Quantitative detection of 6-thioguanine in body fluids based on a free-standing liquid membrane SERS substrate.

The adverse reactions caused by 6-thioguanine (6-TG) in anti-cancer treatment are closely related to the dose, leading to the urgent need for clinical monitoring of its concentration. In this work, a highly reproducible free-standing liquid membrane (FLM) surface-enhanced Raman spectroscopy (SERS) substrate was developed to detect 6-TG in human urine and serum quantitatively. Briefly, a prepared sample was adjusted to pH 2 and mixed with concentrated core-shell bimetallic nanoparticle (AgcoreAushell NP) suspension. The Au/Ag ratio of the AgcoreAushell NPs was optimized. Then the mixture was formed into an FLM using a custom mold. The relative standard deviation (RSD) of the experimental results can be stabilized below 10% (n ≥ 10). The R2 of the calibration curve in the range of 10 ~ 100 µg kg-1 was 0.988. In addition, the limit of detection (LOD) (3σ/k) of 6-TG was 5 µg kg-1. The FLM SERS platform has been successfully applied to the rapid and reliable analysis of 6-TG spiked in body fluids.

Effects of NT5C2 Germline Variants on 6-Mecaptopurine Metabolism in Children With Acute Lymphoblastic Leukemia.

6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide (TGN) metabolites. A recent genomewide association study has identified germline polymorphisms (e.g., rs72846714) in the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 588 children with ALL and identified 121 single nucleotide polymorphisms nominally associated with erythrocyte TGN during 6-MP treatment (P < 0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to interpatient variability in thiopurine drug disposition.

Análisis de la determinación de niveles de tiopurínicos en pacientes pediátricos con enfermedad inflamatoria intestinal / Laboratory determination of thiopurine levels in paediatric patients with inflammatory bowel disease

INTRODUCCIÓN Y OBJETIVOS: Las tiopurinas son fármacos muy empleados para el mantenimiento de la remisión en pacientes con enfermedad inflamatoria intestinal. Se conocen cuáles son los niveles plasmáticos óptimos, y existe controversia acerca de si reducen la necesidad de otros fármacos o son coste-efectivos. El objetivo de nuestro estudio fue describir el uso del tratamiento optimizado con tiopurínicos en pacientes pediátricos con enfermedad inflamatoria intestinal seguidos en nuestra unidad desde la implementación de la determinación de niveles de fármaco. MATERIAL Y MÉTODOS: Estudio descriptivo retrospectivo en el que se analizaron valores en plasma mediante cromatografía líquida de 6-tioguanina (6-TGN), 6-metilmercaptopurina (6-MMP) y sus cocientes, así como estado clínico y variables analíticas y demográficas de pacientes con enfermedad inflamatoria intestinal en seguimiento en nuestra unidad. RESULTADOS: Se incluyeron 72 pacientes y se realizaron 140 determinaciones de metabolitos. En el 61,5% de las determinaciones los niveles de 6-TGN se encontraban por debajo del rango terapéutico (en 7 casos debido a falta de adherencia terapéutica), y en el 7,4% de las de 6-MMP estaban en rango de toxicidad. Tras la determinación de 77 muestras se tomó alguna actitud derivada, procediéndose a la modificación de dosis, al cambio de formulación o a la suspensión del fármaco. Únicamente 9 pacientes escalaron a fármaco biológico (13,4% del total que estaban en monoterapia). No se encontró relación entre la actividad de la enfermedad y los niveles de tiopurínicos. CONCLUSIONES: En nuestra experiencia la monitorización de niveles de tiopurinas ayudó a modificar la dosis de fármaco que recibía el paciente, adecuando sus niveles terapéuticos y evitando potencialmente la adición de nuevos fármacos

