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摘要
Imaging agents based on peptide probes have desirable pharmacokinetic properties provided that they have high affinities for their target in vivo. An approach to improve a peptide ligand's affinity for its target is to make this interaction covalent and irreversible. For this purpose, we evaluated a Cu-64-labeled affinity peptide tag, Cu-64-L19K-(5-fluoro-2,4-dinitrobenzene) (Cu-64-L19K-FDNB), which binds covalently and irreversibly to vascular endothelial growth factor (VEGF) as a PET imaging agent. We compared the in vivo properties of Cu-64-L19K-FDNB in VEGF-expressing tumor xenografts with its noncovalent binding analogs, Cu-64-L19K-(2,4-dinitrophenyl) (Cu-64-L19K-DNP) and Cu-64-L19K. Methods: The L19K peptide (GGNECDIARMWEWECFERK-CONH2) was constructed with 1,4,7-triazacyclononane-1,4,7-triacetic acid at the N terminus for radiolabeling with Cu-64 with a polyethylene glycol spacer between peptide and chelate. 1,5-difluoro-2,4-dinitrobenzene was conjugated at the C-terminal lysine for cross-linking to VEGF, resulting in L19K-FDNB. Cu-64-L19K-FDNB was assayed for covalent binding to VEGF in vitro. As a control, L19K was conjugated to 1-fluoro-2,4-dinitrobenzene, resulting in L19K-DNP. PET imaging and biodistribution studies of Cu-64-L19K-FDNB, Cu-64-L19K-DNP, and the native Cu-64-L19K were compared in HCT-116 xenografts. Blocking studies of Cu-64-L19K-FDNB was performed with a coinjection of excess unlabeled L19K-FDNB. Results: In vitro binding studies confirmed the covalent and irreversible binding of Cu-64-L19K-FDNB to VEGF, whereas Cu-64-L19K-DNP and Cu-64-L19K did not bind covalently. PET imaging showed higher tumor uptake with Cu-64-L19K-FDNB than with Cu-64-L19K-DNP and Cu-64-L19K, with mean standardized uptake values of 0.62 +/- 0.05, 0.18 +/- 0.06, and 0.34 +/- 0.14, respectively, at 24 h after injection (P < 0.05), and 0.53 +/- 0.05, 0.32 +/- 0.14, and 0.30 +/- 0.09, respectively, at 48 h after injection (P < 0.05). Blocking studies with Cu-64-L19K-FDNB in the presence of excess unlabeled peptide showed a 53% reduction in tumor uptake at 48 h after injection. Conclusion: In this proof-of-concept study, the use of a covalent binding peptide ligand against VEGF improves tracer accumulation at the tumor site in vivo, compared with its noncovalent binding peptide analogs. This technique is a promising tool to enhance the potency of peptide probes as imaging agents.
- 出版日期2014-6
