Rat and human liver microsomal incubations, baboon plasma and rat brain extracts were analyzed by radio-HPLC and LC-MS-MS.
In vitro experiments demonstrated the formation by P450s of five polar metabolites. The main routes of LBT-999 metabolism proposed were N-dealkylation, tolyl-hydroxylation and dealkylation plus tolyl-hydroxylation. In vivo in baboons, [18F]LBT-999 was rapidly converted into a [18F]hydroxylated metabolite likely oxidized in plasma into a [18F]carboxylic acid and into unlabeled N-dealkyl-LBT-999. The latter was detected in baboon plasma and in rat brain by LC-MS-MS. The time course of unchanged [18F]LBT-999 decreased rapidly in plasma and was higher than that of [11C]LBT-999 due to the formation of unlabeled N-dealkyl-LBT-999. In rats, striatum-to-cerebellum ratios of [18F]LBT-999, [18F]hydroxylated and [18F]acidic metabolite were 20, 4.2 and 1.65, respectively, suggesting a possible accumulation of the hydroxylated compound in the striatum.
P450s catalyzed the formation of dealkylated and hydroxylated metabolites of LBT-999. In baboons, an extensive metabolism of [18F]LBT-999, with formation of unlabeled N-dealkyl-LBT-999, [18F]fluorobutenaldehyde (or its oxidation product) and [18F]hydroxy-LBT-999 able to penetrate the brain, prevented an easy and accurate estimation of the input function of the radiotracer. CYP3A4 being the main P450 involved in the metabolism of LBT-999, a similar pathway may occur in humans and confound PET quantification.