Lack of low Km diazepam N-demethylase in livers of poor metabolizers for S-mephenytoin 4'-hydroxylation.
Metabolism of diazepam was studied in vitro to identify the forms of cytochrome P450 (CYP) responsible for N-demethylation (nordazepam formation) and 3-hydroxylation (temazepam formation), using liver microsomes obtained from extensive (EM) and poor metabolizers (PM) for S-mephenytoin 4'-hydroxylation. Involvement of at least two P450 forms in diazepam N-demethylation was suggested by a biphasic pattern in Lineweaver-Burk and Eadie-Hofstee plots from the EM, whereas a monophasic pattern was observed from the PM liver microsomes. The kinetic parameters for the N-demethylation in the EM group were: Km 1, 19.4 +/- 0.4 microM; Vmax 1, 0.27 +/- 0.04 nmol min-1 per mg protein; Km 2, 346 +/- 34 microM; Vmax2, 1.82 +/- 0.63 nmol min-1 per mg protein (n = 3, mean +/- SD). The PM group showed the mean values of Km and Vmax (Km, 319 +/- 30 microM; Vmax, 1.49 +/- 0.62 nmol min-1 per mg protein) (n = 3) similar to those of Km2 and Vmax2 in the EM group. An antibody raised against CYP2C9 (anti-human CYP2C) strongly inhibited diazepam N-demethylation in EM liver microsomes at a low substrate concentration (20 microM). However, the anti-human CYP2C showed no clear inhibition of N-demethylation in EM liver microsomes at a high substrate concentration (200 microM). Diazepam N-demethylation in PM liver microsomes was not clearly inhibited by the anti-human CYP2C at either the low or high substrate concentrations. These data suggest that different P450 forms mediated diazepam N-demethylation in EM and PM liver microsomes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Yasumori
T
T
Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
Li
Q H
QH
Yamazoe
Y
Y
Ueda
M
M
Tsuzuki
T
T
Kato
R
R
D000906:Antibodies; D001189:Aryl Hydrocarbon Hydroxylases; D065731:Cytochrome P-450 CYP2C19; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003975:Diazepam / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007700:Kinetics; D008617:Mephenytoin / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010641:Phenotype
D000906:Antibodies; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D010089:Oxidoreductases, N-Demethylating; C067539:diazepam N-demethylase; D003975:Diazepam; D008617:Mephenytoin