from vesicles, the drug uptake into membrane vesicles can reach a maximum
level and then decrease (‘‘overshoot’’ phenomenon). Therefore, the kinetic or
inhibition assay must be used during the initial uptake phase, which is reflective
of transporter activity. Sometimes, an ATP regeneration system is added in the
buffer to ensure a sufficient energy supply for the duration of the incubation
(Adachi et al., 1991).
As a functional assay, the membrane vesicle transport assay has been used
to investigate binding sites (Shapiro et al., 1997, 1999), interspecies differences in
transporter activity (Ishizuka et al., 1999; Ninomiya et al., 2005), polymorph-
isms in transporter activity (Center et al., 1998; Hirano et al., 2005), and substrate
or inhibitor specificity for a given efflux pump (Keppler et al., 1998; Volk and
Schneider, 2003).
6.6.1.2 Cell-Based Assays Most cell-based studies are functional transporter
assays. Due to the intact cell structure, cell-based assays may provide more
definitive information about the interaction between drugs and transporters
and can be employed to assess kinetic parameters, such asKmandVmaxfor
substrates as well asKior IC 50 for inhibitors. These assays can also predict
transporter-related DDI that may occur in the clinic. With automation and cell
culture in a multiwell plate, cell-based assays can be adapted to a high
throughput mode. Several drawbacks of cell-based assays include (1)
expression of multiple transporters in a particular cell line including cell lines
that have been engineered to express a given transporter; (2) the expression
level of transporters changes with culture conditions and the number of cell
passages; (3) cells need to be maintained under culture condition prior to use.
This assay is more labor and time intensive than the ATPase assay and
membrane vesicular transport studies.
Cell-based assays include uptake and transport studies. The transport assay
is the most direct assay for evaluating a given transporter’s function and
measures the permeability of a test compound across cell monolayer. Cells can
be seeded on the Transwell inserts and are ready to be used once they reach
confluence and have completely differentiated. Transport experiment is
initiated by the addition of a solution containing the test compound to either
the apical (or upper chamber, for apical-to-basolateral (A-to-B) transport) or
basolateral (or lower chamber, for basolateral-to-apical (B-to-A) transport)
compartment and an aliquot of the solution is removed from the lower
chamber (for A-to-B transport) or from the upper chamber (for B-to-A
transport) at desired time points. The cumulative amount of drug (Q) in the
receiving side is plotted as a function of time. The steady-state flux rateJis then
estimated from the slope (dQ/dt). The apparent permeability coefficient (Papp)
of unidirectional flux for the test compound is estimated by normalizing the
flux rateJ(mol/s) against the nominal surface areaAand the initial drug
concentration in the donor chamberC 0 (mol/mL), orPapp=J/(A C 0 ). The
B/A ratio is equal to thePappvalue for B-to-A transport (Papp, B-to-A) divided
by thePappvalue for A-to-B transport (Papp, A-to-B). If an efflux transporter is
178 DRUG TRANSPORTERS IN DRUG DISPOSITION, DRUG INTERACTIONS