tailieunhanh - Báo cáo khoa học: Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes Kinetics and sensitivity to cytochrome P450 2D inhibitors

Liver microsomal preparations are routinely used to predict drug interactions that can occurin vivo as a result of inhi-bition 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 sub-strates 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 | Eur. J. Biochem. 270 3768-3777 2003 FEBS 2003 doi Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes Kinetics and sensitivity to cytochrome P450 2D inhibitors Annalise Di Marco1 Dan Yao2 and Ralph Laufer1 1 Department of Pharmacology Istituto di Ricerche di Biologia Molecolare P. Angeletti IRBM Merck Sharp and Dohme Research Laboratories Rome Italy 2Labeled Compound Synthesis Department of Drug Metabolism Merck Research Laboratories Rahway NJ USA 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 ite intrahepatic site of cciion. a amiable 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 vs. M and nmol-min-1 per mg microsomal protein vs. nmol-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