Metabolic Stability of the Demyelination Positron Emission Tomography Tracer [¹⁸F]3-Fluoro-4-Aminopyridine and Identification of Its Metabolites

Abstract

D 2 O, deuterium oxide; DCl, deuterium chloride; 3F4AP, 3-fluoro-4-aminopyridine; HPLC, high-performance liquid chromatography; HRMS, high resolution mass spectrometry; IC 50 , half maximal inhibitory concentration; K i , inhibition constant; KIE, kinetic isotope effect;

Chemical and biophysical characterization of novel potassium channel blocker 3-fluoro-5-methylpyridin-4-amine

“…3 B). We also compared these results with our previous results for 4AP, 3F4AP and the positive control tranylcypromine 30 . This analysis showed that 5Me3F4AP has an IC 50 about two times higher than 3F4AP and about 23 times lower than 4AP (Fig.…”

“…A limitation of this study is that we did not test other possible metabolic enzymes, which may play a role in vivo. Nevertheless, it is reasonable to test CYP2E1 given that prior studies strongly suggest this enzyme is primarily responsible for the metabolism of 3F4AP and 4AP 27 , 30 . Furthermore, the high in vivo stability of 4AP (~ 70% parent fraction in plasma 24 h post oral administration 42 ) compared to 3F4AP (< 50% parent fraction in plasma 60 min post intravenous administration 27 ) is consistent with the measured CYP2E1 oxidation rates in vitro for these compounds 30 .…”

“…To synthesized radiolabeled [ 18 F]5Me3F4AP, one may consider using similar methods to the ones previously developed to synthesize [ 18 F]3F4AP such as 18 F/ 19 F exchange of a fluorinated electron-deficient pyridine N-oxide derivative followed by palladium-mediated reduction 24 or denitroradiofluorination of a 4-methylester pyridine derivative followed by conversion of the ester group to an amino group through rearrangement 25 . In order to test the in vivo stability of [ 18 F]5Me3F4AP, one can consider collecting blood at certain times post injection and analyzing for radiometabolites using a radioHPLC 26 , 30 . Finally, to assess the imaging properties of the tracer, one could consider performing PET imaging studies in rodent models of demyelination and comparing the binding to the lesioned areas with that of [ 18 F]3F4AP 22 .…”

“…This reduction in metabolic stability was found to arise from the inhibitory effect of isoflurane on the metabolism of [ 18 F]3F4AP as evidenced by the fact that awake mice metabolize the tracer faster than anesthetized mice, with 17.7 ± 1.7 PPF (n = 11) vs 74.8 ± 1.6 PPF (n = 10) respectively 35 min post-injection 29 . Additional studies indicate that [ 18 F]3F4AP is oxidized at the 5-position to 5-hydroxy-3F4AP by the cytochrome P450 enzyme CYP2E1 30 , which prompted us to look for more stable derivatives with suitable binding affinity and brain permeability.…”

Common anesthetic used in preclinical PET imaging inhibits metabolism of the PET tracer [18F]3F4AP

“…CYP2E1 plays an integral part in the breakdown and clearance of a variety of small molecules including acetaminophen and halogenated anesthetics 23 . Recent in vitro studies from our laboratory have shown that CYP2E1 is also able to metabolize (nonradioactive) 3F4AP very efficiently 24 . In an in vitro competition assay the affinity of CYP2E1 for 3F4AP was 50 times higher than the affinity for 4AP, suggesting a primary role for CYP2E1 in metabolizing 3F4AP.…”