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Mol Pharm. 2015 Apr 6;12(4):1121-30. doi: 10.1021/mp500601z. Epub 2015 Mar 13.

Synthesis and in vitro and in vivo evaluation of an (18)F-labeled neuropeptide Y analogue for imaging of breast cancer by PET.

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†Faculty of Biosciences, Pharmacy and Psychology, Institute of Biochemistry, Universität Leipzig, 04109 Leipzig, Germany.
‡Department of Nuclear Medicine, Molecular Imaging and Radiochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany.


Imaging of Y1R expression in breast cancer is still a challenging task. Herein, we report a suitable (18)F-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 [F(7),P(34)]NPY analogue was functionalized with an alkyne-bearing propargylglycine (Pra) in position 4. The corresponding fluoroglycosylated (FGlc) peptide analogue [Pra(4)(FGlc),F(7),P(34)]NPY and its (18)F-labeled analogue were synthesized by click chemistry-based fluoroglycosylation. The radiosynthesis was performed by (18)F-fluoroglycosylation starting from the 2-triflate of the β-mannosylazide and the alkyne peptide [Pra(4),F(7),P(34)]NPY. The radiosynthesis of the(18)F-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 [F(7),P(34)]NPY, in vitro Y1R and Y2R activation studies with the cold [Pra(4)(FGlc),F(7),P(34)]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 (18)F-labeled glycopeptide, when binding was reduced by 95% ([Pra(4),F(7),P(34)]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 (18)F-labeled glycopeptide in the tumor region at 60 min p.i., whereas only negligible tumor uptake was observed in animals injected with a nonbinding (18)F-labeled glycopeptide pendant as a measure of nonspecific binding. In conclusion, PET imaging experiments with the (18)F-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 (18)F-glycopeptides with shorter amino acid sequences that would further facilitate PET imaging studies of Y1R-positive breast tumors.


NPY; fluorine-18; glycosylation; neuropeptide Y; peptide; positron emission tomography

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