Human Primary Cell-Based Organotypic Microtissues for Modeling Small Intestinal Drug Absorption

Abstract

Purpose: The study evaluates the use of new in vitro primary human cell-based organotypic small intestinal (SMI) microtissues for predicting intestinal drug absorption and drug-drug interaction.

Methods: The SMI microtissues were reconstructed using human intestinal fibroblasts and enterocytes cultured on a permeable support. To evaluate the suitability of the intestinal microtissues to model drug absorption, the permeability coefficients across the microtissues were determined for a panel of 11 benchmark drugs with known human absorption and Caco-2 permeability data. Drug-drug interactions were examined using efflux transporter substrates and inhibitors.

Results: The 3D-intestinal microtissues recapitulate the structural features and physiological barrier properties of the human small intestine. The microtissues also expressed drug transporters and metabolizing enzymes found on the intestinal wall. Functionally, the SMI microtissues were able to discriminate between low and high permeability drugs and correlated better with human absorption data (r² = 0.91) compared to Caco-2 cells (r² = 0.71). Finally, the functionality of efflux transporters was confirmed using efflux substrates and inhibitors which resulted in efflux ratios of >2.0 fold and by a decrease in efflux ratios following the addition of inhibitors.

Conclusion: The SMI microtissues appear to be a useful pre-clinical tool for predicting drug bioavailability of orally administered drugs.

Keywords: caco-2; drug metabolizing enzymes; drug permeation; drug transporters; drug-drug interaction; organotypic small intestinal microtissues.

Fig. 1

Monolayer culture of primary human small intestinal cells harvested from the: (a) duodenum, (b) jejunum, and (c) ileum of primary human small intestine tissue. Epithelial cells were stained for the epithelial tight junction marker ZO-1 (d), and primary small intestine fibroblasts were stained with the fibroblast marker, Vimentin (e).

Fig. 2

H&E stained histological cross-section of the small intestine microtissues showing the epithelium surface with villi, the basolateral fibroblast extracellular matrix substrate, and the underlying microporous membrane support (pore diameter = 0.4 um).

Fig. 3

Immmuno-stained cross-sections of the in vitro reconstructed SMI microtissues showing cytokeratin (CK)-19 stained columnar epithelial cells (red), villin stained apical surface of epithelium (marker for brush border, green) and vimentin stained fibroblasts in the underlying ECM substrate (white). Additional images show de novo synthesis of the extracellular matrix proteins, (b) collagen IV (green) and (c) fibronectin (red). Human explant small intestinal tissue control tissues stained for villin (green, apical surface), CK19 positive epithelium (red) and fibroblasts (white) are shown (d). Nuclei were stained with DAPI (blue). EC = Epithelial cell layer.

Fig. 4

Transmission electron micrographs (TEM) of small intestine microtissues showing brush border membranes with tight junctions (TJ) in: (a) the SMI microtissues and (b) human intestinal explant tissue. Scanning electron microscopy (SEM) was used to visualize villi structures on the SMI microtissue surface (c).

Fig. 5

Immmuno-stained cross-sections of: (a) In vitro intestinal microtissues and (b) Human small intestine explant tissue. Both tissues express villin (marker of brush border), and the main drug transporter, P-glycoprotein (P-gp). An overlay of the villin and P-gp cross-sections show that both villin and P-gp are expressed on the apical surface of the in vitro and explant tissues.

Fig. 6

The effect of extended culture on the barrier properties of the SMI microtissues. As shown, TEER values remain constant over the 6-week experiment.

Fig. 7

RT-PCR results showing gene expression of efflux transporters, P-glycoprotein (MDR1), BCRP, MRP1, and MRP2, by: (a) human small intestinal explant tissue, (b) full-thickness intestinal microtissues (SMI-FT; epithelial cells plus fibroblasts), and c) partial-thickness microtissues (SMI-PT; epithelial cells only).

Fig. 8

Schematic representation of small intestine tissue showing lumen side (apical) and blood side (basolateral) localization of drug metabolizing enzymes and drug transporters, and A-to-B and B-to-A transport of compounds.

Fig. 9

Correlation of human drug absorption with drug permeability results (P_app) for: (a) SMI-FT microtissues (and b) Caco-2 monolayers. Triplicate tissues were exposed to each model drug (n = 11) for 2 h. Average P_app values from two independent lots of SMI-FT microtissues were used.

Fig. 10

Effect of drug transporter inhibitors on drug permeation and the efflux ratio for the 3D intestinal microtissues. As shown, the presence of substrate inhibitors increases A→B permeability/bioavailability of the drug (a) and decreases the efflux ratio (b). P-gp substrates: Digoxin, Talinolol, Loperamide, and Rosuvastatin. BCRP substrate: Nitrofurantoin. P-gp inhibitor: Verapamil. BCRP inhibitor: Novobiocin.

Fig. 11

CYP450 enzyme metabolic activity of intestinal microtissues. Isozyme-specific substrates were applied to the small intestine microtissues and the metabolic activity was monitored using isozyme-specific fluorogenic substrates of CYP450 isozymes involved in drug metabolism. The CYP450 substrates are metabolized by a specific intestinal microtissue CYP450 enzyme and the products are highly fluorescent are released into the aqueous medium. The presence of fluorescent metabolites in the culture supernatants collected from the apical surfaces was measured after 1, 2, or 3 h. Fluorescent intensities were measured using a fluorescent plate reader.