Phase II - Kinetic Analysis of Test Article Metabolism
The rates of how quickly the test article disappears are monitored in the incubation samples with at least eight concentrations of the test article in triplicates under optimized conditions. Data are analyzed using Michaelis-Menten curve fitting, and kinetic parameters Km and Vmax are determined for the test article.
Phase III - Inhibition Analysis Using CYP-specific Chemical Inhibitors
The test article is incubated in triplicate at a concentration <Km with pooled human hepatic microsomes in the absence or presence of the enzyme-selective chemical inhibitors. Control incubations are performed in the absence of the inhibitor using vehicle solvent only.
Phase IV - Metabolism Using Recombinant Human CYPs
The test article is incubated in triplicate at a concentration <Km with a range of recombinant human CYPs and UGTs, and other DME (see above). The relative concentration of the test article at 0 and 60 minutes is measured.