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The following message was posted to: PharmPK
Hi everyone,
I've been screening novel drug candidates for Phase I metabolism using
liver microsomes (rat and human). I also want to screen my compounds to
see if they are substrates for glucuronidation and (maybe) other Phase
II processes. Are there any methods, eg. in-vitro microsomal or perfused
liver, which can be used as a general screen of Phase II metabolism for
novel compounds? Does the pharmaceutical industry screen drug candidates
for Phase II metabolism and if so how do they do it? I have over 100
compounds to study.
Thanks in advance,
Dr Seetal Dodd
Research Fellow
Department of Pharmaceutics,
Victorian College of Pharmacy, Monash University
381 Royal Parade, Parkville, Victoria 3052
Australia.
Tel: +61 3 9903 9053
Fax: +61 3 9903 9583
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The following message was posted to: PharmPK
Greetings Dr. Dodd...
We have implemented the use of liver S9 (cytosolic) fractions, which
are available commercially, to evaluate Phase II metabolism in vitro. This
assay has a similar incubation as do microsomes, can be formatted for higher
throughput and is an adequate approach to screen for Phase II metabolism.
Ultimately a better way to evaluate such processes is in liver cells/slices
which are also available commercially but are less amenable to higher
throughput. Though the desire is to evaluate metabolism earlier in the drug
discovery/optimization process, the evaluation of Phase II metabolism has
traditionally been done later in this process and its utility has been the
comparison/prediction of metabolism and clearance rates in multiple species.
Hope this helps....
Good Luck!!
Tom Stephan, Ph.D.
ICOS Corporation
tstephan.-a-.icos.com
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[Two replies - db]
From: Espie Pascal
Date: Mon, 16 Jul 2001 11:15:35 +0200
To: david.-a-.boomer.org
Subject: RE: PharmPK Glucuronidation
The following message was posted to: PharmPK
Dear Seetal,
I don't know if the pharmaceutical industry world screens drug candidates
for phase II metabolism at all, but you can easily build up an HTS-assay to
measure the glucuronidation rate of a series of closely related compounds
using human liver microsomes (or rat, dog, ...).
For example, incubating the NCEs with HLM in the presence of 14C-labelled
UDP-GA (ca. 100 000 dpm/incub., 1 mM cold) could be the way. Further, you
may isolate the synthesized glucuronides (generic SPE method, avoid basic
pHs in order to eliminate the risk of hydrolysis of the glucuronides) and
collect your eluates in 96-well plates (OptiPlates, Packard).
To quantify the amount of 14C-glucuronides formed you can use a TopCount
Microplate Scintillation counter (Packard, too). In your incubation, you may
add a detergent (concentration to be validated before) to increase the
interaction between your NCEs and the (embedded) glucuronotransferase.
At the end, you will be able to evaluate the intrinsic clearance towards
glucuronidation.
Hope it helps, and good luck ...
Pascal ESPIE
Product Safety and Metabolism department
UCB Pharma
Belgium
pascal.espie.-at-.ucb-group.com
---
From: Sam.Rebello.at.aventis.com
Date: Mon, 16 Jul 2001 07:58:26 -0500
To: david.at.boomer.org
Subject: RE: PharmPK Re: Glucuronidation
The following message was posted to: PharmPK
Tom:
In response to your reply on the use of S9, I would like to know whether
you add any cofactors in your S9 preparation to examine Phase II
metabolic pathways. We buy S9 fractions from Xenotech but we fortify
our assay by adding PAPS (for sulfation) and UDPGA (for glucuronidation)
to enhance Phase II reactions. I am curious to know whether you add
these or some other cofactors?
Sam Rebello
Pilot PK - DMPK/US
Aventis Pharmaceuticals
(908)-231-4108
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