Experimental Factors Effecting Efflux Ratio

Currently there is no universally accepted standard study protocol for conducting the cell based bidirectional permeability assay. Different laboratories perform these studies under different experimental conditions that often showed a large interlaboratory variability. Standardization of experimental conditions (e.g., pH, drug concentration and duration of incubation, etc.) can provide better consistency of the results and significantly minimize the occurrence of false negatives (i.e., unable to identify a true substrate). The use of an optimal substrate concentration is a key parameter for attaining accurate results from this model. Performance of bidirectional studies at high concentrations (50 |M or more) often lead to saturation of the efflux transporter and result in efflux ratio of unity even for well-known P-gp substrates. Figure 5.8 demonstrates the dramatic effect of using the lower (3 |M) concentration for improving the utility of this model. Classical

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50 uM 3 uM

Verapamil Quinidine BMS-1 BMS-2

Figure 5.8. Effect of substrate concentration (3 |M vs. 50 |M) on the efflux ratio (ratio of B to A/A to B) in the Caco-2 cell bidirectional study. The efflux ratio was calculated using the mean data. The coefficient of variation in the permeability data (both direction) is typically less than 25% amongst replicates

P-gp substrates such as verapamil, quinidine and two internal research compounds are shown to have efflux ratio (ratio of B to A/A to B) of ~ 1 when studied at 50 |M. However, when they were studied at lower concentration of 3 |M, they demonstrated unequivocal efflux characteristics with efflux ratio >2. The P-gp is saturable at high substrates concentrations and thus at 50 |M, the P-gp is knocked out functionally leading to false negative data. using lower substrate concentrations ( < 5 |M) could potentially minimize the saturation of the efflux transporters and can significantly improve the predictability of the assay. It should be recognized that lowering the substrate concentration imposes an analytical challenge, and one must make sure that the analytical technique is capable of measuring the sample concentrations with adequate accuracy.

The preliminary screen at a low substrate concentration is an efficient approach to evaluate a large number of compounds and maintain sufficient throughput. However, "yes or no" classification may not be very useful because it is difficult to quantify (if possible) the clinical significance of the potential role of P-gp in the drug disposition. As a follow-up study, it is recommended that a range of substrate concentrations to be tested, and the estimated Km value can be related to in vivo relevant drug concentrations either in the intestine (during absorption) or systemic circulation (during distribution and excretion). One of the common limitations in conducting concentration dependency experiment is the lack of adequate aqueous solubility of new drug candidates (i.e., poor solubility does not allow a wide range of concentration to be tested).

Another potential issue with cell-based models is that the integrity of tight junctions in the Caco-2 cell monolayers can be compromised (i.e., becomes leaky) when incubated with test compounds, and the extent of damage is typically concentration dependent. The permeability across the compromised monolayer is often much higher compared to the intact monolayers, and the permeability becomes artificially high in both direction. In that case, the efflux ratio often becomes unity, and the test compound is classified as a nonsubstrate even if it is a true substrate (again false negative). To detect cell damage during the incubation, one can measure TEER value before and after the study. But the change in the TEER value is not very sensitive to detect a minor cell damage. More definitive way to detect cell damage is to coincubate a paracellular route marker such as radiolabeled mannitol in the test run. It is also possible to see false positives. It appears that the basolateral membrane of Caco-2 cell monolayer is more sensitive than the apical membrane (i.e., the cell damage occurs more frequently in the B to A direction than the A to B direction). When this occurs, the B to A permeability is significantly greater than A to B permeability, and the test compound is classified as a P-gp substrate even if it is a nonsubstrate (false positives). Figure 5.9 shows that the cell damage due to BMS-X is concentration dependent because the mannitol permeability was significantly higher when incubated with 50 | M compared to 10 | M BMS-X, and it also illustrated that the only basolat-eral membrane was sensitive to BMS-X. When the substrate concentration was reduced to 10 | M, the B to A permeability was reduced and became similar to A to B permeability. Therefore, lowering the substrate concentration is a good way

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