Probing CYP2C19 and CYP3A4 activities in Chinese liver microsomes by quantification of 5-hydroxyomeprazole and omeprazole sulphone.
OBJECTIVE
To develop an analytical method for simultaneous quantification of 5-hydroxyomeprazole (5-OH-OP) and omeprazole sulfone (OPS), and explore whether omeprazole (OP) is an appropriate phenotypic probe for CYP2C19 and CYP3A4 in Chinese liver microsomes.


METHODS
OP metabolism in vitro was conducted in Chinese liver microsomes, and the major metabolites 5-OH-OP and OPS were determined using high pressure liquid chromatography (HPLC). Monoclonal antibodies anti-CYP2C8/9/19 and anti-CYP3A4 were employed to conduct inhibition experiments. The protein contents of CYP2C19 and CYP3A4 were quantified using Western blot analysis and densitometric scanning.


RESULTS
5-OH-OP and OPS gave a baseline resolution in the HPLC analysis. The detection limits for both compounds were 0.01 nmol and the recovery (98%-102%) had good precision with relative standard deviation of < 9.5%. Both anti-CYP2C8/9/19 and anti-CYP3A4 had a significant inhibitory effect (P < 0.05) on the 5-OH-OP formation in a substrate concentration-dependent manner, and anti-CYP3A4 alone could almost abolish the formation of OPS (> 87%). At a substrate concentration of 2 mumol/L OP, good correlations were found between OP 5-hydroxylation and S-mephenytoin 4'-hydroxylation activities (r = 0.72, P < 0.01), OP 5-hydroxylation activities and CYP2C19 contents (r = 0.82, P < 0.01), and OP sulfoxidation activities and CYP3A4 contents (r = 0.78, P < 0.01) in Chinese liver microsomes.


CONCLUSIONS
OP metabolism is mediated mainly by CYP2C19 and CYP3A4, and OP can be used to probe CYP2C19 and CYP3A4 activities in Chinese liver microsomes at appropriate substrate concentrations with the HPLC method presently developed.
Shu
Y
Y
Pharmacogenetics Research Institute, Hunan Medical University, Changsha 410078, China.
Wang
L S
LS
Xiao
W M
WM
Wang
W
W
Huang
S L
SL
Zhou
H H
HH
D053799:2-Pyridinylmethylsulfinylbenzimidazoles; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D044466:Asian People; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000276:immunology / Q000378:metabolism*; D009853:Omeprazole / Q000031:analogs & derivatives / Q000378:metabolism*
D053799:2-Pyridinylmethylsulfinylbenzimidazoles; D000911:Antibodies, Monoclonal; C040450:omeprazole sulfone; D003577:Cytochrome P-450 Enzyme System; C049963:5-hydroxymethylomeprazole; 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; D009853:Omeprazole