Metabolic Stability of the Demyelination PET Tracer [18F]3F4AP and Identification of its Metabolites

Abstract

Abstract[18F]3-fluoro-4-aminopyridine ([18F]3F4AP) is a PET tracer for imaging demyelination based on the multiple sclerosis drug 4-aminopyridine (4AP, dalfampridine). This radiotracer was found to be stable in rodents and nonhuman primates imaged under isoflurane anesthesia. However, recent findings indicate that its stability is greatly decreased in awake humans and mice. Since both 4AP and isoflurane are metabolized primarily by cytochrome P450 enzymes, particularly CYP2E1, we postulated that this enzyme may be responsible for the metabolism of 3F4AP. Here, we investigated the metabolism of [18F]3F4AP by CYP2E1 and identified its metabolites. We also investigated whether deuteration, a common approach to increase the stability of drugs, could improve its stability. Our results demonstrate that CYP2E1 readily metabolizes 3F4AP and its deuterated analogues and that the primary metabolites are 5-hydroxy-3-fluoro-4-aminopyridine and 3-fluoro-4-aminopyridine N-oxide. Although deuteration did not decrease the rate of the CYP2E1 mediated oxidation, our findings explain the diminished in vivo stability of 3F4AP compared to 4AP and further our understanding of when deuteration may improve the metabolic stability of drugs and PET ligands.Significance StatementUnderstanding the metabolic stability of PET tracers is paramount to its application in humans as metabolism, which varies from person to person, can affect the target-to-background signal. This study identified the predominant enzyme that metabolizes the demyelination PET tracer [18F]3F4AP and its metabolites. These findings may allow assessment of whether the radiometabolites can get into the brain and potentially lead to tracers with enhanced stability. Furthermore, this study furthers our understanding of when deuteration can improve metabolic stability.