6.6 METHODS TO EVALUATE TRANSPORTER SUBSTRATE,
INHIBITOR, OR INDUCER
Given the important role of transporters in ADMET and drug-drug
interactions, the screening of drugs for their affinity as substrates or inhibitors
of transporters should be a standard component of the ADME package in the
discovery (Balani et al., 2005) and development stages.
6.6.1 In vitroModels
6.6.1.1 Membrane-Based Assays Membranes prepared from cells expres-
sing transporters have been widely used to study the function of ABC efflux
pumps and to identify their substrates or inhibitors. Currently, there are two
major membrane-based assays: the ATPase assay and the membrane vesicular
transport (uptake) assay. Compared to the cell-based assay, the membrane-
based assay has several advantages including: (1) the assay can be used to
characterize the effect of a xenobiotic on one specific efflux transporter; (2)
the assay can be easily employed in a high throughput mode; (3) membranes
are easy to be maintained after preparation; and (4) the assay is easy to
conduct.
The transport function of the ABC efflux pumps depends on the binding
and the hydrolysis of cytoplasmic ATP within NBD. The ATPase activity of
P-gp, MRP or BCRP expressing in insect cell or mammalian cell membranes is
vanadate sensitive and can be stimulated or inhibited by substrates of these
transporters (Chang et al., 1998; Ozvegy et al., 2001, 2002; Sarkadi et al., 1992;
Senior and Urbatsch, 1995). The released phosphate can be determined by a
sensitive colorimetric reaction under mild acidic conditions (Druekes and
Palm, 1995) or the amount of ATP can be quantified by a luciferase-generated
luminescent signal (Promega). Since the profile of altered ATPase activity by P-
gp substrates has been shown to reflect the nature of the interaction between P-
gp and a substrate (Ramachandra et al., 1996; Rao, 1995), ligand screening
assays have been developed for P-gp and BCRP substrates/inhibitors using the
ATPase assay (Polli et al., 2001; Scarborough, 1995; Xia et al., 2004). In
addition to the stimulation of the basal ATPase activity of P-gp, it has been
suggested to incorporate the inhibition of the stimulation of the ATPase
activity by reference compounds, such as verapamil, progesterone, or
vinblastine in screening studies (Garrigues et al., 2002; Scarborough, 1995).
Coincubation of test agents with P-gp ATPase activators or inhibitors can
provide information about the potential binding site to P-gp (Litman et al.,
1997). ATPase can also be used to evaluate the interspecies difference of an
ABC pump (Xia et al., 2006). The kinetic constant obtained from ATPase
assay can be used to rank order the binding affinity between an ABC pump and
its modulators (Boulton et al., 2002). The membrane ATPase assay is
compound-independent, easy to perform and does not require the use of
radiolabeled compounds. Therefore, it can easily be applied in a high
176 DRUG TRANSPORTERS IN DRUG DISPOSITION, DRUG INTERACTIONS