A novel neutrophil-specific PET imaging agent: cFLFLFK-PEG-64Cu

The synthesis and validation of a new, highly potent (64)Cu-labeled peptide, cFLFLFK-PEG-(64)Cu, that targets the formyl peptide receptor (FPR) on leukocytes is described. The peptide ligand is an antagonist of the FPR, designed not to elicit a chemotactic response resulting in neutropenia. Evidence for the selective binding of this synthesized ligand to neutrophils is provided. PET properties of the compound were evaluated in a mouse model of lung inflammation.

The FPR-specific peptide, cinnamoyl-F-(D)L-F-(D)L-FK (cFLFLF), was sequentially conjugated with a bifunctional polyethylene glycol moiety (PEG, 3.4 kD) and a 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) through a lysine (K) spacer and finally labeled with (64)Cu-CuCl(2) to form cFLFLFK-PEG-(64)Cu. The binding affinity and stimulation potency of the ligand toward human neutrophils were assessed in vitro. Blood kinetic and organ biodistribution properties of the peptide were studied in the mouse. Ten male C57BL/6 mice were used in this study; 4 control mice and 6 administered Klebsiella pneumonia. PET/CT scans were performed to assess the localization properties of the labeled peptide in lungs 18 h after tracer administration. Lung standardized uptake values (SUVs) were correlated with lung neutrophil activity as measured by myeloperoxidase assays. Immunohistochemistry was performed to confirm that neutrophils constitute the majority of infiltrating leukocytes in lung tissue 24 h after Klebsiella exposure.

In vitro binding assays of the compound cFLFLFK-PEG-(64)Cu to the neutrophil FPR yielded a dissociation constant of 17.7 nM. The functional superoxide stimulation assay exhibited negligible agonist activity of the ligand with respect to neutrophil superoxide production. The pegylated peptide ligand exhibited a blood clearance half-life of 55 +/- 8 min. PET 18 h after tracer administration revealed mean lung SUVs and lung myeloperoxidase activities for Klebsiella-infected mice that were 5- and 6-fold higher, respectively, than those for control mice. Immunohistochemistry staining confirmed that the cellular infiltrate in lungs of Klebsiella-infected mice was almost exclusively neutrophils at the time of imaging.

This new radiolabeled peptide targeting the FPR binds to neutrophils in vitro and accumulates at sites of inflammation in vivo. This modified peptide may prove to be a useful tool to probe inflammation or injury.