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I have a problem in measuring protein binding of drug. This drug
reported to have 99% protein bound and we noticed approximately 80% of
the drug adsorbs onto the Amicon ultrafiltration devise. Our concern is
even if use equilibrium dialysis, we are not sure the extent of non
specific binding in the equilibrium dialysis cell.
Can somebody advise me where I can procure equilibrium dialysis cells
made of glass (Not of Poly carbonate or Polypropylene). or How to
minimize and document extent of adsorption on to the surface of
equilibrium dialysis cell.
Any input is highly appreciated.
Prasad Tata, Ph.D.
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[db - a few replies]
Date: Wed, 04 Feb 1998 16:14:12 -0600
From: "Steven J. Weber"
To: PharmPK.-a-.pharm.cpb.uokhsc.edu
Subject:
Mime-Version: 1.0
Do you believe compound "sticks" to the Centrifree housing or the filter
membrane? You may want to try using a Swinney Filter Holder with
appropriate memebrane (available from Fisher). Place plasma sample in
a sigmacoted (i.e., silinized) glass syringe attached to swinney filter
holder. Place a small sigmacoted glass HPLC injection vial into a larger
polypropylene test tube. Then place enter syringe/swinney holder into
larger tube so that end of swinney holder fits into and rests on the HPLC
injection vial. Centrifuge as you would Centrifrees and collect ultrafiltra=
te
from HPLC vial.
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Date: Thu, 5 Feb 1998 08:02:58 +0200 (IST)
From: Zvi Benzvi
To: PharmPK.aaa.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK Protein Binding Question
MIME-Version: 1.0
You could try to use gel chromatography.this method of measuring protein
binding in cases like yours could be very efficient.
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Date: Thu, 05 Feb 1998 10:45:28 +0100
From: "Dr. Bernhard Ladstetter"
Reply-To: Lstetter.-a-.merck.de
Organization: merck.de
MIME-Version: 1.0
To: PharmPK.-a-.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK Protein Binding Question
Dear Prasad,
I am personally not involved in measuring protein binding, but a
colleague had an idea which might help you: for equilibrium dialysis you
could try Dianorm-cells which are made of Teflon. We don=B4t know any
substances being adsorbed to Teflon. Glass is difficult because it might
be not watertight. Ultrafiltration should work after silanizing the
tubes and by using the Amicon microfiltration system MPS1 (Art. No.
4010) which should be silanized as well.
We use 1,1,1,3,3,3-hexamethyldisilazan to silanize our devise.
I hope that helps.
Regards,
Bernhard J. Ladstetter, PhD
(Institute of Pharmacokinetics and Metabolism, Merck KGaA)
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Date: Thu, 5 Feb 1998 09:53:08 -0500 (EST)
X-Sender: boultond.aaa.popmail.musc.edu
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To: PharmPK.-at-.pharm.cpb.uokhsc.edu
From: boultond.-a-.musc.edu (Dave Boulton)
Subject: Re: PharmPK Protein Binding Question
We had a similar problem with one of the highly protein bound bioflavonoids
(quercetin). After evaluating several methods we settled on
ultracentrifugation (160 000 g for 16 hours at various temperatures) which
sediments plasma proteins to virtually undetectable concentrations in a
"protein-free" water layer - a colourless layer under a top layer of LDLs
(we were also able to measure drug in this LDL layer). We used
polycarbonate centrifuge tubes which we showed did not adsorb our compound.
The drug is assayed the "protein-free" layer to determine % unbound.
Unfortunately, in cases where you have high binding you need a very
sensitive assay method (eg radiolabeling) to detect the small free
fraction. However, such an assay is also required for other the other
common protein binding methods which measure unbound drug.
A further consideration with the ultracentrifugation method is that binding
may be altered with the protein concentration gradient in the centrifuge
tube - this is also one of the limitations of ultrafiltration. You also
need to establish that your drug is chemically and/or enzymatically stable
for the relatively long duration of the ultracentrifugation spin. Stability
should also be checked (but normally isn't!) when using the other two
methods, particularly with the high temperatures and long equilibrium times
normally required for dialysis. In short, none of these methods are ideal,
but ultracentrifugation avoids the use of membranes which would seem to be
the major problem you are facing at the moment.
Some potentially useful references:
Barre J. Chamouard JM. Houin G. Tillement JP. Equilibrium dialysis,
ultrafiltration, and ultracentrifugation compared for determining the
plasma-protein-binding characteristics of valproic acid. Clinical
Chemistry. 31(1):60-4, 1985.
Paxton JW. Jurlina JL. Foote SE. The binding of amsacrine to human plasma
proteins. Journal of Pharmacy & Pharmacology. 38(6):432-8, 1986.
Boulton DW. Walle UK. Walle T. Extensive binding of the bioflavonoid
quercetin to human plasma proteins. Journal of Pharmacy and Pharmacology
50: 243-250, 1998.
Good luck
Dave
Dave Boulton PhD MPS
Postdoctoral Fellow
Department of Psychiatry
Medical University of South Carolina
171 Ashley Avenue
Charleston, SC 2942 Phone: +1 803 792 5831
USA Fax: +1 803 792 2475
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Date: Thu, 5 Feb 1998 13:38:12 -0500
From: DERVIEUX Thierry
Subject: PharmPK Protein Binding Question
Sender: DERVIEUX Thierry
To: "INTERNET:PharmPK.aaa.pharm.cpb.uokhsc.edu"
MIME-Version: 1.0
Hi I see your problem,
Not easy, but i think that if the compound absord the AMICON
It is ionisated and then lead to ionic strengh with the AMICON.
You should try different pH or different concnetration of salt in your
sample
to allow the compound beeing not ionizated.
Enjoy research, if it is difficult, it is find
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In following up the protein binding determination question raised by Prasad
has
anyone tried ultracentrifugation method to avoid adsorption problem. Does any
one know any references comparing the ultracentrifugation method with
equilibrium dialysis or ultrafiltration method? Which one is more accurate?
Frank W. Lee
DuPont Merck Pharmaceutical Company
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When you purchase a pack of Centrifrees, Amicon encloses an
instruction book that includes numerous references to the question you
ask. You should be able to request the booklet by calling Amicon at
800-343-0696 (or -1397 for technical) or 508-777-3622.
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Dear Dr. Lee:
Please refer to the publications:
1. Measurement and analysis of Unbound drug concentrations.
J.D. Wright, F.D. Boudinot, M.R. Ujhelyi
Clin. Pharmacokinetics. 1996, 30(6), 445-462.
2. Ultrafiltration Vs. Equilibrium Dialysis for determination of Free
Fraction.
William F. Bowers, Scott Fulton, Judith Thompson
Clinical Pharmacokinetics, 1984, 9(suppl. 1) 49-60.
There are couple of publications in Journal of Pharmaceutical and
Biomedical analysis within the last six - eight months where they
discussed protein binding in details.
Regards,
Prasad Tata
Otsuka America Pharmaceutical, Inc.
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)