title: Microsomal binding of amitriptyline: effect on estimation of enzyme kinetic parameters in vitro.
abstract: The effect of binding of amitriptyline to human liver microsomes and to microsomes from human B-lymphoblastoid cells on the estimation of enzyme kinetic parameters describing N-demethylation to nortriptyline was investigated using a combination of microsomal binding and in vitro enzyme kinetic studies. Quantitative binding in both matrices increased with higher microsomal protein concentrations (free fractions 0.88-0.32 at 100-500 microg protein/ml in human liver microsomes and 0.82-0.26 at 250-1000 microg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 microM. Addition of heat-inactivated microsomal protein (50-450 microg/ml) to native human liver microsomes (50 microg/ml) reduced the amitriptyline N-demethylation rate in a protein concentration dependent manner. This effect was greater at lower substrate concentrations and was overcome by saturating concentrations of substrate, thereby decreasing the apparent affinities of the high- and low-affinity components of the N-demethylation process, with minimal effect on the net V(max). Addition of metabolically inactive microsomes from untransfected human lymphoblastoid cells (750 microg/ml) to CYP2C19 (250 microg/ml protein) increased the apparent K(m) value for amitriptyline N-demethylation by 3.5-fold and increased the uncompetitive substrate inhibition constant (K(s)) by 2.2-fold, making substrate inhibition essentially undetectable. A similar effect was seen with CYP3A4, with a 1.8-fold increase in the S(50) (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter the V(max) of either CYP isoform to any appreciable extent. These findings emphasize the importance of incorporating microsomal binding in the estimation of enzyme kinetic parameters in vitro and making appropriate corrections for unbound drug concentrations.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: K
authors.initials: K
authors.lastname: Venkatakrishnan
authors.forename: L L
authors.initials: LL
authors.lastname: von Moltke
authors.forename: R S
authors.initials: RS
authors.lastname: Obach
authors.forename: D J
authors.initials: DJ
authors.lastname: Greenblatt
mesh_terms: D000465:Algorithms; D000639:Amitriptyline / Q000378:metabolism*; D000929:Antidepressive Agents, Tricyclic / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003640:Dealkylation; D003956:Dialysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007945:Leukemia, Lymphoid / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000929:Antidepressive Agents, Tricyclic; D000639:Amitriptyline; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; C104464:CYP3A protein, human; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human