INTRODUCTION AND OBJECTIVES: Thiopurines are drugs widely used in patients for the maintenance of remission in inflammatory bowel disease. The optimal plasma levels are known, but there is controversy about whether the need for other drugs is reduced or is cost-effective. The aim of this study is to describe the use of the optimised treatment with thiopurines in paediatric patients with inflammatory bowel disease followed up in this Unit since the introduction of determining the drug levels. MATERIAL AND METHODS: A descriptive retrospective study was conducted in which the plasma values of 6-thioguanine (6-TGN), 6-methyl-mercapto-purine (6-MMP), and their ratios were analysed using liquid chromatography. Other variables were collected, such as clinical status, analytical and demographic variables of patients with inflammatory bowel disease followed up in this Unit. RESULTS: A total of 72 patients were included, and 149 determinations of metabolites were performed. The 6-TGN levels were found to below the therapeutic range in 61.5% of patients (in 7 cases due to lack of adherence to therapy), and 6-MMP was in the toxicity range in 7.4%. After the determination of 77 specimens, some action was taken, such as modifying the dose, change of formula, or withdrawing the drug. Only 9 patients were scaled to a biological drug (13.4% of the total on single therapy). No association was found between the activity of the disease and the thiopurine levels. CONCLUSIONS: In our experience, the monitoring of thiopurine levels helped to modify the drug dose that the patient received, adjusting their therapeutic levels, and potentially avoiding the addition of new drugs

[Laboratory determination of thiopurine levels in paediatric patients with inflammatory bowel disease]. / Análisis de la determinación de niveles de tiopurínicos en pacientes pediátricos con enfermedad inflamatoria intestinal.

INTRODUCTION AND OBJECTIVES: Thiopurines are drugs widely used in patients for the maintenance of remission in inflammatory bowel disease. The optimal plasma levels are known, but there is controversy about whether the need for other drugs is reduced or is cost-effective. The aim of this study is to describe the use of the optimised treatment with thiopurines in paediatric patients with inflammatory bowel disease followed up in this Unit since the introduction of determining the drug levels. MATERIAL AND METHODS: A descriptive retrospective study was conducted in which the plasma values of 6-thioguanine (6-TGN), 6-methyl-mercapto-purine (6-MMP), and their ratios were analysed using liquid chromatography. Other variables were collected, such as clinical status, analytical and demographic variables of patients with inflammatory bowel disease followed up in this Unit. RESULTS: A total of 72 patients were included, and 149 determinations of metabolites were performed. The 6-TGN levels were found to below the therapeutic range in 61.5% of patients (in 7 cases due to lack of adherence to therapy), and 6-MMP was in the toxicity range in 7.4%. After the determination of 77 specimens, some action was taken, such as modifying the dose, change of formula, or withdrawing the drug. Only 9 patients were scaled to a biological drug (13.4% of the total on single therapy). No association was found between the activity of the disease and the thiopurine levels. CONCLUSIONS: In our experience, the monitoring of thiopurine levels helped to modify the drug dose that the patient received, adjusting their therapeutic levels, and potentially avoiding the addition of new drugs.

Determination of 6-thioguanine and 6-methylmercaptopurine in dried blood spots using liquid chromatography-tandem mass spectrometry: Method development, validation and clinical application.

BACKGROUND: Therapeutic drug monitoring of azathioprine metabolites is required for pharmacotherapy individualisation in patients with inflammatory bowel disease. Currently mainly hemolysates are used, requiring long sample preparation and showing limited analytes stability. Therefore, a quantitative LC-MS/MS method for determination of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in dried blood spot samples (DBS) was developed. METHODS: Analysis involves liquid extraction from 30 µL blood spot, hydrolysis and quantification with LC-MS/MS. RESULTS: Method met the validation criteria in terms of selectivity, linearity, accuracy, and precision in a range from 50 to 5300 pmol/8 × 108 Ery for 6-TG and from 260 to 5300 pmol/8 × 108 Ery for 6-MMP. Range can be increased to 8000 pmol/8 × 108 Ery. No matrix effect was observed and the recovery was >80%. DBS specific validation parameters were confirmed: spot homogeneity, no influence of blood spot volume (>30 µL) on 6 mm DBS disk, and absence of haematocrit effect. DBS samples were stable for at least one month at temperatures from -20 to 40 °C. Clinical validation confirmed that DBS method and routine clinical method with hemolysate samples give comparable results and enable similar clinical decisions. CONCLUSIONS: The newly developed DBS method is simple and presents an alternative to conventional methods for therapeutic drug monitoring of azathioprine metabolites.

Thiopurine Therapy in Patients With Inflammatory Bowel Disease: A Focus on Metabolism and Pharmacogenetics.

