Michiba Kazuyoshi, Namai Mana, Hashimoto Yoshiki, Shimomura Osamu, Miyazaki Yoshihiro, Hashimoto Shinji, Ohara Yusuke, Enomoto Tsuyoshi, Oda Tatsuya, Maeda Kazuya, Kusuhara Hiroyuki
Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
Drug Metab Dispos. 2025 May;53(5):100075. doi: 10.1016/j.dmd.2025.100075. Epub 2025 Apr 7.
This study aimed to characterize the functional properties of human spheroid-derived differentiated cells for absorption, distribution, metabolism, and excretion studies. Three-dimensional human intestinal spheroids were successfully established from crypts isolated from fresh surgical specimens of the terminal ileum. The mRNA expression of major intestinal transporters and drug-metabolizing enzymes was tested. Region-specific functional expression of proton-coupled folate transporter/solute carrier (SLC) SLC46A1 and apical sodium-dependent bile acid transporter (ASBT)/SLC10A2 was confirmed in freshly isolated human proximal jejunal and terminal ileal tissue sections mounted onto Ussing chambers. Proximal jejunal and terminal ileal spheroid-derived differentiated cell monolayers showed H- and Na-coupled uptake of methotrexate and [H]-taurocholic acid, respectively. The functional expression of CYP3A, CYP2C9, UGT1A, P-glycoprotein, and breast cancer resistance protein (BCRP) was confirmed based on the formation of metabolites (1'-hydroxy midazolam, 4'-hydroxy diclofenac, raloxifene-4'-glucuronide, and raloxifene-6-glucuronide) and the efflux ratio of typical substrates (digoxin, sulfasalazine, rosuvastatin, dantrolene, and furosemide). Terminal ileum-derived differentiated cell monolayers showed extensive taurocholic acid-d5 transport in the apical-to-basal direction, which was markedly inhibited by the ASBT inhibitor elobixibat. One of the terminal ileal spheroids was identified as homozygous for the mutant allele of ABCG2 (rs2231142). The transport activity of BCRP in the differentiated cells, when dantrolene was used as a probe, might reflect this genetic variation. In conclusion, the functional expression of region-specific uptake transporters (proton-coupled folate transporter and ASBT) was successfully reproduced in human intestinal spheroid-derived differentiated cells, which also maintain the activities of P-glycoprotein, BCRP, and metabolic enzymes. Human intestinal spheroid-derived differentiated cells would be useful for investigating region-specific functions in drug transport and metabolism. SIGNIFICANCE STATEMENT: The expression levels of some transporters are not homogeneous along the longitudinal axis of the intestine. To date, there is no in vitro model that can reproduce regional differences in the transporter-mediated transport of nutrients and drugs in the intestine. This study successfully demonstrated that the functional expression of region-specific transporters, the proton-coupled folate transporter and the apical sodium-dependent bile acid transporter, was maintained in human proximal jejunal and terminal ileal spheroid-derived differentiated cell monolayers.
本研究旨在表征人球状体来源的分化细胞在吸收、分布、代谢和排泄研究中的功能特性。从回肠末端新鲜手术标本中分离的隐窝成功建立了三维人肠球状体。检测了主要肠道转运蛋白和药物代谢酶的mRNA表达。在安装于尤斯灌流小室的新鲜分离的人近端空肠和回肠末端组织切片中，证实了质子偶联叶酸转运体/溶质载体（SLC）SLC46A1和顶端钠依赖性胆汁酸转运体（ASBT）/SLC10A2的区域特异性功能表达。近端空肠和回肠末端球状体来源的分化细胞单层分别显示甲氨蝶呤和[H]-牛磺胆酸的H-和Na-偶联摄取。基于代谢物（1'-羟基咪达唑仑、4'-羟基双氯芬酸、雷洛昔芬-4'-葡萄糖醛酸苷和雷洛昔芬-6'-葡萄糖醛酸苷）的形成以及典型底物（地高辛、柳氮磺胺吡啶、瑞舒伐他汀、丹曲林和呋塞米）的外排率，证实了CYP3A、CYP2C9、UGT1A、P-糖蛋白和乳腺癌耐药蛋白（BCRP）的功能表达。回肠末端来源的分化细胞单层显示牛磺胆酸-d5在顶端到基底方向上的广泛转运，这被ASBT抑制剂依洛比昔巴特显著抑制。其中一个回肠末端球状体被鉴定为ABCG2（rs2231142）突变等位基因的纯合子。当使用丹曲林作为探针时，分化细胞中BCRP的转运活性可能反映了这种基因变异。总之，区域特异性摄取转运体（质子偶联叶酸转运体和ASBT）的功能表达在人肠球状体来源的分化细胞中成功再现，这些细胞还维持了P-糖蛋白、BCRP和代谢酶的活性。人肠球状体来源的分化细胞将有助于研究药物转运和代谢中的区域特异性功能。意义声明：一些转运蛋白的表达水平在肠道纵轴上并不均匀。迄今为止，尚无体外模型能够再现肠道中转运体介导的营养物质和药物转运的区域差异。本研究成功证明，区域特异性转运体，即质子偶联叶酸转运体和顶端钠依赖性胆汁酸转运体的功能表达，在人近端空肠和回肠末端球状体来源的分化细胞单层中得以维持。
