Lochman Lukáš, Kahiya Ellen Tanaka, Saade Bechara, Smutný Tomáš, Tebbens Jurjen Duintjer, Pávek Petr, Bernhauerová Veronika
Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, Czech Republic.
Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, Czech Republic.
PLoS Comput Biol. 2025 Apr 15;21(4):e1012886. doi: 10.1371/journal.pcbi.1012886. eCollection 2025 Apr.
Xenoreceptors of the nuclear receptor superfamily, such as pregnane X receptor (PXR), are liver-enriched ligand-activated transcription factors regarded as crucial sensors in xenobiotic exposure and detoxification. PXR controls transcription of many drug-handling genes and influx/efflux transporters, thus playing a crucial role in drug metabolism and excretion. Liver functions have been studied using primary human hepatocytes (PHHs), which, when conventionally cultured, undergo rapid de-differentiation, leaving them unsuitable for long-term studies. Recently, 3D PHHs called spheroids have emerged as an in vitro model that is similar to in vivo hepatocytes regarding phenotype and function and represents the first in vitro model to study the long-term regulation of drug-handling genes by PXR. In this study, we used mathematical modelling to analyze the long-term activation of PXR in 3D PHHs through expression kinetics of three key PXR-regulated drug-metabolizing enzymes, CYP3A4, CYP2C9, and CYP2B6 and the P-glycoprotein efflux transporter encoding gene, MDR1. PXR action in 3D PHHs was induced by the antibiotic rifampicin at two clinically relevant concentrations. The results confirmed that high rifampicin concentrations activated PXR nearly to its full capacity. The analysis indicated the highest PXR-induced transcription rate constant for CYP2B6. The rate constant dictating mRNA degradation associated with activated PXR was highest for CYP3A4. Moreover, we measured the metabolic activity of CYP3A4, CYP2C9, and CYP2B6 and quantified their metabolic rate constants. Metabolic activity rate constant of CYP3A4 was found to be the highest whereas that of CYP2B6 was found to be the lowest among the studied enzymes. Our results provide important insight into the regulation of PXR-target genes in 3D PHHs and show that mRNA expression and metabolic activity data can be combined with quantitative analysis to reveal the long-term action of PXR and its effects on drug-handling genes.
核受体超家族的异源受体，如孕烷X受体（PXR），是肝脏富集的配体激活转录因子，被视为外源性物质暴露和解毒的关键传感器。PXR控制许多药物处理基因和流入/流出转运蛋白的转录，因此在药物代谢和排泄中起着关键作用。人们利用原代人肝细胞（PHH）对肝功能进行了研究，传统培养时，这些细胞会迅速去分化，使其不适用于长期研究。最近，一种名为球体的3D PHH作为一种体外模型出现，在表型和功能上与体内肝细胞相似，是首个用于研究PXR对药物处理基因长期调控的体外模型。在本研究中，我们通过三种关键的PXR调节药物代谢酶CYP3A4、CYP2C9和CYP2B6以及P-糖蛋白流出转运蛋白编码基因MDR1的表达动力学，用数学模型分析了3D PHH中PXR的长期激活情况。在两个临床相关浓度下，抗生素利福平诱导了3D PHH中的PXR作用。结果证实，高浓度利福平几乎能将PXR激活至其最大能力。分析表明，CYP2B6的PXR诱导转录速率常数最高。与激活的PXR相关的决定mRNA降解的速率常数在CYP3A4中最高。此外，我们测量了CYP3A4、CYP2C9和CYP2B6的代谢活性，并对它们的代谢速率常数进行了量化。在所研究的酶中，发现CYP3A4的代谢活性速率常数最高，而CYP2B6的代谢活性速率常数最低。我们的结果为3D PHH中PXR靶基因的调控提供了重要见解，并表明mRNA表达和代谢活性数据可与定量分析相结合，以揭示PXR的长期作用及其对药物处理基因的影响。
