Efflux pump (e.g., P-gp, MRP1, and BCRP) inhibition has been recognized as a strategy to overcome multi-drug resistance and improve drug bioavailability. Besides small-molecule inhibitors, surfactants such as Tween 80, Cremophor EL, several Pluronics, and Vitamin E TPGS (TPGS 1000) are known to modulate efflux pump activity. Competitive inhibition of substrate binding, alteration of membrane fluidity, and inhibition of efflux pump ATPase have been proposed as possible mechanisms. Focusing on TPGS 1000, the aim of our study was to unravel the inhibitory mechanism by comparing the results of inhibition experiments in a Caco-2 transport assay with data from electron spin resonance (ESR) and from ATPase activity studies. ESR results, on Caco-2 cells using 5-doxyl stearic acid (5-SA) as a spin probe, ruled out cell membrane fluidization as a major contributor; change of membrane fluidity was only observed at surfactant concentrations 100 times higher than those needed to achieve full efflux inhibition. Concurrently, TPGS 1000 inhibited substrate induced ATPase activity without inducing significant ATPase activity on its own. By investigating TPGS analogues that varied by their PEG chain length, and/or possessed a modified hydrophobic core, transport studies revealed that modulation of ATPase activity correlated with inhibitory potential for P-gp mediated efflux. Hence, these results indicate that ATPase inhibition is an essential factor in the inhibitory mechanism of TPGS 1000 on cellular efflux pumps.

While epithelial cell culture models (e.g., Caco-2 cell line) are widely used to assess the absorption of drug molecules across healthy intestinal mucosa, there are no suitable in vitro models of the intestinal barrier in the state of inflammation. Thus development of novel drugs and formulations for the treatment of inflammatory bowel disease is largely bound to animal models. We here report on the development of a complex in vitro model of the inflamed intestinal mucosa, starting with the selection of suitable enterocyte cell line and proinflammatory stimulus and progressing to the setup and characterization of a three-dimensional coculture of human intestinal epithelial cells and immunocompetent macrophages and dendritic cells. In the 3D setup, controlled inflammation can be induced allowing the mimicking of pathophysiological changes occurring in vivo in the inflamed intestine. Different combinations of proinflammatory stimuli (lipopolysaccharides from Escherichia coli and Salmonella typhimurium, interleukin-1β, interferon-γ) and intestinal epithelial cell lines (Caco-2, HT-29, T84) were evaluated, and only Caco-2 cells were responsive to stimulation, with interleukin-1β being the strongest stimulator. Caco-2 cells responded to the proinflammatory stimulus with a moderate upregulation of proinflammatory markers and a slight, but significant, decrease (20%) of transepithelial electrical resistance (TEER) indicating changes in the epithelial barrier properties. Setting up the coculture model, macrophages and dendritic cells derived from periphery blood monocytes were embedded in a collagen layer on a Transwell filter insert and Caco-2 cells were seeded atop. Even in the presence of immunocompetent cells Caco-2 cells formed a tight monolayer. Addition of IL-1β increased inflammatory cytokine response more strongly compared to Caco-2 single culture and stimulated immunocompetent cells proved to be highly active in sampling apically applied nanoparticles. Thus the 3D coculture provides additional complexity and information compared to the stimulated single cell model. The coculture system may serve as a valuable tool for developing drugs and formulations for the treatment of inflammatory bowel diseases, as well as for studying the interaction of xenobiotics and nanoparticles with the intestinal epithelial barrier in the state of inflammation.

No abstract

No abstract