Abstract

The effects of microsomal concentration on the inhibitory potencies of four compounds—fluoxetine, quinidine, imipramine, and ezlopitant—on heterologously expressed recombinant CYP2D6-catalyzed bufuralol 1′-hydroxylase activity were determined. Increasing microsomal concentration from 0.0088 to 2.0 mg/ml, using additional microsomes not containing cytochrome P450, resulted in a marked increase in IC₅₀ and K_I values for fluoxetine, ezlopitant, and imipramine, when inhibition constants were calculated using the nominal concentration of inhibitor added to the incubation mixture. The extent of nonspecific binding of these inhibitors to microsomes was determined using equilibrium dialysis. The extent of binding increased with increasing microsomal concentration. Binding was greatest for ezlopitant, followed by fluoxetine, imipramine, and quinidine. Correcting inhibition constants for the extent of nonspecific binding resulted in greater consistency of these values with differing microsomal protein concentrations. This effect was also studied with added phospholipid. Inhibition constants increased with increasing phospholipid, and nonspecific binding was also observed for these four drugs to phospholipid. This suggests that the phospholipid component of microsomes possesses some or all of the responsibility for nonspecific binding, and its effect on inhibitors of drug-metabolizing enzymes. These findings suggest that inhibition constants for drugs as inhibitors of microsomal drug-metabolizing enzymes, such as cytochrome P450, should be corrected for the extent of nonspecific binding to components of the in vitro matrix. The implications of this on the prediction of drug-drug interactions from in vitro data are discussed.