11

Figure 5.9. Effect of drug concentrations on the integrity of Caco-2 cell monolayer to minimize the potential cell damage, and consequently the incidence of both false negatives and false positives.

Another experimental factor that needs to be fully optimized is the pH used in the bidirectional permeability studies. Optimization of the apical pH is not as straightforward as the substrate concentration. Based on the pKa of the test compounds, the change in apical pH can lead to dramatically different extent of ionized and unionized fractions in the apical compartment and consequently significant changes in the permeability value. Figure 5.10 demonstrates the effect apical pH on the efflux ratio observed for well-known P-gp substrates. Digoxin, a neutral P-gp substrate, had no ionization changes with pH and showed a uniform efflux ratio ~10 at all three pH. However, saquinavir, a weak base, showed a significant change in the efflux ratio where the ratio was greater than 50 at an apical pH of 5.5 as compared to the ratio of ~ 10 at pH 7.4. On the contrary, sulfasalazine (contains a carboxylic acid) demonstrated a higher efflux ratio at an apical pH of 7.4. The higher efflux ratio observed with saquinavir and sulfasalazine at an apical pH of 5.5 and 7.4, respectively, is primarily due to lower apical to basolateral permeabilities (i.e., smaller denominator in the ratio calculation) rather than the changes in the basolateral to apical permeability. Therefore, the apical pH needs to be optimized depending on the type of ionizable function group of the compounds of

Digoxin

Saquniavir

Sulfasalazine

Figure 5.10. Effect of apical pH (5.5, 6.5 and 7.4) on the efflux ratio of acidic, basic and neutral P-gp substrates 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

interest. Prior information on the pKa and acidic/basic nature of the compound will be very useful in the P-gp substrate assay. Other experimental variables such as incubation duration, composition of transport buffer, presence/absence of serum, cell-culturing conditions, etc. need to be calibrated using a set of known P-gp substrates. Factors such as culturing conditions, composition of media, cell plate architecture (12-well vs. 24-well Transwell) all have a significant effect on the expression of P-gp and the interlaboratory variability is often significantly large. Therefore, a direct comparison of the results obtained from different laboratories should be done cautiously.

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