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Vol. 293, Issue 2, 343-350, May 2000
Department of Pharmacology and Experimental Therapeutics, Tufts
University School of Medicine, Boston, Massachusetts (K.V., L.L.v.M.,
D.J.G.); Division of Clinical Pharmacology, New England Medical Center
Hospital, Boston, Massachusetts (K.V., L.L.v.M., D.J.G.); and
Department of Drug Metabolism, Pfizer Central Research, Groton,
Connecticut (R.S.O.)
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 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 Vmax. Addition of
metabolically inactive microsomes from untransfected human
lymphoblastoid cells (750 µg/ml) to CYP2C19 (250 µg/ml protein) increased the apparent Km value for
amitriptyline N-demethylation by 3.5-fold and increased
the uncompetitive substrate inhibition constant
(Ks) 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 the
Vmax 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.
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