Yamada Naoki, Negoro Ryosuke, Watanabe Keita, Fujita Takuya
Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, 525-8577, Japan.
Laboratory of Molecular Pharmacokinetics, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, 525-8577, Japan.
Drug Metab Pharmacokinet. 2023 Jun;50:100497. doi: 10.1016/j.dmpk.2023.100497. Epub 2023 Feb 18.
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.
Caco-2细胞被广泛用作体外肠道模型。然而，这些细胞中药物代谢酶CYP3A4和UGT1A1的表达水平低于肠道细胞。此外，当今使用的大多数前药都是含酯类的，而羧酸酯酶（CES）1和CES2是将前药转化为药物的酶之一。在人类小肠中，CES1几乎不表达，而CES2高度表达，但Caco-2细胞中的CES表达模式则相反。在本研究中，我们使用PITCh系统生成了CYP3A4-POR-UGT1A1-CES2敲入（KI）和CES1敲除（KO）的Caco-2（基因组编辑的Caco-2）细胞。基因组编辑的Caco-2细胞显示出功能性CYP3A4、POR、UGT1A1和CES2的表达，而CES1蛋白的表达被完全敲除。我们使用替莫卡普利进行了转运试验。替莫卡普利在基因组编辑的Caco-2细胞中的表观渗透系数（Papp）值高于野生型（WT）Caco-2细胞。有趣的是，基因组编辑的Caco-2细胞顶端侧的替莫卡普利拉量低于WT Caco-2细胞。这些结果表明，基因组编辑的Caco-2细胞作为预测肠道药物吸收和代谢的模型比WT Caco-2细胞更合适。