Thiopurines have been widely used for the maintenance of remission or steroid sparing in patients with inflammatory bowel disease. However, potential drug-related adverse events frequently interfere with their use. Indeed, drug withdrawals associated with adverse reactions have been reported in approximately 25% of patients. To balance the efficacy, safety, and tolerability of thiopurines, regular monitoring of biomarkers (complete blood cell count, liver function test, and metabolic profiles), steady dose escalation, and pretreatment thiopurine S-methyltransferase (TPMT) genotype screening have been routinely recommended. However, the complex thiopurine metabolic pathway and individual differences attributed to pharmacogenetic diversity limit the effectiveness of these strategies in the optimization of thiopurine therapy. Recently, in an effort to facilitate more accurate and personalized prediction of thiopurine response or toxicity, novel genetic markers including NUDT15 and FTO genes were discovered. These discoveries are remarkable because TPMT screening has minimal efficacy for predicting myelosuppression especially in Asian populations, despite the fact that thee populations have a higher frequency of myelosuppression than Western populations. This review focuses on the current understanding of the metabolic pathway and the pharmacogenetics of thiopurines and suggests a personalized preventive strategy against potential adverse drug reactions to optimize their therapeutic application.

A sensitive colorimetric probe for detection of 6-thioguanine based on its protective effect on the silver nanoprisms.

In this work a non-aggregated colorimetric probe for detection of chemotherapeutic drug, 6-thioguanine (6-TG), is introduced. It is based on the protective effect of 6-TG on silver nanoprisms (AgNPRs) against the iodide-induced etching reaction. Iodide ions can attack the corners of AgNPRs and etch them, leading to the morphological transition from nanoprisms to nanodiscs. As a consequence, the solution color changes from blue to pink. However, in the presence of 6-TG, due to its protective effect on the corners of AgNPRs, I- ions cannot etch the prisms and the blue color of solution remains unchanged. Using this effect, selective sensor was designed for detection of 6-TG in the range of 2.5-500 µg L-1, with a detection limit of 0.95 µg L-1. Since with varying the concentration of 6-TG in this range, the color variation from pink to blue can be easily observed, the designed sensing scheme can be used as a colorimetric probe. The method was used for analysis of human plasma samples.

Quantification of Thiopurine Nucleotides in Erythrocytes and Clinical Application to Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: Concentrations of 6-thioguanine (6TG) nucleotides and 6-methylmercaptopurine (6MMP) nucleotides in RBCs were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay was validated for clinical use and was applied to blood samples from patients taking mercaptopurine (6MP). METHODS: RBCs were hemolyzed and deproteinized using perchloric acid, followed by heating for the hydrolysis of nucleotides, and the resultant base was measured using LC-MS/MS. Precision, recovery, linearity, matrix effect, and limit of quantification was validated for clinical application. Our results were compared with another institution's established LC-MS/MS assay. We measured the concentrations of 6TG and 6MMP in RBCs of pediatric patients with acute lymphoblastic leukemia (ALL), and the clinical impact of those metabolites was investigated. RESULTS: The imprecision coefficient of variations of 6TG and 6MMP were 5.7%-8.1%, and the bias was within 5%. Lower limits of quantification were set at 54 ng/mL for 6TG and 1036 ng/mL for 6MMP. Correlation coefficients for 6TG and 6MMP were 0.997 and 1.0 in a comparison study. For clinical proof-of-concept, 74 blood samples were collected from 37 pediatric ALL patients receiving maintenance therapy. Concentration of 6TG ranged from 16.1 to 880 pmol/8 × 10 RBCs and that of 6MMP from 55 to 20,937 pmol/8 × 10 RBCs. The 6MP metabolites were not correlated with WBC or absolute neutrophil count. On the other hand, the higher 6MMP level was associated with elevated alanine aminotransferase and aspartate aminotransferase. CONCLUSIONS: In this study, an assay for the quantification of 6TG and 6MMP in RBCs was established and applied to pediatric ALL patients. Interindividual variability in 6MP metabolite concentrations was considerable and associated with elevation of liver enzymes, which may be useful in the clinical monitoring of 6MP maintenance therapy in pediatric ALL patients.

Clinical experience of optimising thiopurine use through metabolite measurement in inflammatory bowel disease. / Experiencia en práctica clínica de optimización de tiopurinas mediante determinación de sus metabolitos en la enfermedad inflamatoria intestinal.

