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

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [¹¹C]CS1P1 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 [¹¹C]CS1P1 detected a radiometabolite peak that is more lipophilic than [¹¹C]CS1P1. 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 [¹¹C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 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 CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [¹⁸F]TZ82121 by the oxidation of [¹⁸F]FS1P1. Radio-HPLC analysis confirmed that [¹⁸F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [¹⁸F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [¹⁸F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [¹¹C]TZ82121, detected in human plasma post injection of [¹¹C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [¹¹C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.