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The following message was posted to: PharmPK
I would like to see if someone else has experience with the long time
required to reach equilibrium.
We are trying to determined the in vitro plasma protein binding of a
development compound. Because of the high nonspecific binding of the
compound to ultrafiltrate device, we decided to use equilibrium
dialysis.
We run a preliminary time-to equilibrium experiment by dialyzing
plasma spiked with compound against phosphate buffer. However, the
concentration values of the compound in the buffer indicated that the
system did not achieve equilibrium even after 24 hours. One
typically avoids long time for dialysis because of concerns about
volume shit, protein denaturing, compound degradation, etc.
Has anyone had experience with this kind of observations? I'm also
looking for suggestions as to what can be done to get good in vitro
protein binding data in such a case.
Thanks for your comments!
Regards,
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The following message was posted to: PharmPK
Dear Yongqing,
Your observation that long dialysis times may be needed to reach
equilibrium agrees with our experience. We routinely dialyze for 20
hours, and include controls for recovery and plasma stability of the
test compound. With highly bound compounds and/or compounds with slow
off rates, we often observe that equilibrium is not achieved even after
20 hours (one example is warfarin). In our experimental design, we
include a plasma-to-plasma condition to check for equilibrium.
Adrian Sheldon
In Vitro ADMET
Charles River Labs
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Hi
I had also faced this problem long ago and i could not even use
equilibrium dialysis hence i used a method where protein binding was
estimated using charcoal adsorption. The method worked very well for
me and for some of my colleagues. You should be able to see the
original paper sited in the paper below.
Khurana M, Paliwal JK, Kamboj VP, Gupta RC.
Binding of centchroman with human serum as determined by charcoal
adsorption method.
Int J Pharm. 1999 Dec 10;192(2):109-14.
hope this helps.
Manish Issar, Ph.D
Sandoz (Formerly Eon Labs Inc.)
4700 Eon Drive
Wilson, NC 27893
email: m.issar.aaa.eonlabs.com
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