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The following message was posted to: PharmPK
Hi all,
Have a question about how to verify an made
observation in CYP3A4 activity induction (for now it
seems to) using human microsomes. I use a system that
consists of 3uM midazolam, 0.05mg/ml microsomes
together with 10mM MgCl2, 100mM potassium phosphate,
2.6mM NADP, 6.6mM G-6-P, 2u/ml G6P dehydrogenase. The
formation of hydroxymidazolam was monitered by
LC-MS/MS (342>324). Instead of using purified CYP3A4,
is there any other way to verify this result with my
current microsomal system? Like preincubating the
reaction with ketoconazole to see if it can bat down
the induction? Or the best suggestion you have in mind
to this question? Thanks a lot.
Hugo Lee
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The following message was posted to: PharmPK
Hi Hugo:
Microsomes are usually used to study P450 activation/inhibition.
Hepatocytes are the model of choice to evaluate P450 induction, since
induction of P450 is a receptor mediated process. If you are
evaluating activation/inhibition, then your method can be used with
microsomes as well. Preincubation will help determine if the test
compd causes any mechanism based inhibition/TDI of 3A4. Hope this helps.
Regards
Parnali Chatterjee
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The following message was posted to: PharmPK
Dear Lee,
Ideally CYP induction studies were done in cell based systems, like
hepatocytes as it
needs transcription of CYP genes normally occurs in about 2-3 days.
The increased activity of CYP3A4 in your microsome studies is
probably due to
autoactivation by multiple binding sites of CYP3A4.
This can be confirm by using Eadie-Hofstee plot, an atypical kinetic
model, if resulted
cuve is hyperbolic, then it should be due to autoactvation.
Ketoconazole is a potent non-competitive inhibitor, hopefully will
not seve the purpose.
However the best way to confirm is by treating the test compound for
3-4 days in
human/rat hepatocytes and then incubate with midazolam for 1-2 hours
and measure the
levels of hydroxy-medazolam. The increased metabolite levels
indicates the CYP3A4
induction.
Good luck.
Sripal
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The following message was posted to: PharmPK
Dear Lee,
Midazolam (MDZ) metabolism by CYP3A4/3A5 is a little complex; Its
metabolism is known to produce two hydroxy products: 1'OH and 4OH,
although at lower MDZ conccentration it mostly produces only 1'OH.
Further, MDZ is also known to cause mechanism-based inacivation of
CYP3A4 (but not of CYP3A5). Therefore, it is suggested that the
incubation time for MDZ metabolism should be kept as short as possible.
There are not many reports of CYP3A4 auto-activation by MDZ or at least
not as prominient as see with some of other CYP3A4 substrates. However,
1'OH formation is significantly activated (and 4OH is partially
inhibited) in the presence of 7,8-benzoflavone. Opposite is true when
testosterone is added to MDZ incubation.
Thanks and I hope it helps.
Kishore
Ref:
Regina W. Wang, Deborah J. Newton, Nini Liu, William M. Atkins, and
Anthony Y. H. Lu Human Cytochrome P-450 3A4: In Vitro Drug-Drug
Interaction Patterns Are Substrate-Dependent Drug Metab. Dispos. 2000
28: 360-366
Kishore K. Khan, You Qun He, Tammy L. Domanski, and James R. Halpert
Midazolam Oxidation by Cytochrome P450 3A4 and Active-Site Mutants: an
Evaluation of Multiple Binding Sites and of the Metabolic Pathway That
Leads to Enzyme Inactivation Mol. Pharmacol. 2002 61: 495-506.
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