Abstract：
Thiopurine drugs are immunomodulatory antimetabolites relevant for pediatric patients characterized by dose-dependent adverse effects such as myelosuppression and hepatotoxicity, often related to inter-individual differences, involving the activity of important enzymes at the basis of their biotransformation, such as thiopurine S-methyltransferase (TPMT). Surface Enhanced Raman Scattering (SERS) spectroscopy is emerging as a bioanalytical tool and represents a valid alternative in terms of affordable costs, shorter analysis time and easier sample preparation in comparison to the most employed methods for pharmacokinetic analysis of drugs. The aim of this study is to investigate mercaptopurine and thioguanine pharmacokinetics by SERS in cell lysates of a B-lymphoblastoid cell line (NALM-6), that did (TPMT*1) or did not (MOCK) overexpress the wild-type form of TPMT as an in vitro cellular lymphocyte model to discriminate between cells with different levels of TPMT activity on the base of the amount of thioguanosine nucleotides (TGN) metabolites formed. SERS analysis of the cell lysates was carried out using SERS substrates constituted by Ag nanoparticles deposited on paper and parallel samples were used for quantification of thiopurine nucleotides with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A direct SERS detection method has been set up that could be a tool to study thiopurine drug pharmacokinetics in in vitro cellular models to qualitatively discriminate between cells that do and do not overexpress the TPMT enzyme, as an alternative to other more laborious techniques. Results underlined decreased levels of TGN and increased levels of methylated metabolites when TPMT was overexpressed, both after mercaptopurine and thioguanine treatments. A strong positive correlation (Spearman\'s rank correlation coefficient rho = 0.96) exists between absolute quantification of TGMP (pmol/1 x 106 cells), obtained by LC-MS/MS, and SERS signal (intensity of TGN at 915 cm-1). In future studies, we aim to apply this method to investigate TPMT activity in pediatric patients\' leukocytes.
摘要：
硫嘌呤药物是与儿科患者相关的免疫调节抗代谢药物，其特征是剂量依赖性不良反应，如骨髓抑制和肝毒性。通常与个体间的差异有关，在生物转化的基础上涉及重要酶的活性，如硫嘌呤S-甲基转移酶(TPMT)。表面增强拉曼散射（SERS）光谱正在成为一种生物分析工具，并且在负担得起的成本方面是一种有效的替代方法。与药物药代动力学分析最常用的方法相比，分析时间更短，样品制备更容易。这项研究的目的是通过SERS研究B淋巴母细胞样细胞系（NALM-6）的细胞裂解物中巯基嘌呤和硫鸟嘌呤的药代动力学，确实（TPMT*1）或没有（MOCK）过表达野生型TPMT作为体外细胞淋巴细胞模型，以根据形成的硫代鸟苷核苷酸（TGN）代谢物的量来区分具有不同TPMT活性水平的细胞。使用由沉积在纸上的Ag纳米颗粒构成的SERS基底进行细胞裂解物的SERS分析,并使用平行样品用液相色谱-串联质谱法(LC-MS/MS)定量硫代嘌呤核苷酸。已经建立了一种直接的SERS检测方法，该方法可以成为在体外细胞模型中研究硫嘌呤药物药代动力学的工具，以定性区分过表达和不过度表达TPMT酶的细胞。作为其他更费力的技术的替代。结果强调TPMT过表达时，TGN水平降低，甲基化代谢物水平升高，都是在巯基嘌呤和硫鸟嘌呤治疗后。TGMP的绝对定量(pmol/1×106个细胞)之间存在强正相关(Spearman的等级相关系数rho=0.96),通过LC-MS/MS获得，和SERS信号(TGN在915cm-1处的强度)。在未来的研究中,我们的目的是应用这种方法来研究儿童患者白细胞的TPMT活性。
