Comparison of rat and porcine jejunum as in vitro models for P–glycoprotein mediated efflux using the Sweetana–Grass diffusion method / H.J. Oosthuizen
Oosthuizen, Hendrik Jacobus
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Absorption of drug substances across the intestinal epithelium is a complex and dynamic process. Counter transport proteins are responsible for the efflux of specific drug molecules after they have been absorbed. One of the key counter transport efflux proteins, which is of importance in this study, is P–glycoprotein. The efflux pump P–glycoprotein plays a major role in altering the pharmacokinetics of a wide variety of drugs limiting their absorption and therefore also bioavailability. Many flavonoids have been shown to interact with P–glycoprotein mediated efflux in vitro studies. Numerous in vitro methods have been used to study drug absorption across the intestinal membranes, but it is often not possible to use only one in vitro model to accurately predict permeability characteristics. The purpose of this study was to determine the effect of four selected hydroxy– and methoxy– flavonoids on the in vitro transport of Rhodamine 123, a known P–gp substrate, across excised rat and pig intestinal tissue using the Sweetana–Grass diffusion apparatus. The results were further used to determine if the two different animal tissue models corresponded with regard to the flavonoids' effects on P–glycoprotein related efflux. Two control groups were included in the experimental design. In the negative control group, the transport of Rhodamine 123 was tested alone and no modulator was added. In the positive control group, the transport of Rhodamine 123 was determined in the presence of Verapamil, which is a known P–glycoprotein inhibitor. The experiments with the flavonoids Morin, Galangin, 6–Methoxyflavone and 7–Methoxyflavone were done in triplicate to determine repeatability of the results. The transport of Rhodamine 123 was evaluated in both the apical to basolateral (absorptive) and basolateral to apical (secretory) directions. The relative transport of Rhodamine 123, the apparent permeability coefficient (P app) values and flux (J) values in both directions as well as the efflux ratio (ER) and net flux (J net) were calculated. The concentration Rhodamine 123 present in the acceptor chamber was determined by means of a validated HPLC method. Statistical analysis was used to compare the results of the test groups with the control groups in order to indicate significant differences. It has been found that Morin, Galangin and 6–Methoxyflavone have a significant inhibitory effect on the Rhodamine 123 efflux (probably P–glycoprotein related) in both the rat and pig intestinal tissue models with p–values smaller than 0.05. On the other hand, 7–Methoxyflavone showed a significant effect on the efflux of Rhodamine 123 in the pig intestinal tissue model (p < 0.05) but not in the rat intestinal tissue model (p > 0.05). These flavonoids may increase the bioavailability of drugs that are substrates for P–glycoprotein and thereby cause clinically significant pharmacokinetic interactions, however, this should be confirmed with in vivo studies. On the other hand, these flavonoids may be used for drug absorption enhancement when applied under controlled circumstances. With regard to the different animal tissue models used it can be concluded that data obtained from the rat intestinal tissue model cannot be compared and extrapolated to data obtained from the pig intestinal tissue model. It is recommended that the in vitro results be correlated to in vivo findings to identify the most suitable model.
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