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Hello,
Omeprazole 5-hydroxylation is an acceptable in vitro experiment to
monitor CYP2C19 activity by FDA. When we tried to develop a CYP2C19
inhibition assay with omeprazole as substrate and human liver
microsome as enzyme source, we noticed that many CYP3A4 inhibitors
showed inhibitory activities. It=92s known that both CYP2C19 and CYP3A4
are involved in omeprazole 5-hydroxylation. Even though CYP2C19 may
play a major role, but its enzyme content in HLM is much lower than
CYP3A4 (10-60 fold). Therefore, most of 5-hydroxyomeprazole was
probably made through CYP3A4 pathway at our conditions.
What is the relative contribution of the two enzymes to omeprazole 5-
hydroxylation when HLM is used? How can we optimize assay conditions
to make it more CYP2C19 specific?
Thanks for your help,
Sean Wu
Exelixis
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The following message was posted to: PharmPK
Dear Mansi,
http://www.pharmgkb.org/do/serve?objId=PA152530846&objCls=Pathway
Enantiomer/Enantiomer Interactions between the S- and R-Isomers of
Omeprazole in Human Cytochrome P450 Enzymes: Major Role of CYP2C19 and
CYP3A4, JPET 315:777-787, 2005
Thanks,
Sean
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Dear Sean Wu,
Omeprazole hydroxylation shows biphasic kinetic. Suugest that at
least two enzymes are involve in this reaction. It has been reported
by Andersson et al. that CYP2C19 is the high
affinity enzyme nd CYP3A4 is the low affinity enzyme. So it depends
on the substrate concentration of omeprazole that you used in the
incubation. KM for high affinity 5-omeprazole hydroxylation is around
9 micro Molar where as Km for low afinity is around 175 micro Molar.
Thus, you need to use low concentration of omeprazole (5-10 uM) if you
want to measure activity of CYP2C19. You could find details in the
article below.
1. Andersson TA, Miners JO, Tassaneeyakul W, Tassaneeyakul W, Veronese
ME, Meyer, UA. Birkett DJ. Identification of human liver cytochrome
P450 isoforms mediating omeprazole metabolism. Br J Clin Pharmacol
1993; 36: 521-530.
2. Tassaneeyakul W, Guo L, Fukuda K, Ohta T, Yamazoe Y. Inhibition
selectivity of grapefruit juice components on human cytochromes P450.
Arch Biochem Biophys 2000; 378: 356-363.
Wichittra
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Dear Wichittra,
Thanks for your explanation and references. We did try to adjust
omeprazole concentration to shift contribution towards CYP2C19.
Unfortunately, we still observed good correlation between HLM and
recombinant CYP3A4 even when omeprazole concentration was 0.1-1 uM.
Sean
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