Radiolabeling of a chemokine receptor CCR5 targeting peptide with ¹¹¹In and evaluation in an atherosclerosis model

Objectives Chemokine receptor CCR5 and antagonist peptide DAPTA are involved in atherosclerosis progression. Our objective was to develop a ¹¹¹In labeled SPECT tracer targeting CCR5 for imaging inflammation in atherosclerosis.

Methods DAPTA was labeled with ¹¹¹In through DOTA conjugation. Cold indium compound was characterized by mass spectroscopy. Cell uptake studies were conducted in: a) primary spleen cells and HEK-293 cells; b) U87-CD4-CCR5 and U87-MG cells. Biodistribution was determined in C57BL/6 mice. The tracer was injected into: 1) 9 month old ApoE^-/- mice with normal diet; 2) 4 month old ApoE^-/- mice with high fat diet (HFD); 3) 4 month old C57BL/6 mice as controls. The mice were sacrificed at 1 hr p.i.. Aortas were dissected and autoradiography images were collected and compared with en face and Oil Red O images. The lesion uptake (%IDxkg/m²) in autoradiography was quantified using standards.

Results DAPTA was radiolabeled with ¹¹¹In with high radiochemical purity (> 98%) and specific activity (2030 mCi/µmol). The identification of the tracer was confirmed by HPLC and mass spectroscopy of the cold analog. ¹¹¹In-DOTA-DAPTA (DD) exhibited fast pharmacokinetics and renal clearance and high spleen uptake. Cell uptake studies showed: i) the uptake in primary spleen cells was higher than the control HEK-293 cells (ratio: 2.1 at 1 hr incubation); ii) U87-CD4-CCR5 cells had significantly higher uptake than the control U87-MG cells (4.1 fold at 1 hr and 3.3 fold at 2 hr). The uptake in U87-CD4-CCR5 cells was reduced 3 times by block (DAPTA), indicating the uptake is receptor specific. Autoradiography of 9 month ApoE^-/- and 4 month ApoE^-/- HFD mice showed similar lesion uptake (~0.15%), while the uptake of ApoE^-/- mice was significant higher that the control mice (0.02%).

Conclusions ¹¹¹In-DD, specifically targeting CCR5, is a potential SPECT agent for imaging inflammation in atherosclerosis.