Synthesis and in Vitro and in Vivo Evaluation of an ¹⁸F‑Labeled Neuropeptide Y Analogue for Imaging of Breast Cancer by PET

Imaging of Y₁R expression in breast cancer is still a challenging task. Herein, we report a suitable ¹⁸F-labeled high-molecular-weight glycopeptide for imaging of peripheral neuropeptide Y (NPY) Y₁ receptor (Y₁R)-positive tumors by preclinical small-animal positron emission tomography (PET). The Y₁R-preferring NPY [F⁷,P³⁴]­NPY analogue was functionalized with an alkyne-bearing propargylglycine (Pra) in position 4. The corresponding fluoroglycosylated (FGlc) peptide analogue [Pra⁴(FGlc),F⁷,P³⁴]­NPY and its ¹⁸F-labeled analogue were synthesized by click chemistry-based fluoroglycosylation. The radiosynthesis was performed by ¹⁸F-fluoroglycosylation starting from the 2-triflate of the β-mannosylazide and the alkyne peptide [Pra⁴,F⁷,P³⁴]­NPY. The radiosynthesis of the¹⁸F-labeled analogue was optimized using a minimum amount of peptide precursor (40 nmol), proceeding with an overall radiochemical yield of 20–25% (nondecay corrected) in a total synthesis time of 75 min with specific activities of 40–70 GBq/μmol. In comparison to NPY and [F⁷,P³⁴]­NPY, in vitro Y₁R and Y₂R activation studies with the cold [Pra⁴(FGlc),F⁷,P³⁴]­NPY on stably transfected COS-7 cells displayed a high potency for the induction of Y₁R-specific inositol accumulation (pEC₅₀ = 8.5 ± 0.1), whereas the potency at Y₂R was significantly decreased. Internalization studies on stably transfected HEK293 cells confirmed a strong glycopeptide-mediated Y₁R internalization and a substantial Y₁R subtype selectivity over Y₂R. In vitro autoradiography with Y₁R-positive MCF-7 tumor tissue slices indicated high specific binding of the ¹⁸F-labeled glycopeptide, when binding was reduced by 95% ([Pra⁴,F⁷,P³⁴]­NPY) and by 86% (BIBP3226 Y₁R antagonist) in competition studies. Biodistribution and small-animal PET studies on MCF-7 breast tumor-bearing nude mice revealed radiotracer uptake in the MCF-7 tumor of 1.8%ID/g at 20 min p.i. and 0.7%ID/g at 120 min p.i. (n = 3–4), increasing tumor-to-blood ratios from 1.2 to 2.4, and a tumor retention of 76 ± 4% (n = 4; 45–90 min p.i.). PET imaging studies with MCF-7 tumor-bearing nude mice demonstrated uptake of the ¹⁸F-labeled glycopeptide in the tumor region at 60 min p.i., whereas only negligible tumor uptake was observed in animals injected with a nonbinding ¹⁸F-labeled glycopeptide pendant as a measure of nonspecific binding. In conclusion, PET imaging experiments with the ¹⁸F-labeled NPY glycopeptide revealed Y₁R-specific binding uptake in MCF-7 tumors in vivo together with decreased kidney uptake compared to DOTA-derivatives of this peptide. We consider this glycopeptide to be a potent lead peptide for the design of improved ¹⁸F-glycopeptides with shorter amino acid sequences that would further facilitate PET imaging studies of Y₁R-positive breast tumors.