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.