Ccr2-Targeted Pet Imaging Of Injured Heart

98 Objectives: The heart contains distinct macrophage populations with divergent origins and functions. Among these subsets, CCR2+ macrophages are a particularly inflammatory population derived from circulating Ly6ChighCCR2+ blood monocytes that infiltrate heart following various forms of tissue injury. Based on these findings, CCR2+ monocytes and macrophages have been considered a new therapeutic target to modulate cardiac inflammation. However, no techniques are currently available to detect these cells in intact organisms. The goal of this study was to evaluate gallium-68 (68Ga) radiolabeled CCR2 targeting peptide as a PET radiotracer to noninvasively assess recruitment of CCR2+ inflammatory monocytes and macrophages to the heart. Methods: Extracellular loop 1 inverso (ECL1i) peptide is a 7 amino acid peptide which allosterically binds to CCR2. ECL1i was conjugated to a DOTA chelator for 68Ga radiolabeling. Two independent mouse models of cardiac injury were evaluated in this study including diphtheria toxin (DT)-induced cardiomyocyte ablation and reperfused myocardial infarction. Accumulation of CCR2+ monocytes and macrophages in the injured heart in each model was characterized by flow cytometry and immunostaining. Biodistribution, PET, and ex vivo autoradiography were conducted to assess suitability of 68Ga-DOTA-ECL1i for detection of CCR2+ inflammatory cells. In the ischemia reperfusion injury model, serial PET imaging of 18F-FDG and 68Ga-DOTA-ECL1i was performed to demonstrate feasibility of serial imaging of CCR2+ monocytes and macrophages. The binding and targeting specificity was verified in mouse models established in CCR2 KO mice. Translational potential of the tracer was examined in ex vivo human heart tissues using autoradiography. Results: 68Ga-DOTA-ECL1i was prepared with specific activity of ca. 6.05 MBq/nmol and radiochemical purity of ≥95%. The tracer was stable in mouse serum for up to 4 hours. Biodistribution studies demonstrated that 68Ga-DOTA-ECL1i displays minimal cardiac uptake in control mice and is quickly cleared from the blood pool. PET imaging in DT-mediated cardiomyocyte ablation models showed ca. 7-fold higher myocardial radioactivity compared to the control mice (Wild type + DT). The lower radioactivity in DT-treated CCR2 KO mice confirmed the targeting specificity. In ischemia reperfusion injury models, serial PET imaging and ex vivo autoradiography of 68Ga-DOTA-ECL1i revealed robust radiotracer uptake in the sites of injury within the heart at time points correlating with CCR2+ monocyte and macrophage recruitment characterized by flow cytometry and immunohistochemistry. While 18F-FDG PET can noninvasively detect infarct area, tracer uptakes in heart provide little information. Tracer uptake ratio (heart/blood, heart/muscle, and heart/aorta) verified that the accumulation in the heart was independent of blood pool radioactivity. Ex vivo autoradiography in human tissues with ischemic cardiomyopathy showed heterogeneous 68Ga-DOTA-ECL1i signals, which was significantly reduced with competitive receptor blocking indicating its specific binding to human CCR2. Conclusions: These studies demonstrated 68Ga-DOTA-ECL1i can noninvasively detect recruitment of CCR2+ inflammatory cells to the injured heart in two independent mouse models and binds to human CCR2 in ex vivo human specimens. The future translation to humans may visualize CCR2+ monocytes and macrophages in patients and provide new insights into the dynamics of cardiac inflammation, heterogeneity between individual patients, and prognostic relevance of CCR2+ inflammatory cell recruitment.