Development and initial evaluation of a novel 11C-labeled PET tracer to image GPR84 expressing-myeloid cells during neuroinflammation

Purpose/Background: Neuroinflammation is a hallmark of many central nervous system (CNS) diseases including Alzheimer’s disease, multiple sclerosis, and stroke. Unfortunately, most previously investigated biomarkers for quantifying neuroinflammation in vivo using positron emission tomography (PET) imaging have several limitations. For example, the most widely evaluated PET tracers for neuroinflammation target the translocator protein 18 kDa (TSPO)— expressed indiscriminately on activated myeloid lineage cells, reactive astrocytes, and endothelial cells. In contrast, GPR84 is a G-protein coupled receptor (GPCR) expressed predominately on myeloid cells.1,2 Importantly, GPR84 is significantly upregulated specifically on pro-inflammatory myeloid cells following CNS injury or insult.3,4 Here we report the synthesis of a new PET tracer for GPR84 (11C-MGX-10S), based on a dihydropyrimido isoquinolinone inhibitor scaffold5, and its initial assessment in cells and mice. Methods: First, we synthesized the cold version of compound MGX-10S, an allosteric inhibitor of GPR84 and determined its kinetic inhibition constant (Ki) via a competition binding assay with 3H-G9543 (known GPR84 inhibitor). We then generated 11C-MGX-10S by alkylating the phenolic precursor with 11C-methyl iodide in DMF (500 L) for 3 min at 65 °C, using 1M NaOH as a base (Fig 1A). Subsequently we evaluated the binding specificity of 11C-MGX-10S using stable human-GPR84-expressing human endothelial kidney (hGPR84-HEK293) cells versus parental control cells in quadruplicate. Lastly, we assessed the in vivo kinetics, distribution, and blood-brain-barrier (BBB) permeability of 11C-MGX-10S in healthy C57BL/6 mice (n=4) using dynamic PET/CT imaging. Results: The 1,4-dioxane ring linked compound (MGX-10S) was shown to have a Ki of 8.22 nM in the competitive binding assay. Automated module synthesis of 11C-MGX-10S was completed in 60 minutes, with a radiochemical yield of 5.07  1.42% (non-decay corrected), radiochemical purity >99%, and molar activity of 6328  396 mCi/mol. Cell binding studies revealed 14.5 fold higher binding of 11C-MGX-10S to hGPR84-HEK293 cells compared to parental HEK293 cells after 40 minutes of incubation (P <0.0001, n= 4). Blocking with GPR84 antagonist (GLPG1205, 35 M), significantly reduced tracer binding to hGPR84-HEK293 cells by 91.7%, demonstrating high specificity of 11C-MGX-10S (P <0.0001, n= 4). A similar pattern of binding was observed for tracer binding after 20 minutes incubation (Fig 1B). Whole brain time-activity curves (Fig 1C), generated by analyzing PET/CT images co-registered with a mouse brain atlas, demonstrate rapid entry of 11C-MGX-10S into the brain with an average peak uptake of 5.90  0.89 %ID/g (n=4) within 22.5 seconds, decreasing to 1.89  0.35 %ID/g by the end of the 60 minute scan. Whole body 3D maximum intensity projection PET/CT images (Fig 1D) illustrate robust brain signal at 0-5 min with increasing renal/hepatic clearance, in addition to low background signal in all other areas of the body, by the later time point (30-60 min). Conclusion: We have successfully synthesized and are currently evaluating a novel PET tracer for the measurement of GPR84 expression. Our data demonstrate the promise of 11C-MGX-10S as a highly specific tracer for detecting GPR84-expressing myeloid cells in vitro and in vivo. Further studies are currently underway to explore the utility of 11C-MGX-10S for imaging GPR84-associated innate immune activation in rodent models of CNS diseases.