INTRODUCTION: Thiopurine therapy can be optimised by determining the concentration of the drug's metabolites. PATIENTS AND METHODS: Retrospective analysis on a prospective database of 31 patients with inflammatory bowel disease who failed therapy with thiopurines. Thiopurine metabolites (6-thioguanine, 6-TGN and 6-methylmercaptopurine, 6-MMP) were measured by high-performance liquid chromatography (Laboratorios Cerba, Barcelona) and treatment was duly adjusted in accordance with the results. Clinical response was reassessed after six months. RESULT: Despite the appropriate theoretical dose of thiopurines being administered, the dose was insufficient in 45.6% of patients (nonadherence to treatment suspected in 6.45%) and 16.2% received an excessive dose or the drug was metabolised by other metabolic pathways. After treatment was optimised based on metabolite levels, only 25.8% (8/31) were prescribed a biological agent, while 74.2% of cases (23/31) were managed through dose optimisation alone. DISCUSSION: Monitoring thiopurine metabolite levels may help clinicians to assess non-responsive patients before adding or switching to another drug (generally a biological agent), thereby avoiding any additional costs or potential toxicity. This strategy may also help to identify patients receiving an insufficient dose and those with an alternative metabolic pathway, who could be candidates for low-dose AZA with allopurinol, as well as patients who are suspected of being non-adherent. In three out of four patients, switching to a biological agent can be avoided.

Optimized thiopurine therapy before withdrawal of anti-tumour necrosis factor-α in patients with Crohn's disease.

OBJECTIVE: Two meta-analyses have found that the risk of relapse in Crohn's disease (CD) was ~40 and 50% 1 and 2 years, respectively, after withdrawal of anti-tumour necrosis factor-α (anti-TNFα). The aim of this study was to evaluate relapse rates in CD when thiopurine therapy was optimized before anti-TNFα withdrawal. PATIENTS AND METHODS: An observational study was conducted including patients with CD in remission with optimized thiopurine therapy before anti-TNFα withdrawal. We defined optimized thiopurine therapy as 6-thioguanine levels of at least 150 nmol/mmol haemoglobin (∼300 pmol×10 red blood cells) and clinical/biochemical remission as Harvey-Bradshaw Index of 5 or less and faecal calprotectin of 200 µg/g or less. RESULTS: We included 33 patients (median age: 31 years, 55% males, and median disease duration: 7 years) followed for a median of 36 months. A total of three (9%) patients relapsed during the first year and six patients (in total 27%) relapsed after 2 years. After 2 years, none of the additional patients relapsed. The disease duration and duration of anti-TNFα treatment and faecal calprotectin levels before inclusion did not predict relapse. Calprotectin levels of at least 180 after 1 year predicted relapse at year 2. CONCLUSION: This study found that 73% of patients with CD maintained remission (>2 years) when thiopurine therapy was optimized before withdrawal of anti-TNFα. Additional prospective evidence is needed to confirm the findings.

Magnetic molecularly imprinted polymer nanoparticles for simultaneous extraction and determination of 6-mercaptopurine and its active metabolite thioguanine in human plasma.

Cytotoxic drugs used in cancer chemotherapy require the continuous monitoring of their plasma concentration levels for dose adjustment purposes. Such condition necessitates the presence of a sensitive technique for accurate extraction and determination of these drugs together with their active metabolites. In this study a novel solid phase extraction technique using magnetic molecularly imprinted nanoparticles (MMI-SPE) is combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) to extract and determine the anti-leukemic agent; 6-mercaptopurine (6-MP) and its active metabolite thioguanine (TG) in human plasma. The magnetic molecularly imprinted nanoparticles (Fe3O4@MIP NPs) were synthesized via precipitation polymerization technique and were characterized using different characterization methods A computational approach was adopted to help in the choice of the monomer used in the fabrication process. The Fe3O4@MIPs NPs possessed a highly improved imprinting efficiency, fast adsorption kinetics following 2nd order kinetics and good adsorption capacity of 1.0 mg/g. The presented MMI-SPE provided the optimum approach in comparison to other reported ones to achieve good extraction recovery and matrix effect of trace levels of 6-MP and TG from plasma. Chromatographic separation was carried out using a validated LC-MS/MS assay and recovery, matrix effect and process efficiency were evaluated. Recovery of 6-MP and TG was in the range of 85.94-103.03%, while, matrix effect showed a mean percentage recovery of 85.94-97.62% and process efficiency of 85.54-96.18%. The proposed extraction technique is simple, effective and can be applicable to the extraction and analysis of other pharmaceutical compounds in complex matrices for therapeutic drug monitoring applications.

Evaluation of Stability of Thiopurine Metabolites Using a Validated LC-MS/MS Method.

