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In nonclinical studies, models that can predict the metabolism of drug candidates by cytochrome P450 (CYP), including Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) are helpful. CYP3A4-overexpressing human cells have been used universally to evaluate whether CYP3A4 metabolizes drug-candidate compounds. However, CYP3A4-overexpressing human cell lines are problematic because their activity levels are lower than that of in vivo human CYP3A4. Heme plays a paramount role in CYP activity. The rate-limiting step in heme biosynthesis is the generation of 5-aminolevulinic acid (5-ALA). In this study, we examined whether treatment with 5-ALA to CYP3A4-POR-UGT1A1-CES2 knockin and CES1 knockout (genome-edited) Caco-2 cells enhances CYP3A4 activity. A 7-day 5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells without cytotoxicity. Moreover, consistent with the increase in intracellular heme content, 5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells. The results of this research are expected to be applied to pharmacokinetic studies using CYP-overexpressing human cells containing CYP3A4., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Caco-2 cells are widely used as an in vitro intestinal model. However, the expression levels of the drug-metabolizing enzymes CYP3A4 and UGT1A1 are lower in these cells than in intestinal cells. Furthermore, the majority of prodrugs in use today are ester-containing, and carboxylesterase (CES) 1 and CES2 are among the enzymes that process the prodrugs into drugs. In the human small intestine, CES1 is hardly expressed while CES2 is highly expressed, but the CES expression pattern in Caco-2 cells is the opposite. In this study, we generated CYP3A4-POR-UGT1A1-CES2 knock-in (KI) and CES1 knock-out (KO) Caco-2 (genome-edited Caco-2) cells using a PITCh system. Genome-edited Caco-2 cells were shown to express functional CYP3A4, POR, UGT1A1 and CES2 while the expression of the CES1 protein was completely knocked out. We performed transport assays using temocapril. The Papp value of temocapril in genome-edited Caco-2 cells was higher than that in WT Caco-2 cells. Interestingly, the amount of temocaprilat on the apical side in genome-edited Caco-2 cells was lower than that in WT Caco-2 cells. These results suggest that genome-edited Caco-2 cells are more suitable than WT Caco-2 cells as a model for predicting intestinal drug absorption and metabolism., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Multiple drug resistance 1 (MDR1) is highly expressed in various organs, including the liver, small intestine, and blood-brain barrier (BBB). Because MDR1 plays important roles in the excretion of many drugs, it is necessary to evaluate whether drug candidates are potential substrates of MDR1. Recently, many researchers have shown that human induced pluripotent stem (iPS) cell-derived differentiated cells such as hepatocytes and enterocytes can be applied for pharmacokinetic testing. Here, we attempted to generate MDR1-knockout (KO) iPS cell lines using genome editing technology. The correctly targeted human iPS cell lines were successfully obtained. The expression levels of pluripotent markers in human iPS cells were not changed by MDR1 knockout. The gene expression levels of hepatic markers in MDR1-KO iPS-derived hepatocyte-like cells were higher than those in undifferentiated MDR1-KO iPS cells, suggesting that MDR1-KO iPS cells have hepatic differentiation capacity. In addition, MDR1 expression levels were hardly detected in MDR1-KO iPS cell-derived hepatocyte-like cells. We thus succeeded in establishing MDR1-KO iPS cell lines that could be utilized for pharmacokinetic testing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)