Bachour-El Azzi Pamela, Chesné Christophe, Uehara Shotaro
Biopredic International SARL, Saint Grégoire, France.
Biopredic International SARL, Saint Grégoire, France.
Adv Pharmacol. 2022;95:285-305. doi: 10.1016/bs.apha.2022.05.009. Epub 2022 Jun 30.
Primary human hepatocytes are an essential in vitro tool for evaluating drug metabolism, drug-drug interactions, and hepatotoxicity. This model is considered as the gold standard in matter of DMPK studies in both industrial and academic research. The primary human hepatocytes are used either in suspension or in monolayer, as fresh or frozen cells. However, the use of this model is limited due to the lack of availability, rapid loss of functionality, high cost as well as the variable hepatocyte plating efficiencies in culture and the limited stock of hepatocytes derived from the same origin. Chimeric TK-NOG mice with humanized livers (humanized liver mice) are an attractive platform for drug metabolism and toxicity, which were produced by transplanting human hepatocytes into immunodeficient mice with injured livers. Here, we show that, using humanized mouse liver, in vivo human hepatocyte repopulation was over ~100-fold enabling the continuous and abundant use of human hepatocytes of the same origin and improving their plateability. In our latest cell preparations, hepatocytes isolated from humanized liver mice (Hu-Liver cells) exhibited high purity (ratio of HLA-positive cells: 92±3%), good viability (75±12%), and yield (1.0×10 cells/mouse). Human hepatic drug metabolizing enzymes, transporters, and nuclear receptors genes were expressed in humanized mouse liver. Drug-metabolizing activities in Hu-Liver cells were comparable to or higher than those in primary human hepatocytes. An extensive P450-dependent human drug metabolism was observed in Hu-Liver cells. CYP1A2, CYP2B6, and CYP3A4/5 activities/mRNA in Hu-Liver cells were induced by the hepatocyte exposure to typical human P450 inducers, omeprazole, phenobarbital, and rifampicin, respectively. Finally, Human albumin secretion and CYP3A-mediated drug oxidation activity were maintained over 4-weeks. Altogether, the expression level of pharmacokinetics-related genes, enzyme activity, human-typed drug metabolism, and inducibility of P450 in Hu-Liver cells make from humanized mouse liver a relevant and robust model for in vitro preclinical studies, including drug metabolism, pharmacokinetics, and toxicology studies.
原代人肝细胞是评估药物代谢、药物相互作用和肝毒性的重要体外工具。在工业和学术研究的药物代谢和药代动力学（DMPK）研究中，该模型被视为金标准。原代人肝细胞可作为悬浮细胞或单层细胞使用，可使用新鲜细胞或冻存细胞。然而，由于肝细胞来源有限、功能快速丧失、成本高昂以及培养中肝细胞接种效率的差异和来自同一来源的肝细胞库存有限，该模型的应用受到限制。具有人源化肝脏的嵌合TK-NOG小鼠（人源化肝脏小鼠）是用于药物代谢和毒性研究的有吸引力的平台，它是通过将人肝细胞移植到肝脏受损的免疫缺陷小鼠中产生的。在此，我们表明，使用人源化小鼠肝脏，体内人肝细胞的再增殖超过100倍，使得能够持续大量使用同一来源的人肝细胞，并提高了它们的可接种性。在我们最新的细胞制备中，从人源化肝脏小鼠分离的肝细胞（Hu-Liver细胞）表现出高纯度（HLA阳性细胞比例：92±3%）、良好的活力（75±12%）和产量（1.0×10个细胞/小鼠）。人肝脏药物代谢酶、转运蛋白和核受体基因在人源化小鼠肝脏中表达。Hu-Liver细胞中的药物代谢活性与原代人肝细胞相当或更高。在Hu-Liver细胞中观察到广泛的依赖细胞色素P450（P450）的人药物代谢。Hu-Liver细胞中的细胞色素P450 1A2（CYP1A2）、细胞色素P450 2B6（CYP2B6）和细胞色素P450 3A4/5（CYP3A4/5）活性/信使核糖核酸（mRNA）分别通过肝细胞暴露于典型的人P450诱导剂奥美拉唑、苯巴比妥和利福平而被诱导。最后，人白蛋白分泌和CYP3A介导的药物氧化活性在4周内得以维持。总之，Hu-Liver细胞中药代动力学相关基因的表达水平、酶活性、人源型药物代谢以及P450的诱导性使得人源化小鼠肝脏成为用于体外临床前研究（包括药物代谢、药代动力学和毒理学研究）的相关且可靠的模型。
