Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Laboratory of Functional Organoid for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
Toxicol Lett. 2024 Nov;401:158-169. doi: 10.1016/j.toxlet.2024.10.003. Epub 2024 Oct 9.
Caco-2 cells, a human colorectal adenocarcinoma cell line, are widely used to model small intestinal epithelial cells in the drug development process because they can predict drug absorption with high accuracy. However, Caco-2 cells have several issues. First, Caco-2 cells have little expression of cytochrome P450 3A4 (CYP3A4), which is a major drug-metabolizing enzyme in the human intestine. We previously developed Caco-2 cells whose expression of CYP3A4 can be controlled using doxycycline (Dox) (CYP3A4-Caco-2 cells) (Ichikawa et al., Sci. Rep, 2021). However, since the Tet-On system was used to regulate CYP3A4 expression in these cells, there was concern about drug-drug interactions. The second issue is that in the human small intestine, carboxylesterase 2 (CES2) is more highly expressed than carboxylesterase 1 (CES1), while in Caco-2 cells CES1 is more highly expressed. The third issue is the low level expression of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a phase II drug-metabolizing enzyme. In this study, we used genome-editing technology to establish CES1-knockout Caco-2 cells whose CYP3A4 and UGT1A1 expression can be regulated by the Tet-Off system. These cell lines would be useful in pharmaceutical researches, including intestinal toxicological studies, as an in vitro model for orally administered drugs.
Caco-2 细胞是人结直肠腺癌细胞系，因其能够高度准确地预测药物吸收，所以被广泛用于药物开发过程中小肠上皮细胞模型。然而，Caco-2 细胞存在几个问题。首先，Caco-2 细胞细胞色素 P450 3A4（CYP3A4）的表达水平较低，而 CYP3A4 是人类肠道中主要的药物代谢酶。我们之前开发了一种可以通过强力霉素（Dox）（CYP3A4-Caco-2 细胞）控制 CYP3A4 表达的 Caco-2 细胞（Ichikawa 等人，Sci. Rep，2021）。然而，由于这些细胞中 CYP3A4 的表达是通过 Tet-On 系统来调控的，因此人们担心会发生药物相互作用。第二个问题是，在人类小肠中，羧酸酯酶 2（CES2）的表达水平高于羧酸酯酶 1（CES1），而在 Caco-2 细胞中 CES1 的表达水平更高。第三个问题是尿苷二磷酸葡萄糖醛酸转移酶 1A1（UGT1A1）的表达水平较低，UGT1A1 是一种 II 相药物代谢酶。在这项研究中，我们使用基因组编辑技术建立了 CES1 敲除的 Caco-2 细胞，这些细胞的 CYP3A4 和 UGT1A1 表达可以通过 Tet-Off 系统进行调控。这些细胞系将在制药研究中（包括肠道毒理学研究）作为口服药物的体外模型非常有用。
