In Vitro Evaluation of Drug Interaction Caused by Enzyme Inhibition —HAB Protocol—

Abstract

Protocols for evaluating drug interaction caused by enzyme inhibition were established by the Drug Interaction Database Working Group of the Human and Animal Bridge Discussion Group (HAB). Basically, procedures for measurements of the inhibition constant (Ki) of the test article were methodically investigated. 7-Ethoxyresorufin (CYP1A1&2), coumarin (CYP2A6), 7-benzyloxyresorufin (CYP2B6), tolbutamide (CYP2C8&9), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), chlorzoxazone (CYP2E1), nifedipine (CYP3A4) and testosterone (CYP3A4) were used as specific substrates for various isoforms of human liver cytochrome P450 (P450). At least 3 concentrations of the test article were appropriately selected in experiment on concentration-dependency of enzyme inhibition, and used in measurement of apparent Ki value by Dixon plot. In Ki measurements, the P450 substrate was added to the assay system at 3 different concentrations (Km, 1/2 Km and 2 Km). To correct the apparent Ki value of the test article by the binding to the liver microsomes, free concentration of the test article in the system for Ki measurements was determined most conveniently by ultracentrifugation. In this HAB-protocol, method to judge the mechanismbased inhibitor by detecting increased inhibition by the test article after preincubation in the presence of NADPH was also included. Magnitude of drug interaction (R) was estimated according to the equation: R=1+I/Ki, where I represents the concentration of the test article, most preferably the C_max value either in the peripheral or the portal blood.