title: Identification of metabolic pathways involved in the biotransformation of tolperisone by human microsomal enzymes.
abstract: The in vitro metabolism of tolperisone, 1-(4-methyl-phenyl)-2-methyl-3-(1-piperidino)-1-propanone-hydrochloride, a centrally acting muscle relaxant, was examined in human liver microsomes (HLM) and recombinant enzymes. Liquid chromatography-mass spectrometry measurements revealed methyl-hydroxylation (metabolite at m/z 261; M1) as the main metabolic route in HLM, however, metabolites of two mass units greater than the parent compound and the hydroxy-metabolite were also detected (m/z 247 and m/z 263, respectively). The latter was identified as carbonyl-reduced M1, the former was assumed to be the carbonyl-reduced parent compound. Isoform-specific cytochrome P450 (P450) inhibitors, inhibitory antibodies, and experiments with recombinant P450s pointed to CYP2D6 as the prominent enzyme in tolperisone metabolism. CYP2C19, CYP2B6, and CYP1A2 are also involved to a smaller extent. Hydroxymethyl-tolperisone formation was mediated by CYP2D6, CYP2C19, CYP1A2, but not by CYP2B6. Tolperisone competitively inhibited dextromethorphan O-demethylation and bufuralol hydroxylation (K(i) = 17 and 30 microM, respectively). Tolperisone inhibited methyl p-tolyl sulfide oxidation (K(i) = 1200 microM) in recombinant flavin-containing monooxygenase 3 (FMO3) and resulted in a 3-fold (p < 0.01) higher turnover number using rFMO3 than that of control microsomes. Experiments using nonspecific P450 inhibitors-SKF-525A, 1-aminobenzotriazole, 1-benzylimidazole, and anti-NADPH-P450-reductase antibodies-resulted in 61, 47, 49, and 43% inhibition of intrinsic clearance in HLM, respectively, whereas hydroxymethyl-metabolite formation was inhibited completely by nonspecific chemical inhibitors and by 80% with antibodies. Therefore, it was concluded that tolperisone undergoes P450-dependent and P450-independent microsomal biotransformations to the same extent. On the basis of metabolites formed and indirect evidences of inhibition studies, a considerable involvement of a microsomal reductase is assumed.
authors.affiliation: Division of Pharmacology and Drug Safety Research, Gedeon Richter Ltd., Budapest, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Dalmadi
authors.forename: János
authors.initials: J
authors.lastname: Leibinger
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Szeberényi
authors.forename: Tímea
authors.initials: T
authors.lastname: Borbás
authors.forename: Sándor
authors.initials: S
authors.lastname: Farkas
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Szombathelyi
authors.forename: Károly
authors.initials: K
authors.lastname: Tihanyi
mesh_terms: D000906:Antibodies / Q000378:metabolism; D019540:Area Under Curve; D019389:Cytochrome P-450 CYP2D6 / Q000276:immunology / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D009125:Muscle Relaxants, Central / Q000378:metabolism* / Q000493:pharmacokinetics; D011485:Protein Binding; D013379:Substrate Specificity; D014049:Tolperisone / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D000906:Antibodies; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009125:Muscle Relaxants, Central; C400322:cytochrome P-450 CYP2C subfamily; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D014049:Tolperisone