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Dear All,
I about to investigate the metabolic stability of a candidate drug using
rat liver microsomes. But I don't know how to decide the proper
concentration of this particular drug as there's no literature
available. I want to know if there's any general guidelines regarding
this issue. Could you suggest any paper, if any, which discusses this
matter?
Thanks a lot.
--
Yours, Xinting Wang
Email: wxinting1986.aaa.gmail.com
Key Laboratory of Drug Metabolism & Pharmacokinetics
China Pharmaceutical University
24, Tongjia Xiang Road
Nanjing, Jiangsu
People's Republic of China, 210009
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The following message was posted to: PharmPK
Dear xinting,
to be honest I am not aware if there is a paper that specifically
address this
issue. However, at discovery level for a screening of metabolic
stability you
can use a drug concentration of 5 microMolar, if you would like to test
a
single concentration. This is a concentrattion that would represent an
accetable compromise between sensitivity of the analytical system and
likelihood of being below saturation of the microsomal enzymes.
Further, if your test compound is highly lipophyllic and you use a LC/MS
detection you can use a concentration of 1 microMolar.
Best Regards,
Roberto Tolando Ph.D.
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The following message was posted to: PharmPK
1 uM (substrate)
0.25 microsomal protein
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Xinting,
The general idea is to use substrate concentrations lower than Km to
ensure linear conditions and to use minimal protein concentration to
minimize nonspecific binding. However, because these information are
not available in most cases, people use generic conditions of 0.5-1 uM
of substrate and 0.1-0.5 mM protein.
Many publications discuss this topic, here are few of my favorites:
Curr Drug Metab. 2003 Oct;4(5):423-59.
Drug metabolism and drug interactions: application and clinical value of
in vitro models.
Venkatakrishnan K, von Moltke LL, Obach RS, Greenblatt DJ.
AAPS J. 2008 Jun;10(2):410-24. Epub 2008 Aug 7.
In vitro evaluation of reversible and irreversible cytochrome P450
inhibition: current status on methodologies and their utility for
predicting drug-drug interactions.
Fowler S, Zhang H.
Curr Drug Discov Technol. 2010 Sep;7(3):199-222.
Emerging in vitro tools to evaluate cytochrome P450 and
transporter-mediated drug-drug interactions.
Lu C, Liao M, Cohen L, Xia CQ.
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There is no simple answer for your question. It all depnds on your exprimental goal and
concentration that correlates with human theraputic drug concentrations. I consider
following points before I pick any concentration
1. 6 times the anticipated Cmax in Human population (rat to human 6-fold differences been
reported).
2. Range of concentrations surrounding #1 that gives an idea about Vmax and Km.
In the nutshell you have to ask what information I am expecting out of the proposed
incubations if you incubate with too low concentrations you may not see any metabolites or
not able to get any quantitative information.
Hope this suggestion helps.
Prasad NV Tata, Ph.D., FCP
2133 Seven Pines Drive
St. Louis, MO 63134
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