throughput mode. However, the ATPase assay is not a functional transporter
assay and can not distinguish substrates from inhibitors (Boulton et al., 2002;
Polli et al., 2001; Ramachandra et al., 1996; Rao, 1995). Other drawbacks of
this assay include large interday or intraday variations (Xia et al., 2006) and
potential false negative results.
The membrane vesicular transport assay is another high throughput assay
that can be used to identify substrates and/or inhibitors of efflux pumps, such
as P-gp, BCRP, MRP or BSEP, and uptake transporters, such as NTCP. The
membrane vesicles can be prepared not only from transporter-transfected or
overexpressed cells but also from the brush-border membrane of intestine,
kidney, and choroids plexus; hepatic sinusoidal and canalicular membranes;
and luminal and albuminal membranes of the brain (Boyer and Meier, 1990;
Meier and Boyer, 1990). The membrane vesicles obtained from tissues may
contain many varied transporters that may limit their utility for evaluating the
interaction of a given transporter with a given compound. The purity of these
membrane vesicles from tissues can be evaluated by the enrichment of the
relative activity of membrane marker enzymes, such as dipeptidyl peptidase IV
for canulicular membrane of hepatocytes, for the target plasma membranes
(Meier and Boyer, 1990). Most ABC transporters and some SLC transporters
can be studied using membrane vesicles. For example, NTCP is a sodium
dependent transporter. Thus, the NTCP activity can be assessed by measuring
the difference of drug accumulation in the right-side-out membrane vesicles in
the presence and absence of Na+buffer. Since ABC protein (efflux pump)-
mediated transport is ATP-dependent, and ATP cannot traverse the lipid
membrane due to its hydrophilic nature, only inside-out membrane vesicles can
allow ABC transporters to bind ATP and subsequently pump a substrate into
the vesicles. Utilizing a rapid filtration technique, the membrane vesicles can be
collected on a filter membrane and the substrate trapped inside the vesicles can
be quantitated by LC-MS/MS, fluorescence detector, or liquid scintillation
counting (Tabas and Dantzig, 2002; Xia et al., 2004). The difference of the
uptake of a substrate in the presence or absence of ATP or other driving force
such as Na+and H+is attributed to ABC protein or uptake transporter-
mediated transport, respectively. The membrane vesicular assay is an effective
in vitrosystem without intracellular binding and metabolism to directly reflect
transporter functions and to define the kinetic constants such as Km for
substrates (Keppler et al., 1998; Xia et al., 2004), andKior IC 50 for inhibitors
(Hirano et al., 2005; Horikawa et al., 2002). The assay can also be used to study
the driving force of transporter of interest by manipulating the ionic
composition of or ATP concentration in the assay buffer (Meier et al.,
1984). This assay may give false negative results for highly lipophilic
compounds due to high nonspecific binding to lipid membranes or high
passive diffusion. The adsorption can be distinguished from active transporter-
mediated intravesicular accumulation by measuring the uptake under different
osmolarities since the intravesicular volume decreases with increasing
osmolarity. Due to the consumption of ATP and subsequent drug release
METHODS TO EVALUATE TRANSPORTER SUBSTRATE 177