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Hi
I tried to use ultracentrifugation method to determine the non-
specific protein binding of a sticky compound. The experiment was
conducted with 3 concentrations of the compound (5, 20, 80 micoM). (I
wanted to determine whether the non-specific binding was
concentration dependent). At each concentration, I prepared a control
(without protein). However, after centrifuge the controls at 50,000rpm
for 15 hours, the concentrations of the controls are relatively
similar. I think it might be the compound has bad solubility in
aqueous solution. I think it might be a common problem for many drugs.
In this case, how should I set up the experiment to still get the
information I want.
thanks,
Jane
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Hi Jane,
maybe you need to change the separation technique. Do you think with
equilibrium dialysis you would get to an equilibrium in an acceptable
time (e.g. over night). This can be easily tested in a control
experiment (dialysis of your drug in either plasma or buffer against
the same matrix without drug, sampling at different times to get a
time course and see how much time is needed for equilibration), there
are now different convenient 96 well plate based products on the
market for equilibrium dialysis.
I believe that ultracentrifugation is not an optimal method for many
sticky compounds, since they might bind to lipoproteins which can not
be easily (or at all) separated by UC due to there low density. Please
see this publication (Drug Metab. Dispos. 36, 1812-8.) for more
details on this , as well as description of another separation
technique (equilibrium gel filtration) that I believe is very powerful
in case of lipophilic compounds but maybe a bit more work to implement.
Regards,
Markus
H Markus Weiss, PhD
Novartis Institutes for BioMedical Research, Translational Sciences -
DMPK
CHBS, WSJ-210.4.25
Novartis Pharma AG, Werk St. Johann
CH-4057 Basel, Switzerland
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Or it was sticking to the filter? There are different materials for
the filters, and different protocols, what are the specifics? Have
you tried to backflush the filter after removing the retentate,
similar to a desalting protocol to determine what mass may have been
held up? You may also have a stickness issue not only with the filter
membrane but also with the material of the device itself. Dialysis
using the RED device may be helpful. What were the recoveries in
plasma vs control and what were you using to measure? What is the
stability of the compound?
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Dear jane,
If an adsorption takes place on the wall of the tube a rinsing with an
ethanol methanol or DMSO should answer this problem.
If it is a non specific binding, it is enough to make in parallel a
systematic control in presence of protein, after the
ultracentrifugation you stir the tube in order to homogenise
supernatant and pellet, then you measure the total concentration
recovered after incubation and centrifugation.
If a precipitation takes place during the centrifugation you should
have a gradient of concentration along the tube.
If it is a precipitation of the test compound or if the non specific
binding is very high, then we develop the blood partitioning method,
to determine the binding parameters of a sticky drug on plasma or
isolated plasma proteins.
Hoping that helps you
Best regards
Francoise
--
Francoise BREE
XENOBLIS
francoise.bree.aaa.xenoblis.com
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Hi Jane
I believe instead of using a control without protein, i would suggest
you to use some known drug (whose NS binding is known) in plasma as
your positive control.
You can find lot of references in Literature.
Hope this should help you.
Regards
Khalid
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Dear Jane:
Could you clarify "non-specific protein binding"? Is it plasma
protein? tissue protein? or blood protein? What is the target the
drug hypothetically binds to? What is the purpose of this test?
Thank you.
TK Chen
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