Application of the Ussing chamber technique to assess P-glycoprotein-mediated intestinal drug transport

BALLENT, M.; LIFSCHITZ, A.; VIRKEL, G.; MATÉ, L.; SALLOVITZ, J.; LANUSSE, C.

Leipzig, Alemania

Congreso; 11th International Congress of the European Association for Veterinary Pharmacology and Toxicology; 2009

EAVPT

Introduction: Transport proteins play an important role in the intestinal secretion of different drug molecules widely used in veterinary therapeutics. P-glycoprotein (P-gp) secrets a large number of endo-xenobiotic compounds from the intracellular to the extracellular domain by an ATP-dependent process (1).. Different in vitro models have been developed to predict P-gp activity. Caco-2 cell monolayers, derived from a human colonic adenocarcinoma, are widely used to estimate transepithelial passage for different P-gp drug substrates (2). The use of everted gut sacs has been proposed as a new in vitro model for quantification of the P-gp-mediated intestinal drug efflux (3, 4). However, new methodological tools are required to evaluate the physio-pharmacological features of the intestinal drug secretion process in ruminant species. The goal of the current work was to adapt the Ussing chamber technique to the assessment of the P-gp-mediated drug transport in rat and sheep intestine. Material and Methods: The intestine was rapidly removed from experimental animals and washed with a Krebs buffer solution at 4°C. The ileum gut segment was opened along the mesenteric border and then partially stripped from underlying muscle layers. Resulting flat sheets of gut mucosa were mounted into Ussing-chambers. Both mucosal (M) and serosal (S) sides were filled with warmed (37 C) and oxygenated Krebs buffer at pH 7.4. Transepithelial electrical resistance and potential differences were measured to assure the integrity of the intestinal tissues. After a 20 min equilibration period, digoxin (DGX) (200 µM), albendazole sulphoxide (ABZSO) (30 µM) and Rho-123 (5 µM) were added to both mucosal (M) and serosal (S) sides. PSC833 (30 µM) and ivermectin (IVM) (50 µM) were used as P-gp inhibitors. Samples were taken between 30 and 240 min for determination of drug flux across the mucosal and serosal membranes. The amount of permeated DGX and ABZSO was determined by HPLC. The Rho-123 concentrations were measured by spectrofluorometric detection.. The apparent permeability coefficients per unit of membrane surface area (Peff) (cm/s) were estimated.   Results: The evaluation of electrical parameters assured adequate tissue viability (both animal species) over for 4 h. The efflux transport of DGX, ABZSO and Rho-123 was demonstrated both in rat and sheep intestine. The Peff S-M was significantly higher (P<0.05) than the Peff M-S for the three compounds. The efflux coefficient (PeffS-M/PeffM-S)   in rat intestine was 2.37 (DGX) and 1.49 (ABZSO).  This efflux reached values of 1.76 (ABZSO) and 5.37 (Rho-123) in sheep intestine. These results indicate the existence of a transport process towards the intestinal lumen in both species. The presence of PSC833 and IVM (used as P-gp inhibitors) increased the PeffM-S for ABZSO in rat (44 %) and for Rho-123 (68 %) in sheep, which may indicate the involvement of  P-gp in the ABZSO intestinal secretion.. Discussion: These preliminary results demonstrated the usefulness of the developed approach to investigate the intestinal efflux transport for different drug molecules. The current work showed that P-gp may be involved in the intestinal secretion of ABZSO. The Ussing chamber technique offers a great potential to improve the comprehension of the transport mechanisms involved in the absorption/excretion processes for drugs therapeutically important in veterinary medicine.