PDF(Pubmed)
Abstract：
3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35-52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that can be performed using 3D PHH.
摘要：
原代人肝细胞（3DPHH）的3D球体在培养中保留了数周的分化表型，具有很大程度上保守的代谢功能和蛋白质组学指纹。因此,3DPHH作为机械性肝脏稳态研究的模型以及在药物发现中的体外至体内外推（IVIVIVE）越来越重要。然而,尚未在3DPHH中评估药物转运蛋白的动力学和调节。这里,我们使用有机阳离子转运蛋白1（OCT1/SLC22A1）作为模型来研究转运动力学和通过相关信号通路对转运蛋白活性的长期调节。使用荧光模型底物4-（4-（二甲基氨基）苯乙烯基）-N-甲基吡啶（ASP）和已知的OCT1抑制剂在单个3DPHH中研究了OCT1转运蛋白的动力学。对于长期研究，3DPHH用异生物质处理7天，之后评估蛋白质表达和OCT1功能。全局蛋白质组学分析用于追踪肝脏表型以及其他调节蛋白的原型变化。如P-糖蛋白和细胞色素P450.3A4。ASP+动力学表明来自三个供体的Km值为14±4.0µM的全功能OCT1转运蛋白。与已知OCT1抑制剂的共孵育使3DPHH球状体中ASP+的摄取降低了35-52%。长期暴露研究表明，OCT1在激活核受体信号或暴露于可诱导炎症的化合物后相对稳定，脂肪变性或肝损伤。我们的结果表明，3DPHH球体表达生理相关水平的完全活性OCT1，并且可以在3DPHH构型中准确研究其转运蛋白动力学。我们还证实OCT1在改变其他药物转运蛋白和药物代谢酶的表达和功能的关键代谢途径的激活过程中保持稳定和功能。这些结果将扩大可以使用3DPHH进行的研究范围。