Measurement of thiopurine metabolites is helpful to monitor adverse effects and assess compliance in patients on thiopurine treatment. The purpose of this study was to develop and validate an analytical method for measurement of thiopurine metabolites, thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN), in RBCs. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 6-TGN and 6-MMPN and evaluated the stability of the thiopurine metabolites in RBC and whole blood states without any preprocessing at various storage conditions. The linear range was 0.1-10 µmol/L and 0.5-100 µmol/L for 6-TGN and 6-MMPN, respectively. The mean extraction recovery at the two concentrations was 71.0% and 75.0% for 6-TGN, and 102.2% and 96.4% for 6-MMPN. Thiopurine metabolites in preprocessed RBC samples were stable at 25°C and 4°C after storage for 4 hours and at -70°C for up to 6 months. However, 6-TGN decreased by 30% compared with the initial concentration when stored at -20°C for 180 days. In whole blood states, 6-TGN decreased by about 20% at four days after storage at 4°C. We validated a reliable LC-MS/MS method and recommend that the patient's whole blood sample be preprocessed as soon as possible.

Simple and selective determination of 6-thioguanine by using polyethylenimine (PEI) functionalized carbon dots.

In this work, a new selective and simple fluorescence probe based on polyethylenimine(PEI) functionalized carbon dots (PEI-CDs) for fast determination of 6-thioguanine (6-TG) was developed. We successfully prepared the highly fluorescent PEI-CDs by hydrothermal method, and completed the synthesis and modification processes in one step. The fluorescence quantum yield (QY) of the as-synthesized CDs was 38% and higher than that of most previous reports (5-30%). The fluorescence intensity of PEI-CDs decreased obviously with gradually increased concentration of 6-TG. The interference substances caused a negligible effect on the fluorescence intensity of the (PEI-CDs)-6-TG reaction system with the interference ratios all below 2.8%. Under optimum conditions, a great linear relationship between fluorescence intensity function log(F0/F) and concentration of 6-TG in a wide range from 4µM to 800µM with a detection limit (S/N = 3) of 1.33µM was obtained. And this proposed approach was successfully applied for the determination of 6-TG in human serum and urine samples. Furthermore, the PEI-CDs fluorescence probe has superior potential in practical application of detecting 6-TG due to its inexpensive precursors, simple operation, low toxicity and excellent biocompatibility.

A Systematic Review and Meta-Analysis of 6-Thioguanine Nucleotide Levels and Clinical Remission in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Thiopurines are widely used in the management of inflammatory bowel diseases. However, their minimum effective dose and dose-response relationship remain undefined, and evidence about their use in clinical practice is mostly heterogeneous. This systematic review and meta-analysis aimed: i] to assess the clinical value of 6-thioguanine nucleotide thresholds; and ii] to compare mean 6-thioguanine nucleotide concentrations between patients in clinical remission vs. those with active disease. METHODS: A systematic literature search was carried out using four databases. Statistical heterogeneity was assessed with the I2 statistic followed by subgroup and sensitivity analyses. Odds ratios were computed using the random-effects model. RESULTS: A total of 1384 records were identified in the systematic search, of which 25 were retained for further analysis: 22 were used in the cut-off comparisons and 12 were used in the 6-thioguanine nucleotide mean differences analysis. The global odds ratio for remission in patients with 6-thioguanine nucleotide levels above the predefined thresholds was 3.95 (95% confidence interval [CI], 2.63-5.94; p < 0.001]. When considering the different thresholds individually, the odd ratios were significant for values above 235 pmol/8 × 108 and 250 pmol/8 × 108 red blood cells [2.25 and 4.71, respectively]. Mean 6-thioguanine nucleotide levels were higher among patients in clinical remission, with a pooled difference of 63.37 pmol/8 × 108 red blood cells [95% CI, 31.81-94.93; p < 0.001]. CONCLUSIONS: This study reinforces the link between 6-thioguanine nucleotide levels and clinical remission in inflammatory bowel diseases, also exploring the validity of specific 6-thioguanine nucleotide thresholds to predict clinical outcomes.

Analytical Pitfalls of Therapeutic Drug Monitoring of Thiopurines in Patients With Inflammatory Bowel Disease.

