Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [C] has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [C] detected a radiometabolite peak that is more lipophilic than [C]. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [C] in rats, nonhuman primates, and humans. is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive . To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated mono-oxidation of produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with identified the metabolite as -oxide , also named . The MS fragmentation pattern of -oxide also matched that of the major metabolite in rat plasma. To confirm that metabolite does not enter the brain, we radiosynthesized [F] by the oxidation of [F]. Radio-HPLC analysis confirmed that [F] matched the radiometabolite observed in rat plasma post injection of [F]. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [F] does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite -oxide [C], detected in human plasma post injection of [C], does not enter the brain to confound quantitative PET data analysis. [C] is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.