Investigation of the in Vitro Metabolism of the Analgesic Flupirtine

Abstract

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.Flupirtine maleate (1) (ethyl-N-[2-amino-6-(4-fluoro-phenylmethylamino)pyridin-3-yl]carbamate maleate, Katadolon) (Scheme 1), a centrally acting nonopiate analgesic with muscle tone-reducing activity, does not show the classic side effects of opiates or nonsteroidal anti-inflammatory analgesics (Jakovlev et al., 1985;Szelenyi and Nickel, 1991). The drug has been effectively and safely used in Germany for 20 years. The mode of action is now mostly understood. Modification of prostaglandin formation is of minor importance for the analgesic action of flupirtine (Darius and Schrör, 1985). Evidence has accumulated that flupirtine interacts indirectly with the N-methyl-D-aspartate-responsive subtype of the glutamate receptor, possibly by opening K ϩ channels (Jakob and Krieglstein, 1997; Kornhuber et al., 1999a,b). Clinical interest in the compound is growing as further indications for the drug are found. For example, successful treatment of fibromyalgia has been reported (Stoll, 2000). In particular, the neuroprotective properties attributed to flupirtine are making it a possible candidate for treatment of Parkinson's and Alzheimer's diseases, Creutzfeldt-Jacob disease, and other neurodegenerative ailments (Schuste...

Metabolic activation and analgesic effect of flupirtine in healthy subjects, influence of the polymorphic NAT2, UGT1A1 and GSTP1

“…The spectrum of side effects includes a very rare, mild and transient elevation of liver enzymes, aggravation of preexisting liver disease and, in single cases, severe and fatal liver failure [13][14][15][16][17][18]. There is evidence that the liver injury may be initiated by metabolic formation of hepatotoxic metabolites [19].…”

“…The metabolism of flupirtine is not yet completely understood. In addition to the formation of para-fluorohippuric acid [19,20], the drug undergoes hydrolytic cleavage of the carbamate group followed by subsequent acetylation via the human N-acetyltransferases (NAT) 1 and the polymorphic NAT2 generating the pharmacologically active metabolite D13223 [4,19,20,23]. Both the parent flupirtine and D13223 can be conjugated with glucuronic acid by UDP-glucuronosyltransferases (UGT) most likely by the isoforms UGT1A1, 1A3, 1A4 and 1A9, as shown in vitro with recombinant enzymes for retigabine, an anticonvulsant drug closely related in chemical structure to flupirtine [24,25].…”

Isolation, Structure, Synthesis and Cytotoxicity of an Unprecedented Flupirtine Dimer

“…Thus, commercial samples of 1 (as its maleate) were bought. Their analytical investigation showed the presence of a dimeric compound with m/z = 607.2 (ESI-MS, cation-sensitive mode) corre- [7] were detected in these samples. Isolation of this novel dimer by semipreparative HPLC yielded enough material for its investigation by NMR.…”

“…1); the structure of the dimers was deduced by interpretation of HRMS data. For these dimers quasi-molecular ions m/z = 607.258937 and m/z = 607.259010 matching [M+H] + with an empirical formula M = C 30 H 33 F 2 N 8 O 4 were detected [7].…”

“…Recently, the quantification of 1 and its related compounds (e. g. impurities resulting either from synthesis or by decomposition during storage) in pharmaceutical dosage forms by UPLC has been reported [6]. Several years ago, Bednarski et al [7] re-examined some aspects of the metabolism of 1 and revealed the formation of flupirtine dimers 2 and 3 (Fig. 1); the structure of the dimers was deduced by interpretation of HRMS data.…”