Characterization of dextromethorphan N-demethylation by human liver microsomes. Contribution of the cytochrome P450 3A (CYP3A) subfamily

J. Christopher Gorski, David R. Jones, Steven A. Wrighton, Stephen D. Hall

Research output: Contribution to journalArticle

116 Scopus citations


In an effort to identify the human cytochromes P450 involved in the N-demethylation of dextromethorphan, the kinetics of 3-methoxymorphinan formation were studied in microsomal enzyme systems. Under initial rate conditions, 3-methoxymorphinan formation demonstrated single enzyme Michaelis-Menten kinetics using microsomes obtained from three human livers (Km: 0.52-0.71 mM; Vmax: 375-812 pmol/mg protein/min). B-lymphoblastoid cells expressing CYP3A4 incubated with 0.4 mM dextromethorphan catalyzed the formation of 3-methoxymorphinan at a rate of 22 pmol product/mg protein/min. Midazolam, a prototypic substrate for CYP3A4 and CYP3A5, competitively inhibited dextromethorphan N-demethylation by two human liver microsomal samples with Ki values of 46 ± 10 and 63 ± 8 μM. At a dextromethorphan concentration of 0.4 mM, gestodene (100 μM) inhibited 3-methoxymorphinan formation by approximately 50%. Immunoinhibition of dextro-methorphan N-demethylation using rabbit anti-CYP3A4 antibodies resulted in a 60% decrease in 3-methoxymorphinan formation at a dextromethorphan concentration of 0.4 mM. Additional inhibition studies using furafylline, coumarin, sulfaphenazole, mephenytoin, quinidine, and diethyldithiocarbamic acid, which are selective inhibitors of CYP1A2, CYP2A6, CYP2C8/9, CYP2Cmp, CYP2D6, and CYP2E1, respectively, demonstrated no substantial inhibition of dextromethorphan N-demethylation. Correlation analysis was performed using the rate of 3-methoxymorphinan formation at a concentration of 1mM dextromethorphan and immunoquantified levels of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 and their associated characteristic catalytic activities. A significant correlation was observed between dextromethorphan N-demethylase activity and midazolam 1'- and 4-hydroxylase activity (r2 = 0.77 and 0.69 respectively, N = 19, P < 0.01); the exclusion of those samples containing both CYP3A4 and CYP3A5 increased the correlation significantly (r2 = 0.87 and 0.91 respectively, N = 12, P < 0.01). In the absence of CYP3A5, a significant correlation was observed between 3-methoxymorphinan formation and the sample's erythromycin N-demethylase activity (r2= 0.94, N = 12, P < 0.01), testosterone 6 β-hydroxylase activity (r2 = 0.96, N = 7, P < 0.01) and relative immunoquantified levels of CYP3A4 (r2 = 0.96, N = 12, P < 0.01). Inclusion of those samples expressing CYP3A5 in addition to CYP3A4 reduced the magnitude of the observed correlation. No significant correlation between 3-methoxymorphinan formation and the sample's relative immunoquantified levels of or form-selective activity associated with CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19 (or CYP2Cmp), CYP2D6, and CYP2E1 was observed. In conclusion, dextromethorphan N-demethylation appears to be catalyzed primarily by CYP3A4 and to a lesser extent by CYP3A5 in vitro in humans. Thus, the administration of dextromethorphan to human volunteers may provide a means of simultaneously phenotyping the in vivo activity of CYP2D6 and CYP3A.

Original languageEnglish (US)
Pages (from-to)173-182
Number of pages10
JournalBiochemical Pharmacology
Issue number1
StatePublished - Jul 5 1994


  • CYP3A
  • cytochrome P450
  • dextromethorphan
  • human liver microsomes
  • phenotyping

ASJC Scopus subject areas

  • Pharmacology

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