The bidirectional permeability assay, where the basolateral to apical (secretory direction, B to A) permeability is compared to the apical to basolateral (absorptive direction, A to B) permeability, is regarded as the gold standard in identifying P-gp substrates because it is functionally the most direct method of measuring efflux characteristics of drug candidates. Compounds with an efflux ratio (ratio of B to A/A to B) greater than 2-3 are typically considered as P-gp substrates. However, it is well known that efflux transporters other than P-gp (e.g., MRP2, BCRP, etc.) are also functionally expressed in the Caco-2 cells. Therefore, a simple bidirectional difference may not ascertain that the compounds being tested is indeed P-gp substrates. As a confirmatory study, a follow-up bidirectional experiment is routinely repeated in the presence of known inhibitors of P-gp, BCRP and MRP2. GF120918 at 2-4 |xM is often used to selectively inhibit the P-gp-based efflux transport. However, some recent publications have reported that GF120918 interacts not only with P-gp but also with the BCRP (Maliepaard et al., 2001; Woehlecke etal., 2003). Similarly, MK-571 and FTC are used to selectively inhibit the MRP2 (Chen et al., 1999; Dantzig et al., 1999) and BCRP activity (Volk and Schneider, 2003; Zhang et al., 2004), respectively. Comparison of efflux ratio in the absence and presence of these specific inhibitors can delineate the potential role of the individual efflux transporter. Because it is a cell-based assay with a physical barrier (lipid bilayer), the test compounds must have adequate cell permeability for the bidirectional P-gp assay. One major drawback of the bidirectional permeability assay is that the P-gp substrates with insufficient transcellular permeability cannot be identified. Drugs such as famotidine and ranitidine are substrates for secretory transporter proteins (Lee et al., 2002) but often they fail to be detected as P-gp substrates due to their low passive permeability.
Several efflux (e.g., P-gp, MRP2, and BCRP) and influx transporters (e.g., PEPT1) are expressed in Caco-2 cells under standard cell culture conditions. However, the functional expression of drug transporters in Caco-2 cells may vary significantly depending on the passage number and minor changes in culture conditions. For example, the efflux ratio of digoxin or sulfasalazine (both suggested to be efflux transporter substrate) varied drastically with passage number. It is interesting to note that often the lack of P-gp functional expression becomes
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Figure 5.7. Effect of Caco-2 cell passage number on the permeability of sulfasalazine, mannitol, and metoprolol problematic sporadically. There was no identifiable trend with the change in passage number. Figure 5.7 demonstrates that the levels of P-gp expression in Caco-2 cells can vary significantly depending on the cell passage number. Mannitol (a hydrophilic paracellular marker) and metoprolol (a lipophilic transcellular marker) demonstrated consistent permeability values over the different passage numbers. The reproducibility for these two compounds confirms that the cell monolayers are not compromised. However, sulfasalazine, a P-gp substrate that typically has low A to B permeability value (<30nm/s), showed significantly higher permeability (>120 nm/s) for only certain passage numbers. The variability in the levels of expression of P-gp was directly reflected by the variability in the permeability values of sulfasalazine. Similarly, the levels of P-gp expression have also been known to fluctuate widely amongst the wells even in a single passage study. Digoxin efflux ratio can vary from low of 8 to a high of 20 in different wells of the same passage number Caco-2 cells highlighting the variability in the expression of efflux transporter.
Because of inconsistent P-gp functional expression in Caco-2 cells, the cell line which over-express efflux transporters might be a better in vitro tool to examine the drug-transporter interactions. The engineered cell lines selectively express the transporter of interest and facilitate interaction studies with a specific transporter in isolation. Bidirectional studies performed in these cell lines (over-expressing only one transporter) would specifically demonstrate the involvement of a single transporter without any interplay with other transporters. Follow-up studies can be conducted in the presence of selective inhibitor to further confirm the involvement of the transporter (viz: transporter phenotyping). MDCK and LLC-PK1 cell lines stably transfected with a specific efflux transporter (e.g., MDR1, mdrl, MRP2, and BCRP) may be used to tease out transporter interactions at early discovery stage. The functional expression of transporters in these cell lines appears to be more stable compared to Caco-2 cells.
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