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The following message was posted to: PharmPK
Dear Group Members,
We are facing problems in determining protein binding by equilibrium
dialysis and ultra-filtration for certain compounds. Of the other
methods mentioned in literature, binding by Human Serum Albumin (HSA)
immobilized columns appears useful. Has anybody in the group used
these HSA columns as a screening tool ? I would be thankful if the
group would share some practical issues related to the use of these.
Thanks in advance,
Tripta Bansal
Ranbaxy Research Laboratories.
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The following message was posted to: PharmPK
Tripta,
You are not alone in facing problems in determining/screening %PPB
using ultrafiltration (due to nonspecific binding (NSB)?) or
equilibrium dialysis.
We have begun using an HSA-LC/MS technique in our laboratories.
Obviously it has some limitations, but we have found it particularly
useful in confirmining results from otherwise "suspicious" (esp. high
NSB) experiments. We are beginning experiments to examine it's
usefulness in competitive binding experiments with specific binding
sites.
We did do some validation experiments with a wide range of probe
compounds to "calibrate" the column, and did have to work on some
method development to optimize the mobile phase.
There are a number of papers in the literature on this technique, but
the most helpful (and practical) I have found thus far is:
Cheng Y, Ho E, Subramanyam B, Tseng JL., "Measurements of drug-
protein binding by using immobilized human serum albumin liquid
chromatography-mass spectrometry," **J Chromatogr B** (Analyt Technol
Biomed Life Sci) Sep 25, 2004;809(1):67-73.
Best Regards,
Jim Schmidt
Associate Scientist, Drug Metabolism and Pharmacokinetics
Lexicon Genetics
The Woodlands, TX 77381
jschmidt.at.lexgen.com
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The following message was posted to: PharmPK
Tripta and Group Members,
We have used HSA (human serum albumin) binding columns with LC/MS
detection as a screening tool in our group. Rank order of compounds
matches that seen with other techniques. To get accurate binding
parameters, a fair amount of calibration and verification is needed for
any particular individual compound or class of compounds.
Several compounds can be injected at the same time (in theory and often
in practice) to get rank ordering. Of course, one needs to know
something about the MS characteristics of each in advance.
-Tom Tarnowski
Thomas L. Tarnowski, Ph. D.
Drug Metabolism and Pharmacokinetics
Roche Palo Alto
3431 Hillview Avenue, Palo Alto, CA 94304
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The following message was posted to: PharmPK
Tripta,
One good and relatively simple method of measuring serum protein
binding is
Ultracentrifugation. Our experience shows it works OK for variety of
compounds. I am giving the reference below, see if it helps you out.
Reference:
1. Arnold Louie et al., Antimicrobial Agents & Chemotherapy Mar.2001,
p845-851.
Yati Chugh
Wockhardt Research Centre.
D-4, MIDC, Chikalthana.
Aurangabad, India.
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The following message was posted to: PharmPK
Tripta,
please have a look at this paper, it could be useful for your study
Ermondi et al, J.Med.Chem. 2004, 47, 3949
Giulia Caron
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)