Comenius University in Bratislava, Faculty of Natural Sciences, Department of Analytical Chemistry, Ilkovičova 6, SK-842 15, Bratislava, Slovak Republic.
Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, 845 05, Slovak Republic.
J Pharm Biomed Anal. 2021 Sep 5;203:114168. doi: 10.1016/j.jpba.2021.114168. Epub 2021 May 24.
Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.
基因导向酶/前药治疗代表了一种实验性治疗方法。基于胞嘧啶脱氨酶或融合胞嘧啶脱氨酶：尿嘧啶磷酸核糖转移酶将无毒前药 5-氟胞嘧啶转化为化疗药物 5-氟尿嘧啶的系统是最常用的系统之一。对 5-氟胞嘧啶、5-氟尿嘧啶及其代谢物的详细分析，能够了解肿瘤细胞对治疗的各种反应以及耐药机制。建立了一种基于液相色谱-高分辨质谱（LC-HRMS）的快速、灵敏、准确的方法，用于鉴定和定量 5-氟胞嘧啶、5-氟尿嘧啶及其主要代谢物。使用了两种不同的混合高分辨质谱仪，足以研究代谢途径。LC-ESI IT-TOF MS 方法成功地用于鉴定复杂生物基质（间充质基质细胞和肿瘤细胞培养基）中的 5-氟胞嘧啶、5-氟尿嘧啶及其代谢物，并用于确认 5-氟胞嘧啶在甚至在耐药肿瘤细胞培养基样品中的代谢转化。对于定量，开发并验证了 LC-HESI QExactive MS 方法。所开发的方法对 5-氟胞嘧啶的线性范围非常好，从 1ng/mL 到 1000ng/mL，其主要代谢物的线性范围从 5ng/mL 到 1000ng/mL。检测限和定量限范围分别为 1.1 至 26ng/mL 和 3.6 至 87ng/mL。两种开发的方法均证实了基因导向酶前药治疗能够将 5-氟胞嘧啶代谢转化为肿瘤细胞培养基和间充质基质细胞的真实样本中的 5-氟尿嘧啶及其主要代谢物。
