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 (Ki = 17 and 30 μM, respectively). Tolperisone inhibited methylp-tolyl sulfide oxidation (Ki = 1200 μM) 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.
Footnotes
- Abbreviations used are::
- AUC
- area under the curve
- P450
- cytochrome P450
- FMO
- flavin-containing monooxygenase
- HPLC
- high-performance liquid chromatography
- HLM
- human liver microsomes
- SKF-525A
- proadifen-hydrochloride
- ABT
- 1-aminobenzotriazole
- BI
- 1-benzylimidazole
- MpTS
- methylp-tolyl-sulfide
- MpTSO
- methyl p-tolyl sulfoxide
- LC/MS
- liquid chromatography-mass spectrometry
- V
- reaction rate
- S
- substrate concentration
- M1
- hydroxymethyl-tolperisone
- M2
- carbonyl-reduced form of hydroxymethyl-tolperisone
- Clint
- intrinsic clearance
- Received October 28, 2002.
- Accepted February 10, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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