Evaluation of human primary intestinal monolayers for drug metabolizing capabilities

Jennifer E Speer,Yuli Wang,Philip C Smith,Nancy L Allbritton,John K Fallon

doi:10.1186/s13036-019-0212-1

Abstract

BackgroundThe intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo.ResultsThe monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes.ConclusionsThe use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells.Graphical abstract

Highlights

The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs)
The supernatant above the cells was replaced with medium low in growth factors to assist in cell differentiation to mature enterocytes, the major cell type participating in drug metabolism
Prior work demonstrated that at day 10 of this culture protocol, the monolayer formed on both scaffolds, was impermeable to small polar molecules, comprised largely of polarized enterocytes, and displayed functional properties such as drug transport resembling that of native small intestinal epithelium [38]

Summary

Introduction

The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). A major role of the small intestinal epithelium is to act as an initial site of nutrient and drug metabolism by enzymes within or on the surface of the enterocytes, the predominant cell type of the intestinal epithelium These metabolic processes are supported by the small intestine’s large surface area to volume ratio enabled by a lining of villi projecting into the small intestinal lumen and further enhanced by a dense carpet of microvilli on the enterocyte surface [1]. Phase I DMEs include cytochrome P450s (CYPs) and carboxylesterases (CES) and phase II DMEs comprise enzymes such as uridine diphosphate-glucuronosyltransferases (UGTs) [8, 9] Among these enzymes, cytochrome P450 3A (CYP3A4 in humans) and CES2 are major enzymes involved in intestinal contributions to first-pass oxidation and hydrolysis, respectively, of a variety of xenobiotics and endogenous compounds [10], while UGTs are involved in glucuronidation, a Phase II process that can contribute significantly to intestinal first-pass metabolism. All of these enzymes play major roles in drug metabolism, and/or excretion [1]

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Conclusion