Permeability Assays


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Permeability Assays
Background
The Caco-2 cell line, derived from a human
colorectal carcinoma, has become an established
in vitro model for the prediction of drug absorption
across the human intestine. When cultured on semipermeable membranes, Caco-2 cells differentiate
into a highly functionalized epithelial barrier with
remarkable morphological and biochemical similarity
to the small intestinal columnar epithelium. The
membrane transport properties of novel compounds
can thereby be assessed using these differentiated
cell monolayers. The apparent permeability
coefficients (Papp) obtained from Caco-2 cell
transport studies have been shown to correlate
to human intestinal absorption.1,2
Mandin Darby Canine Kidney (MDCK) cells
are a common model for studying drug transport
mechanisms in distal renal epithelia. Like Caco-2
cells, MDCK cells differentiate into columnar
epithelium and form tight junctions when cultured
on semi-permeable membranes. Primarily for
passively absorbed compounds, drug permeability
data from MDCK cell assays have been shown to
be similar to permeability data from Caco-2 cell
assays.1 As a consequence, MDCK cells are gaining
acceptance within the pharmaceutical industry
as an alternate model to Caco-2 cells for rapidly
screening compounds for absorption potential,
as part of the pre-clinical drug selection process.
In addition, preliminary data from some
laboratories show a good correlation between MDCK
cell and Bovine Brain Endothelial Cell (BBEC) culture
permeabilities for several compounds. BBEC cultures
are used as an in vitro model to predict compound
permeability across the blood brain barrier (BBB).
Key Features of the Assay
• model of drug absorption from the intestine to
blood: mimicking the intestinal environment
• model of drug uptake from the blood into
the brain: mimicking the blood brain barrier
Assay Applications
• mechanistic studies of drug absorption
• screening assay for pre-clinical drug selection
Diagram of assay insert
FIG. 1 Cell monolayers are cultured on a semipermeable membrane to form tight cell-cell junctions.
The inner well containing the cells is called the apical
compartment while the outer well is called the
basolateral compartment.
Assay Principle
Permeability across the Caco-2 or MDCK
cell layer is determined by growing the cells on
a membrane placed between two (donor and
acceptor) chambers. Drug candidates are typically
added to the apical side of the cell layer and their
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Apical side
Caco-2 cells
Basolateral side
Semi-permeable
membrane
appearance in the basolateral side is measured over
an incubation time. Permeability in this direction
represents intestinal absorption. Permeability may
also be determined from the basolateral to the apical
side of the Caco-2/MDCK cells. A higher apical
to basolateral Papp, compared to the basolateral to
apical Papp, is indicative of carrier-mediated transport.
P-gp mediated transport is suggested when a higher
basolateral to apical Papp is observed.
Assay Protocol
Caco-2/MDCK Cells
• seed cells in a semi-permeable membrane
• culture for predetermined time
• add drug to apical and/or basolateral
compartments
• incubate for pre-determined time
• sample from each compartment at
pre-determined times
• quantify drug concentrations by LC/MS/MS analysis
FIG. 2 The intestinal absorption of a compound
may occur via passive diffusion (paracellularly or
transcellularly), or via carrier-mediated active
transport (eg. D-glucose, dipeptides). The intestinal
absorption of a compound may be hindered by
P-glycoprotein (P-gp), an ATP-dependent multidrug
efflux pump. These pathways are also present
for the permeation of compounds across the
Caco-2/MDCK cell monolayer.
Compounds Papp (nm/sec) Papp (B to A)/Papp (A to B)
Apical to Basal Basal to Apical
Caco-2 Cells
Lucifer Yellow 11.3 ± 2.6 10.3 ± 2.5 0.91
Rhodamine 123 7.5 ± 1.5 21.4 ± 2.3 2.9
Warfarin 283 ± 19.4 269 ± 16.6 0.95
MDCK Cells
Lucifer Yellow 30.8 ± 3.8 28.3 ± 4.0 0.92
Rhodamine 123 23.6 ± 3.8 48.8 ± 6.3 2.1
Warfarin 357 ± 11.2 326 ± 24.8 0.91
References
1. J.D Irvine et al. J.Pharm.Sci. 88:28 (1999)
2. P. Artursson. J.Pharm.Sci. 79:476 (1990)
This table shows the apparent permeability coefficient values derived in our laboratory for Caco-2 and
MDCK cells. Passive diffusion of a substance occurs by the paracellular (lucifer yellow) or transcellular
(warfarin) routes. Efflux of compounds by P-glycoprotein (P-gp), was demonstrated by rhodamine 123.


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