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The following message was posted to: PharmPK
We are studying the oxidative metabolism of a drug using Rat Liver
microsomes (RLM) using the following protocol :
1) Drug + RLM + NADPH
2) Drug + RLM - NADPH
3) Drug + Inactivated RLM + NADPH - Inactivation done by heating the
microsomes at 60 degree C for 30 min.
We have observed that in the presence of NADPH 40 - 50% of the parent
compound disappears.
However, the same percentage of the parent drug disappears in the
experiment 3 where we are using inactivated RLM's.
How do we explain this phenomenon ??
Also we have used different concentrations of NADPH - 1.3 mM and 10 mM
using experiment 1. And we have observed that 10 mM NADPH brings about
less disappearance of parent compound as compared to 1.3mM ???
Y.Chugh
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[Two replies - db]
From: "Tata, Prasad N"Date: Fri, 25
Jan 2002 14:03:37 -0600
To: david.-a-.boomer.org
Subject: RE: PharmPK Oxidative metabolism with rat liver microsomes
The following message was posted to: PharmPK
Dear Dr. Chugh:
In your experiment you are getting 40-50% disappearance of parent drug
in the presence of active or inactivated liver homogenate. The lacking
information in your post is nature of the drug. Have you considered
hydrolysis of drug or thermal stability of drug as the reason for your
parent drug's disappearance?
Philosophically many people thinks metabolism meaning one caused by
liver microsomes, though this is true with most of the drugs, other
forms of metabolism caused by esterases and other routes are often
ignored. To gain complete understanding you need to study your drug
molecule and map out potential metabolic pathways and then design your
studies accordingly.
Hope this kind of spin in designing your metabolism studies would be
helpful.
Prasad Tata
Mallinckrodt, Inc.
PS: Always wondering why small structural changes that generate
metabolites play a havoc in pharmacological activity and
pharmacokinetics of a drug--- perhaps that is the challenge of
metabolism studies.
---
From: "AZHER HUSSAIN"Date: Fri, 25 Jan 2002
14:07:10 -0600
To: david.at.boomer.org
Subject: Re: PharmPK Oxidative metabolism with rat liver microsomes
The following message was posted to: PharmPK
Dear Chugh:
The loss of drug you have seen in the third case (incactivation) could
be due to the degradation of the drug, if you are heating the mix
(Drug+RLM+NADPH) instead of inactivating the RLM prior to addition to
the incubation mixture. The other thing you can check is, if the
inactivation is complete or not by looking at the absorbance at 420 nm.
The second observation may be due to the concentration dependent
reducing affect of NADPH.
Regards,
Azher M. Hussain, Ph.D
Post Doctoral Researcher
Drug Inhibition
Xenotech, LLC
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The following message was posted to: PharmPK
hi
with my past experience with metabolism it seems that even though you
inactivate RLM by heating at 60?C for 30 mins there are that chances
that the enzyme system is still active with lower activity. this was
similar to what had happened with me when i heated RLM at 80?C for 30-40
mins. i was surprized to find that my compound (carbonyl group )of
interest was getting reduced to its hydroxy metabolite in the absence of
NADPH. probably the oxidoreductases were quite resistant to degradation.
i could not too find the reason conclusively, why?
i would suggest that u should perform a parallel study where the drug is
spiked in to the buffer solution + cofactors and no RLM. if ur compound
is stable there then it could be possible that other forms of
degradation/or conversion of the parent compound is possible. for
example the esterases etc..
did u find the stability of the compound in whole blood , plasma or in
the RBC's?
hope this helps
with best wishes
manish
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The following message was posted to: PharmPK
YChugh.-a-.wockhardtin.com (by way of David Bourne) wrote:
>"...And we have observed that 10 mM NADPH brings about less
disappearance of parent compound as compared to 1.3mM ??? ...">
There are several possibilities depending on the chemistry of your drug,
but I would suggest one more control: No microsomes, +_ hem-bound iron,
+_ phospholipid, +_ NADPH.
You may have non-enzymatic cooxygenation coupled to lipid peroxidation.
Excess of NADPH may actually act as an antioxidant. It's just a
speculation, but similar artifacts I observed with some quinones. It
would be interesting to see where the parent compound goes.
Best wishes.
Janusz Z. Byczkowski, Ph.D.,D.Sc.,D.A.B.T.
Consultant
212 N. Central Ave.
Fairborn, OH 45324
e-mail januszb.at.AOL.com
homepage: http://members.aol.com/JanuszB/index.html JZB Consulting web
site: http://members.aol.com/JanuszB/consult.htm
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