Buffer compositions strongly impact the in vitro assessment of CYP fraction metabolized using human liver microsomes or expressed isoenzymes

Abstract

We systematically evaluated the effects of different buffer compositions on the activities of cytochrome P450 (CYP)1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A in human liver microsomes and in recombinant human CYP expression systems using the in vitro cocktail method. The activities of all CYP isoenzymes varied depending on the buffer conditions, particularly the activity of CYP1A2, which varied by 27.0-fold in phosphate buffer and HEPES or Tris-HCl buffer. The difference in the metabolic activities of CYP2D6 and CYP3A4 was smaller but significant, showing values about 2.0- and 3.0-fold higher in phosphate buffer than in HEPES or Tris-HCl buffer. A similar trend was observed for recombinant human CYP3A4/5. In contrast, the metabolic activities of CYP2A6 and CYP2B6 in phosphate buffer decreased by less than half as the phosphate concentration was increased. Changes in the metabolic activity of multiple CYP isoenzymes in buffers with different compositions suggest that the fraction metabolized (fm) evaluated in vitro differs depending on the experimental conditions. Specifically, the previously reported fm values of CYP1A2, CYP2B6, CYP2D6, and CYP3A in phosphate buffer may be overestimated. When evaluating the fm of these isoenzymes, we recommend conducting experiments under multiple buffer conditions.