An in vitro safety evaluation model for assessing the effects of a chemical drug on the human intestinal barrier integrity using genetically modified Caco-2 cells expressing drug-metabolizing enzymes.

The human intestinal cell line Caco-2 is widely used as an in vitro human intestinal barrier-integrity model for safety evaluation of xenobiotics including drugs, but the profiles of chemical-metabolizing enzymes differ between Caco-2 and the human intestine. In particular, Caco-2 cells are characterized by the presence of carboxylesterase 1 (CES1), lower expression of cytochrome P450 3A4 (CYP3A4) and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). To overcome these differences, a series of genetically modified Caco-2 cell lines have been generated, but their applicability as a human intestinal model to evaluate effects of xenobiotics on the intestinal barrier integrity remains unclear. In this study, we evaluated the effects of irinotecan (CPT-11, a substrate of CES and CYP3A4) and SN-38 (a CES-mediated active metabolite of CPT-11, a substrate of UGT1A1) on barrier integrity in these Caco-2 cell lines. CPT-11 was less toxic in CES1-knockout CYP3A4-expressing cells than in the parental Caco-2 cells and CYP3A4-expressing cells. Deletion of CES1 attenuated the barrier-loosening and permeability-enhancing activity by treatment with CPT-11. Thus, expression of CES1 led to overestimation of intestinal biotransformation of CPT-11 in the parental Caco-2 cells. When UGT1A1 was introduced in CES1-knockout CYP3A4-expressing cells, cytotoxicity of SN-38 was decreased, and intestinal barrier-loosening and permeability to macromolecules were also attenuated. These findings indicate that the CES1-knockout, CYP3A4- and UGT1A1-expressing Caco-2 cells may be a useful model for evaluating risks of drugs and xenobiotics on intestinal barrier function.