Development of a novel gut microphysiological system that facilitates assessment of drug absorption kinetics in gut

Abstract

There is an urgent need for novel methods that can accurately predict intestinal absorption of orally administered drugs in humans. This study aimed to evaluate the potential of a novel gut microphysiological system (MPS), gut MPS/Fluid3D-X, to assess the intestinal absorption of drugs in humans. The gut MPS/Fluid3D-X model was constructed using a newly developed flow-controllable and dimethylpolysiloxane-free MPS device (Fluid3D-X^®). Human induced pluripotent stem cells-derived small intestinal epithelial cells were employed in this model, which exhibited key characteristics of the human absorptive epithelial cells of the small intestine, including the expression of key gene transcripts responsible for drug transport and metabolism, and the presence of dome-like protrusions in the primary intestinal epithelium under air-liquid interface culture conditions. Functional studies of transporters in the constructed model demonstrated basal-to-apical directional transport of sulfasalazine and quinidine, substrates of the active efflux transporters breast cancer resistance protein and P-glycoprotein, respectively, which were diminished by inhibitors. Furthermore, a cytochrome P450 (CYP) 3A inhibitor increased the apical-to-basal transport of midazolam, a typical CYP3A4 substrate, and reduced metabolite formation. These results suggest that gut MPS/Fluid3D-X has the potential to assess the intestinal absorption of small-molecule drugs.

Fig. 1

MPS hardware. MPS hardware. (a) Hard plastic film lamination fabricated using Fluid3D-X^®. (b) Photograph of Fluid3D-X^®. Blue: Apical channel; Red: Basal channel. (c) Illustration of Fluid3D-X^® and the medium perfusion setup. (d) Photograph of the medium perfusion setup.

Fig. 2

Construction of gut MPS/Fluid3D-X. Cells were cultured for 11 days, and phase-contrast images of gut MPS/Fluid3D-X were captured using an APX100 microscope (EVIDENT CORPORATION). Bright-field images of (a) liquid-liquid interface and (b) air-liquid interface cultures. Bar: 2 mm. (c) Cultured cells under ALI conditions (day 12) were observed using an optical coherence tomography (OCT) imaging system (CELL3 IMAGER ESTIER, SCREEN Holdings Co., LTD.). Top left: Bright field image of the observation area. Blue line indicates the scan direction. Top right: X-Z section image of yellow line (top left). The images were acquired at a 3 μm pitch across a 1 mm² area. Bottom left: 3D dome structure obtained from the top. The image was generated utilizing Image J software (National Institutes of Health, Bethesda, MD, USA) Bottom right: 3D dome structure taken from the side. Scale bars: 250 μm. (d) Time-dependent expression of mRNAs (days 8, 11, and 13) was confirmed by probes that detect transporters, P450 enzymes, intestinal epithelial markers, and stem cell markers. The absolute value of each copy number was divided by that of EpCAM1. * Copy numbers were less than the lower limit of the calibration curve. Each bar represents the mean value (n = 2).

Fig. 3

Assessment of gut transport across the hiSIECs monolayers in gut MPS/Fluid3D-X. Transport of antipyrine (a and b), atenolol (c and d), midazolam (e and f), quinidine (g and h), and sulfasalazine (i and j) across hiSIEC monolayers in gut MPS/Fluid3D-X. Cumulative amount of drugs and transport clearances as transported from apical to basal side in the presence (closed circle) or absence (open circle) of inhibitor cocktail after dosing of test substances to the apical side of gut MPS/Fluid3D-X. For antipyrine, quinidine, and sulfasalazine, transport from the basal to apical side was also examined in the presence (closed square) or absence (open square) of the inhibitor cocktail after dosing with antipyrine (a and b), quinidine (g and h), or sulfasalazine (i and j) to the basal side of the gut MPS/Fluid3D-X. †p < 0.05, ††p < 0.01; significant difference in apical-to-basal transport in the presence and absence of inhibitors. *p < 0.01, **p < 0.01; significant difference between the apical-to-basal transport compared to basal-to-apical transport in the absence of inhibitor. Each bar and error bar represent the mean value and SD, respectively (inhibitor(-): n = 4, inhibitor (+): n = 3).

Fig. 4

Metabolic activities of CYP3A4 in gut MPS/Fluid3D-X. Metabolism of midazolam in the gut MPS/Fluid3D-X. Midazolam was administered to apical side of gut MPS/Fluid3D-X in the presence (closed circles) or absence (open circles) of inhibitors, and concentration of its metabolite, 1-hydroxymidazolam was monitored in apical (a) or basal side (b). The total amount of 1-hydroxymidazolam formed by gut MPS/Fluid3D-X after dosing the apical side with midazolam is shown in Fig. 4c. ^##p < 0.01; significant difference between the presence and absence of inhibitors. Each bar and error bar represent the mean value and SD, respectively (inhibitor(-): n = 4, inhibitor (+): n = 3).