Effect of Crohn's Disease on Villous Length and CYP3A4 Expression in the Pediatric Small Intestine.

Changes in absorptive capacity and first-pass metabolism in the small intestine affect oral drug bioavailability. Characterization of such changes as a consequence of inflammation is important for developing physiologically-based pharmacokinetic (PBPK) models for inflammatory bowel disease. We sought to elucidate the impact of small intestinal Crohn's disease (CD) on villous length and CYP3A4 expression in children. Freshly frozen duodenal and terminal ileum (TI) biopsies from 107 children (1-19 years) with and without CD were evaluated for active inflammation. Villous length and CYP3A4 mRNA/protein expression were compared among regions of active and inactive inflammation in CD and controls. A twofold reduction in villous length was observed in inflamed duodena and ilia of children with CD, but in the absence of regional inflammation, villi in CD were comparable in length to controls. Expression of CYP3A4 mRNA correlated significantly with villous length in the TI (P = 0.0003), with a trend observed in the duodenum that did not reach statistical significance. In the presence of active inflammation, a significant decrease in CYP3A protein expression was confirmed in the duodenum, where protein expression also correlated significantly with villous length across diagnoses (P < 0.0001). Our findings suggest that previous observations of decreased CYP3A4 expression and function in inflamed intestine may not be due solely to downregulation by inflammatory cytokines, but also to villous blunting and subsequent loss of surface area for protein expression. This information is relevant for PBPK model development and could aid with dose adjustment decisions for oral CYP3A4 substrates administered during CD flare (e.g., budesonide).

Reevaluating the role of megalin in renal vitamin D homeostasis using a human cell-derived microphysiological system

The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysio­logical systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.

Development of a microphysiological model of human kidney proximal tubule function.

The kidney proximal tubule is the primary site in the nephron for excretion of waste products through a combination of active uptake and secretory processes and is also a primary target of drug-induced nephrotoxicity. Here, we describe the development and functional characterization of a 3-dimensional flow-directed human kidney proximal tubule microphysiological system. The system replicates the polarity of the proximal tubule, expresses appropriate marker proteins, exhibits biochemical and synthetic activities, as well as secretory and reabsorptive processes associated with proximal tubule function in vivo. This microphysiological system can serve as an ideal platform for ex vivo modeling of renal drug clearance and drug-induced nephrotoxicity. Additionally, this novel system can be used for preclinical screening of new chemical compounds prior to initiating human clinical trials.

Functional Comparison of Human Colonic Carcinoma Cell Lines and Primary Small Intestinal Epithelial Cells for Investigations of Intestinal Drug Permeability and First-Pass Metabolism.

To further the development of a model for simultaneously assessing intestinal absorption and first-pass metabolism in vitro, Caco-2, LS180, T84, and fetal human small intestinal epithelial cells (fSIECs) were cultured on permeable inserts, and the integrity of cell monolayers, CYP3A4 activity, and the inducibility of enzymes and transporters involved in intestinal drug disposition were measured. Caco-2, T84, and fSIECs all formed tight junctions, as assessed by immunofluorescence microscopy for zonula occludens-1, which was well organized into circumscribing strands in T84, Caco-2, and fSIECs but was diffuse in LS180 cells. The transepithelial electrical resistance value for LS180 monolayers was lower than that for Caco-2, T84, and fSIECs. In addition, the apical-to-basolateral permeability of the paracellular marker Lucifer yellow across LS180 monolayers was greater than in fSIECs, T84, and Caco-2 monolayers. The transcellular marker propranolol exhibited similar permeability across all cells. With regard to metabolic capacity, T84 and LS180 cells showed comparable basal midazolam hydroxylation activity and was inducible by rifampin and 1α,25(OH)2D3 in LS180 cells, but only marginally so in T84 cells. The basal CYP3A4 activity of fSIECs and Caco-2 cells was much lower and not inducible. Interestingly, some of the drug transporters expressed in LS180 and Caco-2 cells were induced by either 1α,25(OH)2D3 or rifampin or both, but effects were limited in the other two cell lines. These results suggest that none of the cell lines tested fully replicated the drug disposition properties of the small intestine and that the search for an ideal screening tool must continue.