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

Abstract

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.

Methods: 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.

Results: 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.

Conclusion: 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.

FIGURE 1

A representative saturation curve of cFLFLF-PEG-⁶⁴Cu specifically bound to human neutrophils. The binding affinity was computed to be 17.71 nM. The binding data has also been plotted as a Scatchard plot. Nonspecific binding was computed in the presence of excess cold compound and is estimated to contribute to 20-30% of total binding.

FIGURE 2

Stimulation of the neutrophil oxidative burst by cFLFLF-PEG-Cu and fMLF as detected by luminol-enhanced chemiluminescence. The results are reported as peak chemiluminescence as a percentage of TNF-primed and fMLF-stimulated (10^-6M) control. cFLFLF-PEG-Cu demonstrated no agonist activity towards neutrophils (EC50 value not computable), while fMLF stimulated neutrophil superoxide release with an EC50 of 3.7×10^-7 M. Data are reported as mean ± SEM for at least two independent measurements.

FIGURE 3

Tissue and organ accumulation of cFLFLF-PEG-⁶⁴Cu at 18 hr post injection in control and Klebsiella infected mice, expressed as the percent injected dose per gram of tissue. Mean lung radioactivity is 3.8 times greater in infected lungs compared to controls (*P<0.05).

FIGURE 4

Examples of microCT and microPET images of control and Klebsiella infected mice. PET images were obtained 18 hr after tail vein injection of cFLFLF-PEG-⁶⁴Cu . Both transverse (top row) and coronal (bottom row) images are shown. In mice administered Klebsiella, there is extensive attenuation in the lungs on the CT scans compared to controls. PET scans revealed Klebsiella infected mice had visually more tracer uptake in lung tissue compared to controls, which was quantified by ROI analysis. “H” represents the heart and “L” represents the liver. The color bar along the side indicates increasing tissue radioactivity on the PET images.

FIGURE 5

Lung standardized uptake values (SUVs) for control and Klebsiella infected mice. Lung SUVs were 5.8 times greater in Klebsiella infected mice as compared to controls (*P<0.005).

FIGURE 6

Myeloperoxidase (MPO) activity in lung homogenates, expressed as the change in optical density (15) per minute per milligram of tissue. MPO activity measured in Klebsiella-infected lungs was approximately 6 fold higher than that measured in controls (*P<0.05).

FIGURE 7

Immunohistochemical staining of neutrophils (stained with a rat anti-mouse IgG (MCA771G; Serotec)) and macrophages (stained with anti MAC-2 IgG (ACL8942P; Accurate)) in lung tissue excised from a control and a Klebsiella-infected mouse (42 hr post administration). Immunostained cells appear dark brown. Control lungs revealed no neutrophils (A) or macrophages (B), only normal alveolar wall structure. Infected lungs had significant neutrophil accumulation (C), with very little macrophage infiltration (D, indicated by arrowheads). Images were taken with a magnification of 400x, black bar indicates 100 μm.