Back to the Top
The following message was posted to: PharmPK
Hi
I am currently working with some very lipophilic compounds and am
attempting to determine the plasma binding in mouse plasma. I tried to
use Amicon Centrifree units, but the compounds completely stuck to the
housing and membrane. I am now attempting to use Teflon equilibrium
dialysis chambers and measuring the compound on both sides of the
membrane. The problem is that I am loosing 30-40% of my compounds
somewhere -- I assume they bind to the regenerated cellulose membrane,
since "nothing sticks to Teflon". Does anyone have any ideas on how to
overcome this problem?
Thanks,
-Jodi Wood, Ph.D.
postdoctoral fellow
School of Pharmacy
University of Connecticut
Back to the Top
The following message was posted to: PharmPK
Hello
You can try "Charcoal Adsorption" method. This reference will be of
help to
you.
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.
Venkatesh Atul Bhattaram
CDER, FDA.
Back to the Top
The following message was posted to: PharmPK
Jodi:
If you can collect enough plasma from your mice and you have a sensitive
analytical method, you could try ultracentrifugation which avoids the
use of a
membrane altogether.
There are also some discussions on this issue in the PharmPK archive
(thanks
DB!).
Some potentially useful references for ultracentrifugation:
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.
Regards
Dave
Back to the Top
The following message was posted to: PharmPK
Jodi,
Nonspecific binding of drug during protein binding determination
is definitely a subject close to my heart. I recently spotted the
article by Lee at al. (Pharm. Res. 20: 1015-1021 [2003]) in which
they describe greatly reducing nonspecific binding to regenerated
cellulose membranes by pretreatment with detergent (Tween 80 or
benzalkonium chloride). I haven't yet had a chance to try this
myself but hope that it might help.
Another major cause for loss during equilibrium dialysis (other
than binding) is precipitation of drug. It may be worth checking
to see if that is an issue.
Another alternative is to switch to the ultracentrifugation
technique as there is no membrane involved.
All the very best,
Bernard
Bernard Murray, Ph.D.
Senior Pharmacologist
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
Bernard.Murray.aaa.abbott.com
Back to the Top
The following message was posted to: PharmPK
Assuming that your analytical procedure is robust and accuracy is good,
then you probably have focused on the correct operational issue. You
should use silanized glassware instead of plastic or synthetic labwear.
You may get some insight from reading on similar studies done with Taxol
and Taxotere.
Good luck,
Ving J. Lee
Anacor Pharmaceuticals, Inc.
Palo Alto, California 94303
Phone: (650)-739-0706
Back to the Top
The following message was posted to: PharmPK
Hi Jody:
What you are seeing is non specific binding. I would simply
pre-saturate
this membrane by dipping in simethicone suspension (classical
siliconizing
agent) if you want you can leave the apparatus in pre-saturation
condition
at 37C and then simply rinse it with deionized water before you
determine
your plasma.
If you need any further advise please contact me a personal e-mail or
contact me.
Prasad
Prasad NV Tata, M.Pharm., Ph.D.
Manager-Pharmacokinetics
Mallinckrodt, Inc.
675 McDonnell Blvd.
Saint Louis, MO 63134-5840
Tel: (314) 654-5325
Fax: (314) 654-9325
e-mail: prasad.tata.-a-.tycohealthcare.com
Back to the Top
The following message was posted to: PharmPK
Hi Jodi,
Have u tried the ultra centrifugation technique. this is the best
technique
for determining the protein binding to avoid the non specific membrane
binding. the ultra centrifuge tubes are available form BECKMANN and the
procedure involves 36 h ulracentrifugation of the samples at 30000 rpm.
tell
me if it is a possible technique for u.All the best, Jagannath
Back to the Top
The following message was posted to: PharmPK
Hi,
You can also try medical grade stainless steel cells for equilibrium
dialysis.
After siliconisation of the cells we used it for both cyclosporine and
tacrolimus protein binding study [Akhlaghi et al 1999, Zahir et al
2001].
Whatever you decide to do, its worth checking both the used membranes
and
cells (you are using for dialysis exp) for the drug. Because in our
case drug
was not binding to the membrane.
You should also check the stability of your molecule at 37C for the
period of
your experiment.
Siliconisation of all glass and plastic surfaces which may come in
contact
with your drug samples (including your plasma samples containing drug)
may
also improve your recovery.
