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Dear All:
We all know that how to get Km and Vmax values of test compounds
using in vitro studies (Test compound can be incubated with microsomes
at multiple concentrations), and the intrinsic clearance can be
calculated by Vmax/Km. If we know the free fraction of test compound in
the incubation system, the unbound intrinsic clearance can be calculated
by Vmax/(Km*fu). My question is: In terms of multiple concentrations of
test compound were used in this incubation study, for the protein
binding study, which concentration value should be used to do to get fu?
Any suggestions are appreciated. Thanks!
Jian
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Dear Jian,
If I have understood your question correctly, we are asking about the
substrate concentration you would use for your in vitro assay for fumic
determination (non-specific binding to microsomes). The general
assumption is that nonspecific binding to microsomes is linear , and
fumic should not change with substrate concentration ( at a fixed
microsomal protein concentration). Same concept applies to plasma
protein binding where fu (fraction unbound to plasma proteins) is
usually linear with substrate concentration. You can use fu and fumic
determined from one (intermediate) substrate concentration level for
your clearance prediction. You can also predict fu and fumic from in
silico based on physchem data
In case you have checked and found out concentration dependant fu or
fumic, then prediction of clearance is more difficult. I advise you to
consult the website of SimCYP (www.simcyp.com) for potential
publications on the topic.
Kind Regards, Youssef
Youssef Hijazi, PhD. PharmD, member ACCP
Associate Director Pharmacokinetics
Micromet AG
81477 Munich, Germany
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The following message was posted to: PharmPK
Hi Jian
In case your compound has a concentration dependent protein binding then you
should use that substrate conc. for protein binding which is close to
the therapeutic conc you expect in humans.
Comments from others are welcomed.
Tausif Ahmed Ph.D
Asst. Dir.
Translational Research
Piramal Life Sciences Mumbai
India
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The following message was posted to: PharmPK
Hi jian
The better way is to select 4-5 substrate concentrations within the
range and experimentally determine the Fu. You can then fit the obtained
data to a quadratic equation and determine Fu at other conc. Then apply
Fu correction while predicting vmax and km . Feel free to contact if any
additional info is required.
Cheers,
Ravi
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