Effects of CYP2C19 genotype and CYP2C9 on fluoxetine N-demethylation in human liver microsomes.
OBJECTIVE
The present study was designed to define the kinetic behavior of fluoxetine N-demethylation in human liver microsomes and to identify the isoforms of cytochrome P-450 (CYP) involved in this metabolic pathway.


METHODS
The kinetics of Ne formation of norfluoxetine was determined in human liver microsomes from six genotyped CYP2C19 extensive metabolizers (EM). The correlation studies between the fluoxetine N-demethylase activity and various CYP enzyme activities were performed. Selective inhibitors or chemical probes of various cytochrome P-450 isoforms were also employed.


RESULTS
The kinetics of norfluoxetine formation in all liver microsomes were fitted by a single-enzyme Michaelis-Menten equation (mean Km=32 micromol/L+/-7 micromol/L). Significant correlations were found between N-demethylation of fluoxetine at both 25 micromol/L and 100 micromol/L and 3-hydroxylation of tolbutamide at 250 micromol/L (r1=0.821, P1=0.001; r2=0.668, P2=0.013), respectively, and S-mephenytoin 4'-hydroxylase activity (r=0.717, P=0.006) at high substrate concentration of 100 micromol/L. S-mephenytoin (SMP) (a CYP2C19 substrate) at high concentration and sulfaphenazole (SUL) (a selective inhibitor of CYP2C9) substantially inhibited norfluoxetine formation. The reaction was minimally inhibited by coincubation with chemical probe, inhibitor of CYP3A4 (triacetyloleandomycin, TAO). The inhibition of fluoxetine N-demethylation at high substrate concentration (100 micromol/L) was greater in PM livers than in EM livers (73 % vs 45 %, P < 0.01) when the microsomes were precoincubated with SUL plus TAO.


CONCLUSIONS
Cytochrome P-450 CYP2C9 is likely to be a major CYP isoform catalyzing fluoxetine N-demethylation in human liver microsomes at a substrate concentration close to the therapeutic level, while polymorphic CYP2C19 may play a more important role in this metabolic pathway at high substrate concentration.
Liu
Z Q
ZQ
Pharmacogenetics Research Institute, Hunan Medical University, Changsha 410078, China.
Shu
Y
Y
Huang
S L
SL
Wang
L S
LS
He
N
N
Zhou
H H
HH
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D005473:Fluoxetine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D011110:Polymorphism, Genetic; D017367:Selective Serotonin Reuptake Inhibitors / Q000378:metabolism; D013426:Sulfaphenazole / Q000494:pharmacology
D001189:Aryl Hydrocarbon Hydroxylases; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D005473:Fluoxetine; D006899:Mixed Function Oxygenases; D017367:Selective Serotonin Reuptake Inhibitors; D013426:Sulfaphenazole; C450260:CYP2C9 protein, human; C045793:CYP2C19 protein, human; C036139:norfluoxetine