The use of thiopurines in the treatment of inflammatory bowel disease (IBD) can be optimized by the application of therapeutic drug monitoring. In this procedure, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) metabolites are monitored and related to therapeutic response and adverse events, respectively. Therapeutic drug monitoring of thiopurines, however, is hampered by several analytical limitations resulting in an impaired translation of metabolite levels to clinical outcome in IBD. Thiopurine metabolism is cell specific and requires nucleated cells and particular enzymes for 6-TGN formation. In the current therapeutic drug monitoring, metabolite levels are assessed in erythrocytes, whereas leukocytes are considered the main target cells of these drugs. Furthermore, currently used methods do not distinguish between active nucleotides and their unwanted residual products. Last, there is a lack of a standardized laboratorial procedure for metabolite assessment regarding the substantial instability of erythrocyte 6-TGN. To improve thiopurine therapy in patients with IBD, it is necessary to understand these limitations and recognize the general misconceptions in this procedure.

6-Mercaptopurine modifies cerebrospinal fluid T cell abnormalities in paediatric opsoclonus-myoclonus as steroid sparer.

The purpose of this study was to evaluate the capacity of 6-mercaptopurine (6-MP), a known immunosuppressant, to normalize cerebrospinal fluid (CSF) lymphocyte frequencies in opsoclonus-myoclonus syndrome (OMS) and function as a steroid sparer. CSF and blood lymphocytes were immunophenotyped in 11 children with OMS (without CSF B cell expansion) using a comprehensive panel of cell surface adhesion, activation and maturation markers by flow cytometry, and referenced to 18 paediatric controls. Drug metabolites, lymphocyte counts and liver function tests were used clinically to monitoring therapeutic range and toxicity. In CSF, adjunctive oral 6-MP was associated with a 21% increase in the low percentage of CD4+ T cells in OMS, restoring the CD4/CD8 ratio. The percentage of CD4+ T cells that were interferon (IFN)-γ+ was reduced by 66%, shifting the cytokine balance away from T helper type 1 (Th1) (proinflammatory) predominance. The percentage of natural killer (NK) cells decreased significantly in CSF (-32%) and blood (-67 to -82%). Low blood absolute lymphocyte count was more predictive of improvement in CSF lymphocyte proportions (correlated with % CD4+ T cells) than the 6-thioguanine level (no correlation). 6-MP was difficult to titrate: 50% achieved the target absolute lymphocyte count (< 1·5 K/mm); 20%, the 'therapeutic' 6-thioguanine level; and 40% the non-toxic 6-methylmercaptopurine level. Side effects and transaminase elevation were mild and reversible. Clinical steroid-sparing properties and lowered relapse frequency were demonstrated. 6-MP displayed unique pharmacodynamic properties that may be useful in OMS and other autoimmune disorders. Its steroid sparer capacity is limited to children in whom the therapeutic window can be reached without limiting pharmacokinetic factors or side effects.

The effects of inherited NUDT15 polymorphisms on thiopurine active metabolites in Japanese children with acute lymphoblastic leukemia.

Thiopurines [e.g. mercaptopurine (MP)] are widely used as chemotherapeutic agents in the treatment of pediatric acute lymphoblastic leukemia with dose-limiting hematopoietic toxicity. Recently, germline variants in NUDT15 have been identified as a major genetic cause for MP-related bone marrow suppression, and there is increasing interest in the clinical implementation of NUDT15 genotype-guided MP dose individualization. Therefore, we sought to evaluate the effects of NUDT15 on thiopurine metabolism and identify pharmacologic markers to inform NUDT15 genotype-guided MP dosing. In 55 Japanese children with acute lymphoblastic leukemia, we simultaneously measured both thioguanine nucleotides (TGN) in red blood cells and DNA-incorporated thioguanine (DNA-TG) in white blood cells. TGN levels were significantly lower in patients with NUDT15 deficiency, likely because of toxicity-related MP dose reduction. In contrast, when exposed to the same dose of MP, DNA-TG accumulated more efficiently in vivo with increasing number of risk alleles in NUDT15 (P=4.0×10). Cytosolic TGN and nuclear DNA-TG were correlated positively with each other across genotype groups (P=6.5×10), but the ratio of DNA-TG to TGN was significantly higher in NUDT15-deficient patients (P=3.6×10), consistent with excessive MP activation. In conclusion, our results suggest that DNA-TG is a more relevant MP metabolite than TGN to inform NUDT15 genotype-guided dose adjustments.

DNA-thioguanine nucleotide concentration and relapse-free survival during maintenance therapy of childhood acute lymphoblastic leukaemia (NOPHO ALL2008): a prospective substudy of a phase 3 trial.