Best wishes
Hamim
Hamim Zahir
Post Doctoral Research Fellow
Department of Applied Pharmaceutical Sciences
College of Pharmacy
Fogarty Hall
41 Lower College Road
University of Rhode Island
RI 02881, USA
Ph 1-401-874-5019
Back to the Top
The following message was posted to: PharmPK
Jodi
For drugs that exhibit greater than 98% fraction bound and high
lipophilicities, non-specific binding is a major experimental problem.
Although the various membrane treatments and other palliative measures
will
help but invariably they will not eliminate the problem.
Assuming binding to plasma proteins is an equilibrium (as opposed to a
covalent/coordinate binding) in your case, you need to exploit this
equilibrium condition to your advantage. Recognize that in the body the
drug
encounters blood and not plasma alone. A drug will simultaneously
partition
into erythrocytes while binding and equilibrating with plasma proteins.
This
concept was successfully exploited by E. R. Garrett and subsequently
modified and improved upon by J. Schuhmacher et al. The technique is a
little more labor intensive than your standard
ultrafiltration/equilibrium
dialysis/ultracentrifugation techniques and it also requires you to
have a
sensitive analytical technique or use of a radiolabeled molecule.
The useful references are:
Paul Altmayer and E.R. Garrett in J.Pharm. Sci., Vol. 72(11), 1309-1318,
1983
Joachim Schuhmacher et.al. in J. Pharm. Sci., Vol. 89(8), 1008-1021,
2000
We have successfully employed this technique in our shop. If you have
any
further questions you can call me or e-mail me.
Good Luck
Anup
Anup Zutshi Ph.D.
Pharmacokinetics, Dynamics and Metabolism
Pfizer
Mail Zone: T3O/Lab: T312E
800 N. Lindbergh Blvd.
St. Louis, MO-63141
Tel: (314) 274-8349
Fax: (314) 274-4426
Anup.Zutshi.-a-.Pfizer.com
Back to the Top
The following message was posted to: PharmPK
Dear chaps,
To continue this thread I have a question regarding the relative pros
and cons of ultracentrifugation vs equilibrium dialysis methods for
very lipophillic compounds. Cyclosporin and tacrolimus are good
examples where you get very different results depending on the method
used. From my recollection reported fu for both compounds based on
dialysis is approx 1% or less. The values for ultracentrifugation are
much higher (over 10%). Which values/methods if any are correct?
Cheers
Alex
A.J.MacDonald Ph.D
F. Hoffmann-La Roche Ltd
Pharma Research
Non-Clinical Safety
Modelling and Simulation
PRNS, 70/132
CH-4070 Basel Switzerland
Tel: ++41 61 68 84098
Fax: ++41 61 68 82066
alexander.macdonald.aaa.roche.com
Back to the Top
The following message was posted to: PharmPK
Maybe you can get some inspiration from the work of Philipp Mayer, e.g.
Mayer et al., Establishing and Controlling Dissolved Concentrations of
Hydrophobic Organics by Partitioning From a Solid Phase, Environmental
Science and Technology, 1999, 33, 2284-2290.
Or from some of his co-workers: Wouter HJ Vaes or Joop LM Hermens.
It may also be interesting to consider using the technique based on
SPME to
establish partitioning coefficients, e.g.
Urrestarazu Ramos et al., Using Solid Phase Micro-Extraction (SPME) to
Determine Partition Coefficients to Humic Acids and Bioavailable
Concentrations of Hydrophobic Chemicals, Environmental Science and
Technology, 1998, 32, 3430-3435 and
Holten Lützhøft et al., Influence of pH and Other Modifying Factors on
the
Distribution Behavior of 4-Quinolones to Solid Phases and Humic Acids
Studied by "Negligible-Depletion" SPME-HPLC, Environmental Science and
Technology, 2000, 34, 4989-4994.
Based on the papers of Vaes, WHJ et al., Measurement of the Free
Concentration Using Solid-Phase Microextraction: Bindnig to Protein,
Analytical Chemistry, 1996, 68, 4463-4467 and Vaes, WHJ et al.,
Partitioning
of Organic Chemicals to Polyacrylate-coated Solid Phase Microextraction
Fibers: Kinetic Behavior and Quantitative Structure-Property
Relationships,
Analytical Chemistry, 1996, 68, 4458-4462.
You are welcome to contact me on e-mail: hchl.-at-.dfuni.dk for further
details.
Hans-Christian Holten Lützhøft, Ass. Prof., Ph.D.Pharm.
Department of Pharmaceutics, 13-415B
The Danish University of Pharmaceutical Sciences
Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark
+45 3530 6528 or hchl.-at-.dfuni.dk
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)