Abstract 12854: Translocator Protein Ligand Has Improved Binding Characteristics For Quantification of Macrophages in Atherosclerosis
Carotid atherosclerotic plaque rupture is clinically assessed by vessel stenosis. High macrophage content is a consistent finding in ruptured plaques and the translocator protein (TSPO) is highly expressed in activated macrophages. TSPO ligands exist which are readily labelled by positron emission tomography (PET) radionuclides and thus, may non-invasively quantify macrophage burden. However, many of these have high non-specific binding (NSB) producing disparity between total and specific binding. Since total binding is the measure obtained in a clinical setting, reduction in NSB will improve macrophage quantification. We have previously shown that other TSPO ligands bind with high affinity to macrophages and binding correlates with macrophages in atherosclerotic plaque. However, NSB is typically greater than 50% of total binding on rabbit and human tissues. N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide (DPA-713) was designed with reduced lipophilicity, whilst retaining high affinity and selectivity for the TSPO.We hypothesise that DPA-713 will allow for more accurate determination of atherosclerotic plaque inflammatory burden.Receptor binding analysis and autoradiography were performed with 0.1 to 30nM and 1nM [3H]-DPA-713 (PerkinElmer, USA) respectively. The inclusion of 10∝M unlabelled ligand was used to quantify NSB. Protein concentration was quantified by the Biuret assay and macrophage and TSPO expression were identified by immunohistochemistry with mouse anti-human CD68 (Dako, UK) and goat anti-rabbit TSPO antibodies. Rabbit atherosclerotic carotid arteries were obtained from hypercholesterolemic animals.Saturation binding analysis of [3H]-DPA-713 on rabbit heart tissue indicated high affinity binding to a single site (Kd = 4.1 +/− 0.5, Bmax = 290.1 +/− 12.1fmoles/mg, n = 6). Non-specific binding was 3% of total binding. [3H]-DPA-713 binding in rabbit atherosclerotic plaques co-localised with macrophage presence and TSPO expression.In conclusion, DPA-713 has improved binding characteristics for the TSPO compared with other ligands and should allow for better quantification of atherosclerotic plaque macrophage burden using PET imaging in a clinical setting.
- © 2011 by American Heart Association, Inc.