), and [Lys(Pip-DOTA)⁴, Bip⁵]BVD15 (CCZ01055) had good binding affinity to Y1R (Ki = 23.4 - 32.3 nM). However, the substitution of homoarginine for arginine at position 6 [Lys(DOTA)⁴, Har⁶]BVD15 (P05067) led to poor binding affinity (Ki > 1000 nM) and this compound was not further evaluated. CCZ01035, CCZ01053 and CCZ01055 were labeled with ⁶⁸Ga with > 99% radiochemical purity with average decay-corrected radiochemical yields ranging from 42 – 69%. Their average specific activity were > 2.5 Ci / μmol (n = 3). [⁶⁸Ga]CCZ01035 was very stable in vitro, with more than 95% of intact peptide after 1 h incubation in mouse plasma. The peptides with an Ahx or Pip linker had lower plasma stability in vitro. PET/CT imaging and biodistribution studies for ⁶⁸Ga-labeled CCZ01053, CCZ01035 and CCZ01055 showed that the radioactivity had a predominant renal clearance. HEK293T::hY1R tumors were clearly visualized with minimal background activity with CCZ01035 and CCZ01055. [⁶⁸Ga]CCZ01055 provided lower kidney accumulation and improved tumor visualization, with average uptake ratios of Y1R tumor to wild type tumor, blood and muscle of 3.87 ± 0.83, 4.12 ± 1.14 and 17.6 ± 4.64 %ID/g, respectively. Y1R tumor uptake with [⁶⁸Ga]CCZ01055 was reduced with co-injection of 100 μg peptide YY, confirming the specificity of tumor accumulation was receptor mediated.

Conclusions Key amino-acid substitutions combined with the use of a cationic linker on a modified DOTA-conjugated truncated NPY1R binding peptide allowed clear visualization of NPY1R expressing tumors. [⁶⁸Ga]CCZ01055 is a promising imaging agent to visualize Y1R expressing cancers by positron emission tomography.