BACKGROUND: Adjustment of mercaptopurine and methotrexate maintenance therapy of acute lymphoblastic leukaemia by leucocyte count is confounded by natural variations. Cytotoxicity is primarily mediated by DNA-incorporated thioguanine nucleotides (DNA-TGN). The aim of this study was to establish whether DNA-TGN concentrations in blood leucocytes during maintenance therapy are associated with relapse-free survival. METHODS: In this substudy of the NOPHO ALL2008 phase 3 trial done in 23 hospitals in seven European countries (Denmark, Estonia, Finland, Iceland, Lithuania, Norway, and Sweden), we analysed data from centralised and blinded analyses of 6-mercaptopurine and methotrexate metabolites in blood samples from patients with non-high-risk childhood acute lymphoblastic leukaemia. Eligible patients were aged 1·0-17·9 years; had been diagnosed with non-high-risk precursor B-cell or T-cell leukaemia; had been treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol; and had reached maintenance therapy in first remission. Maintenance therapy was (mercaptopurine 75 mg/m2 once per day and methotrexate 20 mg/m2 once per week, targeted to a leucocyte count of 1·5-3·0 × 109 cells per L). We measured DNA-TGN and erythrocyte concentrations of TGN nucleotides, methylated mercaptopurine metabolites, and methotrexate polyglutamates. The primary objective was the association of DNA-TGN concentrations and 6-mercaptopurine and methotrexate metabolites with relapse-free survival. The secondary endpoint was the assessment of DNA-TGN concentration and 6-mercaptopurine and methotrexate metabolites during maintenance therapy phase 2. FINDINGS: Between Nov 26, 2008 and June 14, 2016, 1509 patients from the NOPHO ALL2008 study were assessed for eligibility in the DNA-TGN substudy, of which 918 (89%) of 1026 eligible patients had at least one DNA-TGN measurement and were included in the analyses. Median follow-up was 4·6 years (IQR 3·1-6·1). Relapse-free survival was significantly associated with DNA-TGN concentration (adjusted hazard ratio 0·81 per 100 fmol/µg DNA increase, 95% CI 0·67-0·98; p=0·029). In patients with at least five blood samples, erythrocyte concentrations of TGN, methylated mercaptopurine metabolites, and methotrexate polyglutamates were associated with DNA-TGN concentration (all p<0·0001). INTERPRETATION: Our results suggest the need for intervention trials to identify clinically applicable strategies for individualised drug dosing to increase DNA-TGN concentration, and randomised studies to investigate whether such strategies improve cure rates compared with current dose adjustments based on white blood cell counts. FUNDING: Danish Cancer Society, Childhood Cancer Foundation (Denmark), Childhood Cancer Foundation (Sweden), Nordic Cancer Union, Otto Christensen Foundation, University Hospital Rigshospitalet, and Novo Nordic Foundation.

Outcomes in Pediatric Autoimmune Hepatitis and Significance of Azathioprine Metabolites.

OBJECTIVES: Autoimmune hepatitis (AIH) is a common pediatric liver disease and long-term remission is usually maintained with azathioprine (AZA). There is no consensus on the target range for AZA active metabolite 6-thioguanine (6-TGN) levels in pediatric AIH. The aim of the present study was to characterize the outcomes of pediatric patients with AIH and determine correlations between AZA dosing or 6-TGN metabolite levels and biochemical remission. METHODS: A retrospective chart review was performed and data on presentation, laboratories including AZA metabolite levels, medication use, and outcomes were collected. RESULTS: Between 2002 and 2013, 66 children with AIH were identified (mean age at diagnosis 9.6 ±â€Š5.1 years) with a mean follow-up period of 2.9 ±â€Š3.2 years. Common presenting symptoms included jaundice, fatigue, and abdominal pain. The majority of subjects received steroids for induction and AZA for maintenance of remission. Seventy-nine percent achieved biochemical remission (mean time to remission 6.2 ±â€Š9.2 months), 14% were in the induction phase of therapy, 6% required liver transplantation, and 18% were weaned off immunosuppression and remained in remission. 6-TGN levels ranging from 50 to 250 pmol/8 × 10 red blood cell count were associated with biochemical remission (alanine aminotransferase levels of ≤50 U/L). CONCLUSIONS: The vast majority of children with AIH maintain a sustained remission with AZA monotherapy. Biochemical remission was maintained with 6-TGN levels much lower than that recommended for inflammatory bowel disease. These findings suggest that patients should be maintained at the lowest AZA dose possible that is associated with biochemical remission.