Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium

Purpose. The purpose of this work was to elucidate the asymmetric effect of P-gp on modulation of absorptive and secretory transport of compounds across polarized epithelium, to develop experimental parameters to quantify P-gp-mediated modulation of absorptive and secretory transport, and to elucidate how P-gp-mediated modulation of transport is affected by passive diffusion properties, interaction of the substrate with P-gp, and P-gp expression. Methods. The permeability of a set of P-gp substrates was determined in absorptive and secretory directions in Madine-Darby Canine kidney (MDCK), Caco-2, and MDR-MDCK monolayers. The transport was also determined in the presence of GW918, a non-competitive P-gp inhibitor, to quantify the permeability without the influence of P-gp. From these two experimental permeability values in each direction, two new parameters, absorptive quotient (AQ) and the secretory quotient (SQ), were defined to express the functional activity of P-gp during absorptive and secretory transport, respectively. Western blot analysis was used to quantify P-gp expression in these monolayers and in normal human intestinal. Results. P-gp expression in Caco-2 and MDR-MDCK monolayers was comparable to that in normal intestine, and much less in MDCK cells. For all models, the substrates encompassed a wide range of apparent permeability due to passive diffusion (PPD). The parameters AQ and SQ, calculated for all compounds, assessed the attenuation in absorptive and enhancement of secretory transport, respectively, normalized to the permeability due to passive diffusion. Analysis of these parameters showed that 1) P-gp affected absorptive and secretory transport differentially and 2) compounds could be stratified into distinct groups with respect to the modulation of their absorptive and secretory transport by P-gp. Compounds could be identified whose absorptive transport was either strongly affected or poorly affected by changes in P-gp expression. For certain compounds, AQ values showed parabolic relationship with respect to passive diffusivity, and for others AQ was unaffected by changes in passive diffusivity. Conclusions. The relationship between attenuation of absorptive transport and enhancement of secretory transport of compounds by P-gp is asymmetric, and different for different sets of compounds. The relationship between attenuation of absorption by P-gp and passive diffusivity of compounds, their interaction potential with P-gp, and levels of P-gp expression is complex; however, compounds can be classified into sets based on these relationships. A classification system that describes the functional activity of P-gp with respect to modulation of absorptive and secretory transport was developed from these results.