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I have been evaluating drug absorption by using Caco-2 cell monolayer. In
the case of high
lipophilic compound, I cannot often reject the influence of adsorption of
drug to the polycarbonate
membrane which supports Caco-2 cell monolayer. So, accurate permeability
coefficient of the
drug cannot be evaluated
I would appreciate if any members of PharmPK can advise me on what
experimental conditions sould be
applied in order to avoid the drug adsorption.
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Excellent question, however, I do not have any clear answer at this
point. I am also using Caco-2 as a screen in drug discovery and have
essentially experienced same problem of adsorption/absorption of
"grease balls" (a term used by Stella for highly lipophilic compounds)
to In Vitro system (transwell and membrane). This problem severely
limits the use of existing In Vitro systems (both transwell and
vertical diffusion chamber system by Costar) as far as lipophilic
compounds are concerned. In a two compartment system such as these,
doing an additional experiment with a blank set (one without Caco-2
monolayers) also presents mathematical challenges as far as correction
due to adsorption/absorption is concerned.
Some other approaches such as the use of solvents to minimize this
problem can affect the integrity of the monolayers. The use of
solvents for recovery form the receiver side at the end of sampling
(basal or serosal side) can be tried but the amount bound to the donor
side (apical of mucosal for forward transport) will still pose a
problem. Again, donor samples can be taken at the beginning and at the
end of experiments to calculate total recovery, but that still does
not completely solve the problem.
I hope I have described the problem in detail and would appreciate
more thoughts on this matter.
Gopal Krishna
______________________________ Reply Separator
_________________________________
Subject: Adsorption of lipophilic drug to polycarbonate membrane
Author: PharmPK.aaa.pharm.cpb.uokhsc.edu at INTERNET
Date: 6/19/97 1:29 PM
PharmPK - Discussions about Pharmacokinetics
Pharmacodynamics and related topics
I have been evaluating drug absorption by using Caco-2 cell monolayer. In
the case of high
lipophilic compound, I cannot often reject the influence of adsorption of
drug to the polycarbonate
membrane which supports Caco-2 cell monolayer. So, accurate permeability
coefficient of the
drug cannot be evaluated
I would appreciate if any members of PharmPK can advise me on what
experimental conditions sould be
applied in order to avoid the drug adsorption.
--
Gopal Krishna, Ph.D.
Senior Scientist, Drug Metabolism and PK, Schering-Plough
Maintainer: PK/Biopharmaceutics web page
URL: http://griffin.vcu.edu/~gkrishna/PK/pk.html
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In reply to Teruaki Okuda-
This is a classic problem in cell physiology. One important class of
experiments that could help is characterization of the kinetics of drug
adsorption to the polycarbonate membrane. A good place to start is to load
the membrane (with no cells) with the drug for several fixed loading
periods and several doses, then evaluate the efflux kinetics by removing
the loading solution, washing quickly, and then sampling the efflux medium
as frequently as practical thereafter. A kinetic model of the adsorption
process can then be constructed based on the loading and efflux data. You
can then fit the transport data with a model of both the polycarbonate
membrane and the cell monolayer. The major difficulty you will face is that
the total binding capacity of the PC membrane may not be the same with and
without cells. Nevertheless, you should be able to place an upper bound on
the influence of adsorption. You will want to fit the data from cell
experiments and the data from no-cell experiments simultaneously. This is
relatively straightforward with modern software tools.
Alternatively, you may want to test other growth surfaces, but my
experience is that adsorption is never negligible, and the best approach is
to include the support membrane in your PB-PK model. Analysis is always
cheaper than more experiments. Indeed, this is one of the principal
motivations for moving to complex kinetic models.
Regards,
Bob
----------
Robert D. Phair, Ph.D. rphair.-a-.ix.netcom.com
BioInformatics Services http://webcom.com/rphair
Partnering and Outsourcing for Computational Biology
-----Original Message-----
From: Teruaki Okuda [SMTP:tokuda.-a-.inetmie.or.jp]
Sent: Thursday, June 19, 1997 2:30 PM
To: Multiple recipients of PharmPK - Sent by
Subject: Adsorption of lipophilic drug to polycarbonate membrane
PharmPK - Discussions about Pharmacokinetics
Pharmacodynamics and related topics
I have been evaluating drug absorption by using Caco-2 cell monolayer. In
the case of high
lipophilic compound, I cannot often reject the influence of adsorption of
drug to the polycarbonate
membrane which supports Caco-2 cell monolayer. So, accurate permeability
coefficient of the
drug cannot be evaluated
I would appreciate if any members of PharmPK can advise me on what
experimental conditions sould be
applied in order to avoid the drug adsorption.
Also see: http://www.cpb.uokhsc.edu/pkin/pkin.html
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)