A. Project Summary/Abstract The goal of this renewal R01 application is to identify an 18F-labeled radiotracer to quantitatively measure sphingosine-1-phosphate receptor 1 (S1PR1) expression in multiple sclerosis (MS) patients. MS is associated with a lymphocyte-mediated autoimmune response that ultimately leads to repeating cycles of demyelination and repair. S1PR1 is extensively expressed on lymphocytes and endothelial cells and participates in this autoimmune inflammatory process by regulating immune cell trafficking in the brain. Currently, four S1P modulators fingolimod, siponimod, ozanimod, and ponesimod have been approved by FDA for treating MS, but the pathophysiologic mechanism of S1PR1 in MS is still not well understood. Positron emission tomography (PET) investigating S1PR1 function in MS and other diseases is limited by the lack of a clinically suitable radiotracer. To address this urgent clinical need, our team successfully transferred [11C]CS1P1, a C-11 S1PR1 radiotracer into clinical investigation. Our human study data confirm that liver is the critical organ for radiation dose absorption and that [11C]CS1P1 is safe for patient who receives a dose of up to 740 MBq (20 mCi). Our proof of concept brain studies showed higher uptake of [11C]CS1P1 in gray matter than in white matter, consistent with S1PR1 mRNA expression in the normal human brain. In MS patients, we observed increased [11C]CS1P1 uptake within both brain lesions and normal appearing white matter (WM). These results are consistent with extant evidence of inflammatory infiltrate in both the brain lesions and normal appearing WM in MS. Together, our data suggest that PET measurement of S1PR1 protein expression could provide a unique method to detect neuroinflammation and response in MS patients. Our initial radiometabolite analysis of human plasma discovered a lipophilic radiometabolite post injection of [11C]CS1P1 and led to initial concerns. However, we recently confirmed the molecular structure of the radiometabolite, and radiometabolite analysis of the rat brain and plasma post-injection of the radiotracer and PET brain studies of the F-18 labeled radiometaobolite demonstrated the radiometabolite does not enter into the brain. Additionally, our team has synthesized ~120 new structurally diverse S1PR1 ligands, and ~30 of them are highly potent (IC50 < 20 nM) and selective for S1PR1. To date, our preliminary data indicate that three F-18 radiotracers are promise for S1PR1 imaging, two of them are F-18 labeled using different procedures and are anticipated to have different pathway compared to [11C]CS1P1. Therefore, in this renewal phase, we will leverage our recent experience with [11C]CS1P1 and develop and transfer an 18F-labeled S1PR1 tracer for clinical investigation. We propose two specific aims: 1) Identify and transfer the most promising 18F-labeled S1PR1 tracer into human investigations. 2) Implement PET imaging human studies in healthy controls and MS patients. Wh...