title: Impact of nonspecific binding to microsomes and phospholipid on the inhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo drug interactions.
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 IC(50) 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.
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut 06340, USA.
authors.forename: Jeannine M
authors.initials: JM
authors.lastname: Margolis
authors.forename: R Scott
authors.initials: RS
authors.lastname: Obach
mesh_terms: D001596:Benzylamines / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004983:Ethanolamines / Q000378:metabolism; D005473:Fluoxetine / Q000494:pharmacology; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D066298:In Vitro Techniques; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology*; D011485:Protein Binding; D011802:Quinidine / Q000494:pharmacology
chemical_list: D001596:Benzylamines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D010743:Phospholipids; D005473:Fluoxetine; C413864:ezlopitant; C039685:Ro 03-7410; D019389:Cytochrome P-450 CYP2D6; D011802:Quinidine; D007099:Imipramine