SERS spectroscopy as a tool for the study of thiopurine drug pharmacokinetics in a model of human B leukemia cells.

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.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AIMS: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation. RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. CONCLUSION: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

Responses of patients with juvenile idiopathic arthritis to methotrexate: a genomic outlook.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is a chronic disease characterized by persistent joint inflammation. JIA is the most common pediatric chronic rheumatic disease and no curative therapy is currently available. Methotrexate (MTX) is an important treatment for JIA even though a high inter-individual variability in response is observed in patients. Among the factors of this variability, genetics and epigenetics might play an important role. AREAS COVERED: This review summarizes the results of pharmacogenetic and pharmacoepigenetic studies regarding MTX response in JIA. Studies considering epigenetic factors in JIA patients are still very limited, therefore this review includes also studies performed in adult patients with rheumatoid arthritis. Moreover, the relevance of biomarkers measured in blood or urine of JIA patients in relation to MTX treatment is discussed. EXPERT OPINION: Nowadays, even though many pharmacogenomics studies have been published, a specific genetic marker predictor of MTX efficacy or adverse events has not yet been identified. Encouraging results are available and great expectations rely on the study of epigenetics. Future studies are needed in order to identify genetic and epigenetic biomarkers that can be implemented in the clinical practice.

Ergothioneine, a dietary amino acid with a high relevance for the interpretation of label-free surface enhanced Raman scattering (SERS) spectra of many biological samples.

Intense SERS spectra of the natural amino acid ergothioneine (ERG) are obtained on different substrates upon 785 nm excitation. A characteristic spectral pattern with a distinctive intense band at 480-486 cm-1 is conserved when substrates of different type and characteristics are used. On the basis of available literature, we propose ERG is adsorbed on the metal surface in its thiolate form via the sulphur and heterocyclic nitrogen. The same spectral pattern is obtained in SERS spectra of filtered erythrocytes lysates, confirming the presence of ERG in those cells. The occurrence of ERG bands in label-free SERS spectra of serum and plasma reported in literature by different authors is discussed, highlighting the importance of this amino acid for the interpretation of SERS spectra of these biofluids.

Pharmacogenetics of thiopurines.

Polychemotherapeutic protocols for the treatment of pediatric acute lymphoblastic leukemia (ALL) always include thiopurines. Specific approaches vary in terms of drugs, dosages and combinations. Such therapeutic schemes, including risk-adapted intensity, have been extremely successful for children with ALL who have reached an outstanding 5-year survival of greater than 90% in developed countries. Innovative drugs such as the proteasome inhibitor bortezomib and the bi-specific T cell engager blinatumomab are available to further improve therapeutic outcomes. Nevertheless, daily oral thiopurines remain the backbone maintenance or continuation therapy. Pharmacogenetics allows the personalization of thiopurine therapy in pediatric ALL and clinical guidelines to tailor therapy on the basis of genetic variants in TPMT and NUDT15 genes are already available. Other genes of interest, such as ITPA and PACSIN2, have been implicated in interindividual variability in thiopurines efficacy and adverse effects and need additional research to be implemented in clinical protocols. In this review we will discuss current literature and clinical guidelines available to implement pharmacogenetics for tailoring therapy with thiopurines in pediatric ALL.