Surrogate assays for drug metabolism and inhibition are traditionally performed in buffer systems at pH 7.4, despite evidence that hepatocyte intracellular pH is 7.0. This pH gradient can result in a pK-dependent change in intracellular/extracellular concentrations for ionizable drugs that could affect predictions of clearance and P450 inhibition. The effect of microsomal incubation pH on in vitro enzyme kinetic parameters for CYP2C9 (diclofenac, (S)-warfarin) and CYP3A4 (midazolam, dextromethorphan, testosterone) substrates, enzyme specific reversible inhibitors (amiodarone, desethylamiodarone, clozapine, nicardipine, fluconazole, fluvoxamine, itraconazole) and a mechanism-based inhibitor (amiodarone) was investigated. Intrinsic clearance through CYP2C9 significantly increased (25% and 50% for diclofenac and (S)-warfarin respectively) at intracellular pH 7.0 compared with traditional pH 7.4. The CYP3A4 substrate dextromethorphan intrinsic clearance was decreased by 320% at pH 7.0, while midazolam and testosterone remained unchanged. Reversible inhibition of CYP2C9 was less potent at pH 7.0 compared with 7.4, while CYP3A4 inhibition potency was variably affected. Maximum enzyme inactivation rate of amiodarone toward CYP2C9 and CYP3A4 decreased at pH 7.0, while the irreversible inhibition constant remained unchanged for CYP2C9, but decreased for CYP3A4 at pH 7.0. Predictions of clearance and drug-drug interactions made through physiologically based pharmacokinetic models were improved with the inclusion of predicted intracellular concentrations based at pH 7.0 and in vitro parameters determined at pH 7.0. No general conclusion on the impact of pH could be made and therefore a recommendation to change buffer pH to 7.0 cannot be made at this time. It is recommended that the appropriate hepatocyte intracellular pH 7.0 be used for in vitro determinations when in vivo predictions are made.
The proton gradient from the intracellular space to plasma creates an unbound drug gradient for weak acids and bases that could modulate apparent drug clearance and drug-drug interactions. Cytochrome P450 intrinsic clearance and inhibitor potency are routinely determined in vitro at the plasma pH of 7.4 rather than the intrahepatocyte pH of 7.0. We determined the impact of pH on in vitro enzyme kinetic parameters and inhibition potency for substrates (bufuralol, dextromethorphan), reversible inhibitors (quinidine, amiodarone, desethylamiodarone, clozapine), and mechanism-based inhibitors (paroxetine, desethylamiodarone) of the major drug metabolizing-enzyme CYP2D6. The lower intracellular pH 7.0 compared with pH 7.4 resulted in a 60 and 50% decrease in intrinsic clearance for the substrates bufuralol and dextromethorphan, respectively. Reversible inhibition constants for three of the four inhibitors tested were unaffected by pH, whereas for the inhibitor quinidine, a 2-fold increase in the inhibition constant was observed at pH 7.0. For time-dependent inhibitors desethylamiodarone and paroxetine, changes in time-dependent inhibition parameters were different for each inhibitor. These results were incorporated into physiologically based pharmacokinetic models indicating that the changes in in vitro parameters determined at pH 7.0 offset the effect of increased unbound intracellular concentrations on apparent clearance and extent of drug-drug interactions. However, this offset between concentration and enzyme activity cannot be generalized for all substrates, inhibitors, and enzymes, as the effect of a lower pH in vitro varied significantly; therefore, it would be prudent to determine in vitro enzyme parameters at the hepatocyte-appropriate pH 7.0.
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