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Hi
I had a question on the metabolism study of compounds done using
commercial
source of rat/mice liver microsomes.
For the study, do we need to add any cofactors like NADPH etc.
Does it make a difference if use microsomes vs S9 fraction?
Do liver microsomes contain both Phase ! and Phase II metabolizing
enzymes
including esterases?
Thanks for your input
Best regards
R.P. Iyer, Ph.D.
Spring Bank Technologies, Inc.,
Suite S-7,
113 Cedar Street, Milford, MA 01757
Ph: 508-473-5993, Ext 13
FAX 508-473-6375
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Dear R.P. Iyer
Answer to your first question; yes we need to add cofactors like
NADPH in case of rat/human liver microsomes. And UDPGA, PAP in case
of rat/human s9 fraction.
There is a difference between %metabolic stability with microsomes
and s9 fraction. We found microsomes are more rich in enzymes compare
to s9 fraction. Microsomes have only phase I enzymes which includes
desacetylation.
rgds,
Jignesh Kotecha
Scientist 1
Torrent Pharm. Ltd.
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Hi Iyer
Yes, you need to add either NADPH or NADPH regenerating system
(available commercially) for CYP reaction. In our lab we use hgih
concentration of NADPH for 15 min incubation.
Using S9 or microsomes does not make any difference for CYP
metabolism becuase CYPs present only in microsomes, but it may make
difference for carboxylesterase metabolism as it is present in both
microsomes and cytosol. Although microsomes contain majority of the
carboxylesterase, cytosolic enzyme might contribute to the metabolism
depending upon the substrate because this enzyme is known to exist in
different isoforms.
Hope this helps
satheesh
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The following message was posted to: PharmPK
Dear Dr. Iyer,
Regenerating system such as NADPH + glutathione or NADP + glucose-6-
phosphate + GDH
+glutathione, should be added in the microsome to produce the active
form of the enzyme.
The cytochrome P450 mixed function oxidation system is located
primarily in the
microsomal membrane fraction while soluble cytoplasmic phase II
enzymes are concentrated
in the S9 fraction. So use of microsome or S9 fraction depends on the
type of study. For
example to gererate active metabolites that are highy unstable, from
their prodrugs such
as clopidogrel, use of micosome fraction is suitable. However some
commercial S9
fraction, is contaminated by microsomes. So both PhaseI and II
metabolizing enzymes are
present.
Hope this help to some extent.
Best Regards
Sripal
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The following message was posted to: PharmPK
Hi there
A nice way to set up a microsomal metabolism experiment might be to have
two or three conditions for each drug substance.
0) set up a control of your drug in just the buffer and run it over time
along side your samples so that you can correct for loss of drug due to
non-metabolic degradation
1) microsomes with no cofactors--this condition will let you observe
what
metabolism occurs that is not P450 (or other stuff requiring cofactors)
dependant--these may be esterases and amidases...
2) microsomes with NADPH--this condition will let you observe what
effect
the P450 enzymes and possibly FMO have on your drug
3) microsomes with UDPGA--this condition will allow you to observe the
direct glucuronidation for your drug in this system--I don't know if you
might actually observe post-oxidative glucuronidation if you also add
NADPH?
maybe ?look into that if it might be of interest..
as for the S9s, as I'm sure you know they contain the micosomal
enzymes plus
the cytosolic enzymes (the S9 is the supernatant for the 9000 x g
spin of
the liver homogenate--the microsomes are the resuspended pellet of the
100,000 x g spin of the S9) If you were, for example, interested in
sulfation of your compound you could add the cofactor PAPS to your assay
with S9s to look for Sulfotransferase activity. I think Glutathione
can be
added to the S9s to look for Glutathione S Transferase activity.
It definitely depends what you are looking for. A basic screen might
just
include microsomes +/- NADPH and then you can progress from there. Good
luck!
-Rachel Mandell
Rigel Pharm
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Dear David Bourne/ Kris Iyer,
I am responding to the questions posted on PharmPK. The answers to
some of your questions
- you do need to add cofactors
- microsomes are generally used for Phase I enzymes
- S9 fractions are used for both Phase I & II enzymes
I have attached a copy of our recent newsletter. If you would like to
discuss further details of the assays do not hesitate to contact me.
Thanks.
Sincerely,
Prabu Devanesan
440-357-3106
[You should contact Prabu directly for the newsletter as attachments
are not allowed on the PharmPK list - db]
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The following message was posted to: PharmPK
Hi Satheesh,
I am not agree with your following sentance
"Using S9 or microsomes does not make any difference for CYP
metabolism"
The reason is that it will not affect it only if you are considering the
total enzyme activity in respect of protein content,and if you are
considering it then you are right. Because the enzyme activity of S9
fraction ( Phase I) is less as compare to microsome.
Rajesh Chavan
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)