Identification of Cytochrome P450 Isoforms Involved in the Metabolism of Paroxetine and Estimation of Their Importance for Human Paroxetine Metabolism using a Population-Based Simulator

We identify here for the first time the low-affinity CYP isoforms that metabolize paroxetine, using cDNA-expressed human CYPs measuring substrate depletion and paroxetine-catechol (product) formation by liquid chromatography tandem mass spectrometry. CYP1A2, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were identified as paroxetine-catechol–forming CYP isoforms, and CYP2C19 and CYP2D6 were identified as metabolizing CYP isoforms by substrate depletion. Michaelis-Menten constants K m and V max were determined by product formation and substrate depletion. Using selective inhibitory studies and a relative activity factor approach for pooled and single-donor human liver microsomes, we confirmed involvement of the identified CYP isoforms for paroxetine-catechol formation at 1 and 20 µM paroxetine. In addition, we used the population based simulator Simcyp® to estimate the importance of the identified paroxetine metabolizing CYP isoforms for human metabolism taking mechanism based inhibition into account. The amount of active hepatic CYP2D6 and CYP3A4 (not inactivated by mechanism based inhibition) was also estimated by Simcyp®. For extensive and poor metabolizers of CYP2D6, Simcyp®-estimated pharmacokinetic profiles were in good agreement with those reported in published in vivo studies. Considering the kinetic parameters, inhibition results, relative activity factor calculations, and Simcyp® simulations, CYP2D6 (high affinity) and CYP3A4 (low affinity) are most likely to be the major contributors to paroxetine metabolism in humans. For some individuals CYP1A2 could be of importance for paroxetine metabolism, whereas the importance of CYP2C19 and CYP3A5 is probably limited. studies in human liver microsomes (HLMs) and comparing the results to those obtained using a relative activity factor (RAF) approach, we investigated the validity of the obtained kinetic parameters. Finally, in order to estimate the importance of the CYP isoforms for human paroxetine metabolism an IVIVE of paroxetine metabolism was performed using the population-based simulator Simcyp® to generate pharmacokinetic simulations that incorporate aspects of MBI. These pharmacokinetic simulations were compared with existing in vivo pharmacokinetic data for paroxetine to validate the simulations in EM and PM CYP2D6 population groups. Comprehensive identification of the CYP isoforms involved in the metabolism of paroxetine, estimation of their kinetic parameters, and subsequent integration into a physiologically based pharmacokinetic model such as Simcyp® will provide a platform for a better fundamental understanding of paroxetine pharmacokinetics in vitro as well as in the clinic. Liquid Mass Spectrometry Analysis for Paroxetine-Depletion Experiments. Analysis was done on an Agilent 1100 (Agilent Technologies, Paroxetine-Catechol Formation Kinetic Studies. Incubations used to derive kinetic constants by paroxetine-catechol formation had the following CYP/HLM and paroxetine concentrations: CYP1A2 45 pmol/ml, CYP2C19 20 pmol/ml, CYP3A4 20 pmol/ml, CYP3A5 50 pmol/ml, and 0.61, 1.3, 2.2, 3.6, 6.1, 9, 12, 15, 20, 30, 51, 73, 103, and 152 µM paroxetine; CYP2D6 1.5 pmol/ml, and 0.0061, 0.010, 0.017, 0.028, 0.047, 0.079, 0.13, 0.22, 0.36, 0.61, 1.21, 2.19, 3.64, and 6.07 µM paroxetine; pHLM and sdHLM(1) (PM2D6 HLM) 0.20 mg protein/ml, and 0.010, 0.028, 0.079, 0.22, 0.61, 1.3, 3.6, 6.1, 15, 30, 51, 73, 103, and 152 µM paroxetine. Incubation time was 5 min, and all experiments were done with duplicates of microsomes and background formation samples. Background formation of paroxetine-catechol was subtracted from the formation in microsomes to obtain the amount of actively formed paroxetine-catechol. The paroxetine concentration was calculated by a geometric mean (eq. 3) if the depletion was above 20%: