low endogenous transporter background and short expression turnaround time
(4–7 days). Additionally, the cells can perform the appropriate posttranslational
modification; the transporters are appropriately localized in the membrane; and
a high proportion of cells express the transferred genetic information (Sigel,
1990). The drawbacks of oocytes as an expression system include (Sigel, 1990) (1)
transient expression of the transporter, which usually lasts no longer than 14
days; (2) seasonal variations in the quality of the oocytes; (3) low to medium
throughput assays; (4) cDNA that has to be transcribed, capped, and
polyadenylatedin vitrounless it is directly injected into the nucleus of the
oocytes and (5) requirement of microinjection skill for transfecting cDNA or
mRNA and for efflux transporter studies.
6.6.1.4 Induction Models Since Caco-2 cells lack steroid xenobiotic receptor
(SXR), a human typical pregnane X receptor (PXR) (Li et al., 2003), it is a not
a good model to evaluate P-gp inducers. SXR ligands, such as rifampicin,
paclitaxel, nifedipine, and St. John’s Wort, increase both P-gp and CYP3A4
expression in LS180 (Li et al., 2003; Perloff et al., 2001), a human intestinal
carcinoma cell line, suggesting that LS180 cells can be used for the evaluation
of P-gp inducers. Currently, all these models have shown an induction at the
mRNA and protein levels but not in the transport function assay yet.
6.6.1.5 Mapping Transporter Substrates or Inhibitors Estimation of a drug
transporter substrate or inhibition properties can help to understand the drug
disposition profile, pharmacological effects, and potential drug-drug interac-
tions. Transporter substrate mapping can be done by using selective inhibitors
or specific transporter transfected systems. Combined with efflux transporter
inhibitor treatment, Caco-2 cells can be used to map efflux transporters to their
substrates. This method is highly depended upon the selectivity of transporter
inhibitors. The selectivity of several commonly used efflux transporter
inhibitors is listed in Table 6.4. It is difficult to identify MRP2 substrate by
using an inhibitor method because of the lack of the selective MRP2 inhibitors.
It is noteworthy that not only the transfected transporters but also endogenous
TABLE 6.4 Selectivity of commonly used transporter inhibitors.
IC 50 (mM)P-gp BCRP MRP2 OATP2
Test system Caco-2 cells Membrane vesicles Membrane vesicles Oocytes
Substrate
(concentration)
Taxol (50 nM) Estrone sulfate
(2mM)Leukotriene C 4
(3.2 nM)Estrone sulfate
(2mM)
GF120918 0.04 0.06 > 100 > 100
MK571 25 0.6 2.2 52.7
LY335979 0.05 12.0 > 100 > 100
Ko143 > 50 0.03 > 100 61.3METHODS TO EVALUATE TRANSPORTER SUBSTRATE 181