Abstract

The in vitro metabolism of flupirtine, ethyl-N-[2-amino-6-(4-fluorophenylmethyl-amino)pyridine-3-yl]carbamate, a centrally acting analgesic with muscle tone-reducing activity, was studied. Two flupirtine metabolites were already known: the N-acetylated analog D13223 and 4-fluorohippuric acid. The structure of flupirtine suggested that redox chemistry may play a role in metabolism, and cyclic voltammetry studies showed that the drug undergoes facile and irreversible redox reactions. Thus, oxidative metabolism was investigated first. With CYP3A1-induced rat liver microsomes an 18% turnover of flupirtine and a 20 to 25% turnover of D13223 took place over 30 min, but less than 5% turnover of flupirtine was observed with all human liver microsomal preparations tested, evidence that cytochrome P450 does not contribute appreciably to the metabolism in humans. Likewise, no involvement of human monoamine oxidase (isoforms A and B) was found for either flupirtine or D13223. In contrast, flupirtine was an excellent substrate for both human myeloperoxidase and horse radish peroxidase (HRP). These enzymes produced detectable amounts of oxidation products. Incubations of flupirtine with HRP produced an oxidation product that could be trapped with glutathione, the resulting glutathione conjugate was characterized by mass spectrometry and NMR. Metabolism of D13223 by both peroxidases was also observed but to a much lesser extent. Porcine liver esterases cleave the carbamate group of flupirtine, and both human N-acetyltransferases 1 and 2 acetylated the hydrolysis product, presumably descarboethoxyflupirtine, with nearly equal efficiencies to yield D13223. Incubations of human liver microsomes with flupirtine or the metabolite D13223 together with UDP-glucuronic acid gave two isomeric N-glucuronides in both cases.