Development of an in vitro incubation procedure for screening of CYP2D6 intrinsic clearance

The in vitro intrinsic clearances (CLint) for the metabolism of p-methoxymethamphetamine (PMMA) and fluoxetine by the CYP2D6 enzyme were calculated using a steady-state (SS) approach and a new general enzyme (GE) method, which measures the formation of product and the depletion of substrate as a function of time. For PMMA, the SS experiment resulted in a CLint of 2.7 ± 0.2 μL pmol 2D6−1 min−1 and the GE experiment resulted in a CLint of 3.0 ± 0.6 μL pmol 2D6−1 min−1. For fluoxetine, the SS experiment resulted in a CLint of 0.33 ± 0.17 μL pmol 2D6−1 min−1 and the GE experiment resulted in a CLint of 0.188 ± 0.013 μL pmol 2D6−1 min−1. We used two kinetic modeling techniques that can accommodate atypical kinetic models. We also show that the addition of fluoxetine results in a 10-fold decrease in the observed intrinsic clearance of PMMA, confirming that fluoxetine is a potent inhibitor of the liver enzyme CYP2D6.