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 Statement The demyelination tracer [¹⁸F]3F4AP was found to undergo rapid metabolism in humans, which could compromise its utility. Understanding the enzymes and metabolic products involved may offer strategies to reduce metabolism. Using a combination of in vitro assays and chemical syntheses, this report shows that cytochrome P450 enzyme CYP2E1 is likely responsible for [¹⁸F]3F4AP metabolism, that 4-amino-5-fluoroprydin-3-ol (5-hydroxy-3F4AP) and 4-amino-3-fluoropyridine 1-oxide (3F4AP N-oxide) are the main metabolites, and that deuteration is unlikely to improve the stability of the tracer in vivo.