Faculty of Pharmacy, Drug Research Program, Division of Pharmaceutical Chemistry and Technology University of Helsinki, Viikinkaari 5 E 00014, Finland.
Anal Chem. 2020 Nov 3;92(21):14693-14701. doi: 10.1021/acs.analchem.0c03258. Epub 2020 Oct 25.
The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the interindividual variation in one of the four model reaction activities, that is, ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 μg microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.
细胞色素 P450（CYP）超家族酶负责人体内大多数治疗药物的内在清除。然而，由于遗传和外部因素，CYP 介导的药物清除动力学在个体之间存在显著差异，并且许多肝脏疾病也会极大地影响酶的数量和功能。在这项研究中，我们开发了一种新的方法，基于数字微流控（DMF），用于快速测定来自活检规模的人源肝样本人 CYP 活性的个体变化。该测定法采用人肝微粒体（HLMs），固定在磁性珠上，以促进在生理温度下平行化系统中测定微粒体 CYP 酶的活性。通过定制的、喷墨打印的微加热器阵列与 DMF 平台的模块化集成来实现热控制。借助预荧光、酶选择性模型化合物，通过基于原位光学读出定量各自的荧光代谢物来确定 CYP 活性。该测定法的选择性和灵敏度已针对四种不同的 CYP 模型反应得到确立，并通过确定四种模型反应之一（即 CYP1A1/2 的乙氧基Resorufin O-去乙基化）的五个供体之间的个体间变异性来验证诊断概念。总体而言，该开发的方案每个测定仅消耗约 15μg 微粒体蛋白。因此，它在技术上适应于自动化筛选活检规模肝样本人 CYP 酶功能的个体差异，以支持医疗疗法的个体化，例如在肝脏疾病诊断的背景下。
