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PAMPAFrom Wikipedia, the free encyclopedia
PAMPA(parallel artificial membrane permeability assay) is a
method which determines thepermeability of substances from a donor
compartment, through alipid-infused artificial
membrane into an acceptor compartment. A multi-well microtitre
plate is used for the donorand a membrane/acceptor compartment is
placed on top; the whole assembly is commonlyreferred to as a
sandwich. At the beginning of the test, the drug is added to the
donorcompartment, and the acceptor compartment is drug-free. After
an incubation period whichmay include stirring, the sandwich is
separated and the amount of drug is measured ineach compartment.
Mass balance allows calculation of drug that remains in the
membrane.To date, PAMPA models have been developed that exhibit a
high degree of correlation withpermeation across a variety of
barriers,
includingCaco-2cultures,[1][2]thegastrointestinaltract,[3]bloodbrain
barrier[4]and skin.
The donor and/or acceptor compartments may contain solubilizing
agents, or additives thatbind the drugs as they permeate. To
improve thein vitro-in vivocorrelation andperformance of the PAMPA
method, the lipid, pH and chemical composition of the system
is often designed with biomimetic considerations in mind.
The popularity of PAMPA inADMEscreening attests to its utility
as an early stage, low-costalternative to cellular models for
research compounds. Althoughactive transportis notmodeled by the
artificial PAMPA membrane, up to 95% of known drugs are
absorbedbypassive transport.[5]Some experts support a lower figure,
so the amount is open to someinterpretation. Microtiter plates with
96 wells can be used for the assay which increases thespeed and
lowers the per sample cost.
Since the first publication by Kansy and coworkers,[6]several
companies developed theirown versions of the assay. Early models
incorporated iso-pH conditions in thecompartments separated by a
simple lipid membrane; subsequently, commercial productswere
introduced which incorporated more sophisticated lipid
membranes.[7]The commercial
products helped ensure that medicinal chemists across different
corporate labs within aworldwide organization used the same
standardized methodology, reagents and obtainedequivalent system
performance as demonstrated with a set of test compounds. This
hasproved very useful as various operational activities have been
outsourced to othercountries.
As financial and other factors in pharmaceutical development
continue to strain timelinesand resources, PAMPA will most likely
continue to play an ever-increasing role inlaboratories wishing to
increase their operating efficiencies and success rate.
Caco-2From Wikipedia, the free encyclopedia
The Caco-2cell line is a continuous
cellofheterogeneoushumanepithelialcolorectaladenocarcinomacells,
developed bytheSloan-Kettering Institute for Cancer Researchthrough
research conducted by Dr.Jorgen Fogh.[1]
Although derived from a colon (large intestine)carcinoma,when
cultured under specificconditions the cells become differentiated
and polarized such that theirphenotype,morphologically and
functionally, resembles theenterocyteslining
thesmallintestine.[2][3]Caco-2 cells express tight
junctions,microvilli,and a number ofenzymesandtransporters that are
characteristic of such
enterocytes:peptidases,esterases,P-glycoprotein,uptake transporters
foramino acids,bile acidscarboxylic acids,etc. They arecommercially
available through theAmerican Type Culture Collection(ATCC;
Manassas,VA, USA).
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intestinehttp://en.wikipedia.org/wiki/Enterocyteshttp://en.wikipedia.org/wiki/Phenotypehttp://en.wikipedia.org/wiki/Carcinomahttp://en.wikipedia.org/wiki/Large_intestinehttp://en.wikipedia.org/wiki/Caco-2#cite_note-1http://en.wikipedia.org/wiki/Memorial_Sloan-Kettering_Cancer_Centerhttp://en.wikipedia.org/wiki/Adenocarcinomahttp://en.wikipedia.org/wiki/Colon_(anatomy)http://en.wikipedia.org/wiki/Epitheliumhttp://en.wikipedia.org/wiki/Humanhttp://en.wikipedia.org/wiki/Heterogeneoushttp://en.wikipedia.org/wiki/PAMPA#cite_note-7http://en.wikipedia.org/wiki/PAMPA#cite_note-6http://en.wikipedia.org/wiki/PAMPA#cite_note-5http://en.wikipedia.org/wiki/Passive_transporthttp://en.wikipedia.org/wiki/Active_transporthttp://en.wikipedia.org/wiki/ADMEhttp://en.wikipedia.org/wiki/In_vivohttp://en.wikipedia.org/wiki/In_vitrohttp://en.wikipedia.org/wiki/Blood%E2%80%93brain_barrierhttp://en.wikipedia.org/wiki/Blood%E2%80%93brain_barrierhttp://en.wikipedia.org/wiki/PAMPA#cite_note-3http://en.wikipedia.org/wiki/Gastrointestinal_tracthttp://en.wikipedia.org/wiki/Gastrointestinal_tracthttp://en.wikipedia.org/wiki/PAMPA#cite_note-1http://en.wikipedia.org/wiki/PAMPA#cite_note-1http://en.wikipedia.org/wiki/Caco-2http://en.wikipedia.org/wiki/Lipid
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When looking at Caco-2 cell cultures microscopically, it is
evident even by visual inspectionthat the cells are heterogeneous.
As a result, over the years the characteristics of the cellsused in
different laboratories around the world have diverged
significantly, which makes itdifficult to compare results across
labs.[4]
Caco-2 cells are most commonly used not as individual cells, but
as a
confluentmonolayeron a cell culture insert filter (e.g.,
Transwell). When cultured in thisformat, the cells differentiate to
form a polarized epithelial cell monolayer that provides aphysical
and biochemical barrier to the passage of ions and small
molecules.[3][5]The Caco-2 monolayer is widely used across the
pharmaceutical industry as an in vitro model of thehuman small
intestinal mucosa to predict the absorption of orally administered
drugs. Thecorrelation between the in vitro apparent permeability
(Papp) across Caco-2 monolayersand the in vivo fraction absorbed
(fa) is well established.[6]Transwell diagram
This application of Caco-2 cells was pioneered in the late 1980s
by Ismael Hidalgo, workingin the laboratory of Ron Borchardt at the
University of Kansas, and Tom Raub, who was attheUpjohn Companyat
the time. Following stints atSmithKline BeechamandRhone-Poulenc
Rorer,Hidalgo went on to co-found a company,Absorption Systems,in
1996,where he remains as Chief Scientist.
The considerable impact of the Caco-2 cell monolayer model can
be measured in at leasttwo ways. First, considering that
poorpharmacokineticproperties accounted for ~40% ofdrug failures in
development in the early 1990s and only ~10% by 2009, an interval
inwhich Caco-2 monolayers were widely used throughout
thepharmaceutical industrytopredict absorption, it is not
unreasonable to attribute some of that shift to this simple
yetpowerful model. Second, the 1989 Gastroenterology paper that
demonstrated the utility ofthe model for this application has been
cited more than 1000 times since its publication.
The versatility of Caco-2 cells is demonstrated by the fact
that, even to this day, they areserving as the basis for the
creation of innovative new models that are contributing to
ourunderstanding of drug efflux transporters such as P-glycoprotein
(ABCB1) and BCRP(ABCG2). RNA interference has been used to silence
the expression of individual effluxtransporters, either
transiently[7]or long-term.[8][9]
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