Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes

Eur J Biochem. 2003 Sep;270(18):3768-77. doi: 10.1046/j.1432-1033.2003.03763.x.


Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.

MeSH terms

  • Animals
  • Benzoflavones / pharmacology
  • Carbon Radioisotopes
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / metabolism
  • Dextromethorphan / analogs & derivatives
  • Dextromethorphan / metabolism*
  • Enzyme Inhibitors / pharmacology*
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism*
  • Inhibitory Concentration 50
  • Kinetics
  • Male
  • Methylation
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / metabolism*
  • Oxidoreductases, O-Demethylating / antagonists & inhibitors
  • Protein Binding
  • Quinidine / metabolism
  • Quinidine / pharmacology
  • Quinine / metabolism
  • Quinine / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Sulfaphenazole / pharmacology
  • Troleandomycin / pharmacology


  • Benzoflavones
  • Carbon Radioisotopes
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Sulfaphenazole
  • alpha-naphthoflavone
  • Dextromethorphan
  • Cytochrome P-450 Enzyme System
  • Quinine
  • Troleandomycin
  • Oxidoreductases, O-Demethylating
  • Quinidine