expressed on the apical cell membrane, thenPapp, A-to-B<Papp, B-to-A; if an uptake
transporterisexpressed ontheapicalcell membranethenPapp, A-to-B>Papp, B-to-A.
These relationships will be reversed if transporter is localized on the basolateral
cell membrane. For passively diffused compounds, thePappvalue is concentra-
tion independent and the flux rate is correlated linearly with concentration. For
actively transported compounds, the flux rate is saturable with increasing
concentration of a test compound and the net transporter-mediated flux can be
calculated by subtracting the passive diffusion flux from the total flux rate. The
passive diffusion flux can be estimated either from the flux at 4C (at this
temperature all active transport processes cease to function) or in the presence of
transporter inhibitors. Kinetic parameters such as Km and Vmax can be
determined for net transporter-mediated flux. Polarized cells with the formation
of tight junctions are suitable to be used in the transport assay. Currently,
Caco-2, Madin–Darby canine kidney (MDCK), LLC-PK1, and certain primary
cultured cells are commonly used. In the Draft FDA Guidance on DDI studies,
transcellular transport assay is recommended as the efflux transporter functional
assay and Caco-2, MDR1-MDCK, and MDR1-LLC-PK1 cells have been
accepted as models to evaluate P-gp substrates or inhibitors.
Uptake assay is used to measure the amount of a compound accumulated in
cells. Concentrations of a compound in cells can be quantified by different
analytical tools based upon a given compound’s properties. By using a
fluorescent or a radiolabeled compound as a substrate, the uptake study can be
set up as a high throughput assay for transporter inhibitory potential
screening. In most cases a transporter substrate is also a competitive inhibitor
although there are exceptions under certain circumstances (e.g., a substrate has
low affinity for the transporter or a substrate is unable to reach sufficiently high
concentrations due to cytotoxicity or poor solubility). Cell viability should be
measured in the inhibitor screening studies in order to overcome the false
negative for efflux pumps or positive results for uptake transporters.
The acetoxymethyl ester of calcein (Calcein-AM) is a highly lipid soluble
and nonfluorescent compound that can rapidly penetrate cell plasma
membranes. Once Calcein-AM is taken up into the cells, the ester bond can
be quickly and irreversibly converted to hydrophilic, nonpermeable and
intensively fluorescent free acid form, Calcein. When traversing the cell plasma
membrane, Calcein-AM can be pumped out of the cells by P-gp. Inhibition of
P-gp causes an increase in the cellular level of Calcein-AM and subsequently
the appearance of fluorescent Calcein. Calcein AM uptake studies in P-gp
expressing cells have been extensively used as a screening tool for P-gp
inhibitors (Polli et al., 2001).
Daunorubicin and rhodamine123 (P-gp substrates), Calcein (a MRP
substrate) (Sarkadi et al., 2001), LysoTracker (Xia et al., 2005b) (a BCRP
substrate), and H 2 FDA and BODIPY (BSEP substrates) have been used as
fluorescent substrates for the screening of transporter inhibitors and their
concentrations can be measured by fluorescence-activated cell sorter (FACS)
flow cytometry (Wang et al., 2000, 2003) or any fluorescence detectors.
METHODS TO EVALUATE TRANSPORTER SUBSTRATE 179