EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 544

Transwell Method The transwell method is useful to study the directional transport
of test compounds in polar cells such as Caco-2 and LLC-PK1. Confluent cells are
washed with Hanks' balanced salt solution (HBSS, pH 7.4, 325 mOsm/kg). Then
0.5- and 1.5-mL aliquots of HBSS are added on the apical and basolateral sides,
respectively, of the cell insert. To study apical-to-basolateral or basolateral-to-apical
flux, the test compound is added to the apical or basolateral solution, respectively.
At appropriate times, samples (0.5 mL of basolateral-side or 0.2 mL of apical-side
solution) are collected from the acceptor compartment and replaced with an equal
volume of HBSS, followed by measurement of the drug concentration with suitable
analytical equipment. 25 , 38 , 39
We have used the Transwell method to study the transport of quinolones,
grepafloxacin, sparfloxacin, and levofloxacin in Caco-2 and LLC-PK1 cells. Direc-
tional transport of [ 14 C]grepafloxacin was studied by using monolayers of Caco-2
cells grown on Transwells. The apical-to-basolateral permeability coefficient was
found to be 2.60

10 − 5

± 0.32 × cm/s, which was 74 times higher than the value

10 − 5 cm/s), representing paracellular transport.
The basolateral-to-apical permeability coefficient of [ 14 C]grepafloxacin was 4.80

of mannitol transport (0.035 ×

±

10 − 5 cm/s and was significantly higher than that in the apical-to-basolateral
directional, suggesting that the secretory-directional transport of grepafloxacin can
be ascribed to carrier-mediated transport. We also used cells of the LLC-PK1 se-
ries to examine the transepithelial transport of [ 14 C]grepafloxacin, [ 14 C]sparfloxacin,
and [ 14 C]levofloxacin. LLC-GA5-COL150 cells showed greater basolateral-to-apical
and smaller apical-to-basolateral permeation of these compounds, indicating that
grepafloxacin, sparfloxacin, and levofloxacin are substrates for MDR1. 25 , 41

0.08 ×

18.5. MEMBRANE VESICLES

Membrane vesicles provide an excellent experimental system for examining transport
functions under various conditions. Generally, membrane vesicles can be prepared
from animal tissues and cultured cells; those derived from tissues are used to inves-
tigate drug transport in organs. In kidney, small intestine, and liver, epithelial cells
have polarity, and the plasma membrane has different transport properties on the
apical side and basolateral sides. Methods have been established for preparing mem-
brane vesicles derived from either apical or basolateral membrane, and they are often
called brush-border membrane vesicles (BBMV) and basolateral membrane vesicles
(BLMVs), respectively. These are powerful tools for the detailed study of directional
transport systems.

18.5.1. Membrane Vesicles from Cultured Cells

Membrane vesicles can be used to investigate in detail the properties of transporters,
including investigating the driving forces for transport, such as energy coupling, ef-
fects of trans -or cis -stimulation, and effects of intravesicular conditions. Membrane