Synthesis and Evaluation of a Novel ⁶⁴Cu- and ⁶⁷Ga-Labeled Neurokinin 1 Receptor Antagonist for <i>in Vivo</i> Targeting of NK1R-Positive Tumor Xenografts

Neurokinin 1 receptor (NK1R) is expressed in gliomas and neuroendocrine malignancies and represents a promising target for molecular imaging and targeted radionuclide therapy. The goal of this study was to synthesize and evaluate a novel NK1R ligand (NK1R-NOTA) for targeting NK1R-expressing tumors. Using a carboxymethyl moiety linked to L-733060 as a starting reagent, NK1R-NOTA was synthesized in a three-step reaction and then labeled with ⁶⁴Cu (or ⁶⁷Ga for <i>in vitro</i> studies) in the presence of CH₃COONH₄ buffer. The radioligand affinity and cellular uptake were evaluated with NK1R-transduced HEK293 cells (HEK293-NK1R) and NK1R nontransduced HEK293 cells (HEK293-WT) and their xenografts. Radiolabeled NK1R-NOTA was obtained with a radiochemical purity of >95% and specific activities of >7.0 GBq/μmol for ⁶⁴Cu and >5.0 GBq/μmol for ⁶⁷Ga. Both ⁶⁴Cu- and ⁶⁷Ga-labeled NK1R-NOTA demonstrated high levels of uptake in HEK293-NK1R cells, whereas co-incubation with an excess of NK1R ligand L-733060 reduced the level of uptake by 90%. Positron emission tomography (PET) imaging showed that [⁶⁴Cu]­NK1R-NOTA had a accumulated rapidly in HEK293-NK1R xenografts and a 10-fold lower level of uptake in HEK293-WT xenografts. Radioactivity was cleared by gastrointestinal tract and urinary systems. Biodistribution studies confirmed that the tumor-to-organ ratios were ≥5 for all studied organs at 1 h p.i., except kidneys, liver, and intestine, and that the tumor-to-intestine and tumor-to-kidney ratios were also improved 4 and 20 h post-injection. [⁶⁴Cu]­NK1R-NOTA is a promising ligand for PET imaging of NK1R-expressing tumor xenografts. Delayed imaging with [⁶⁴Cu]­NK1R-NOTA improves image contrast because of the continuous clearance of radioactivity from normal organs