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Dear All,=20
For a compound (NCE) having in-vitro half life using (rat liver =
microsomes) more than 120 min, but for the same compound half life is 11 =
min in in vivo. Met ID in rat plasma samples we have seen that oxidation =
is the major path of metabolism but we didn't do Met ID with rat liver =
microsomes incubation. Is there any enzyme that responsible for =
metabolism in vivo, but not present in the liver microsomes? or what may =
the reason for this.=20
I will appreciate the comments=20
regards=20
Rammohan
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Dear Rammohan,
There could be many reasons for the short in vivo half-life observed in =
your case, as against rat liver microsomes.
The drug can undergo phase II conjugation (monitor for glucuronides, =
sulphate etc conjugates in plasma), which is not seen in microsomes =
unless you supplement it with cofactors such as UDPGA (only for =
glucuronides).
May be incubation in hepatocytes might provide you more information.
Also there could be role of other enzymes and transporters leading to =
short half-life in vivo.
There could also be a possibility that the drug is not stable in blood =
or partitions into RBC's.
Thoughts from other experts are welcomed.
Tausif Ahmed, Ph.D.=20
Associate Director, DMPK and Tox.
Sai Advantium Pharma Ltd., Pune, India=
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Perhaps another route of elimination than the liver may explain a =
shorter half-life, like the kidney.
Jean=
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Tell us the class we may be able to help. Lots of other enzyme =
differences plasma/red cells, endothelium, lung, kidney, etc, etc, etc.=
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Hello Rammohan,
Without knowing the structure of the molecule, one can only speculate. =
Rats have very efficient esterases in the plasma that are not found in =
the microsomes. I assume that you are not using radiolabeled material, =
so you don't know if urinary or biliary excretion are the major routes =
of elimination. Microsomal stability will not predict other routes of =
elimination. I hope my comments are useful in your search for the =
answer.
=20
Chris
=20
Christopher Town, PhD
EMAIL: town.chris.at.sbcglobal.net=
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Dear Rammohan,=20
Organs other than liver do have metabolic efficiency which could cause =
this discrepancy between in vitro and in vivo results. You have to also =
take into consideration that microsomes do not have any transporters. A =
drug being a substrate for any transporters can also cause this.
Jigar Patel=20
University of Alberta, Canada.=
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Dear Rammohan,
If your in vitro half life is 120 minutes it is unlikely that you will
see many metabolites using an in vitro microsomal preparation. A
hepatocyte preparation may be more beneficial. You state, however, that
the major metabolites in vivo are oxidative so that would suggest a P450
mediated metabolism as a likely site of metabolism. You should look at
the incubation concentration in your microsomal preparation and also
whether your compound is not only a substrate but also an inhibitor of
P450's. It is possible that if your concentration is high enough in the
microsomal incubation you are seeing auto-inhibition. An analysis of Km
and Vmax as well as IC50 or Ki on specific P450's might help explain
this apparent contradiction.
Norman
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Dear rammohan,
Experiment performed using Microsomes invitro doesnt represent the liver metabolism as a whole invivo. So you can try out performing the metabolic stabilty using rat cryo preserved hepatocytes.
With regards,
V. Vijaya Bhaskar
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Dear all,
I am just asking, why cryopreserved hepatocytes, why not freshly isolated rat hepatocytes cultured in sandwich-configuration?
Will not it be more reliable?
Regards
Sagnik
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Hi Sagnik,
You can always go for the freshly isolated hepatocytes, provided you have setup in house to do so. I prefer suspension cultures used immediately after isolation than cultured in sandwich configuration. There will be a huge variation with the data you generate in sandwich cultures, due to change in the CYP-P450 content over a period of time.
Regards
--Naveed Shaik Abdul
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