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 μg protein/ml in human liver microsomes and 0.82–0.26 at 250–1000 μg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 μM. Addition of heat-inactivated microsomal protein (50–450 μg/ml) to native human liver microsomes (50 μg/ml) reduced the amitriptylineN-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 μg/ml) to CYP2C19 (250 μg/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 S₅₀ (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter theV_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.