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In the treatment of diseases such as acute childhood leukaemia (ALL) and inflammatory bowel disease (IBD), the thiopurines azathioprine, 6-mercaptopurine, and 6-thioguanine are used. Thiopurines are antimetabolites and immunomodulators used to maintain remission in patients. They are all prodrugs and must be converted into the competing antimetabolites thioguanosine triphosphate and deoxythioguanosine triphosphate for final incorporation into RNA or DNA. The current therapeutic drug monitoring (TDM) method measures the sum of the formed metabolites in the sample, after acidic hydrolysis at high temperature. In this work, the goal is to measure these drugs closer to their pharmacological endpoints, once incorporated into DNA. After extracting DNA from whole blood, followed by DNA hydrolysis, 2'-deoxythioguanosine (dTG) and the complementary natural nucleobase 2'-deoxycytidine (dC) were measured. Chromatographic separation on a HSS T3 column followed by mass spectrometric detection was performed in multi-reaction monitoring (MRM) mode on a Xevo TQ-XS with ESI in positive mode, within 5 min. The concentration range for dTG was 0.04-5 nmol/L, and for dC, 0.1-12.5 µmol/L. The lower limit of detection was determined to a concentration of 0.003 nmol/L for dTG and 0.019 µmol/L for dC. The intra- and inter-assay imprecision for the quality controls ranged between 3.0 and 5.1% and between 8.4 and 10.9%, respectively. Sample stability for up to 4 years is shown. In summary, a sensitive method to quantify the thiopurines incorporated into DNA as dTG has been developed and will be used in further clinical studies for a better understanding of the mode of action of the thiopurines and the use of this method in TDM., Competing Interests: Declarations. Ethics approval: The study was approved by the Ethics Commission of Linköping University (diary number EPN 2018/47-31) and was conducted in accordance with the Declaration of Helsinki. All subjects who provided blood samples signed informed consent. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease treated by steroids and immunomodulator thiopurine drugs such as azathioprine (AZA). AZA is metabolized in the human body into bioactive forms such as 6-thioguanine (6-TG) and 6-methyl-mercaptopurine (6-MMP). Monitoring the levels of bioactive AZA metabolites is very important for proper treatment of patients. In this study, our aim was to develop and validate a fast and sensitive ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method for the analysis of 6-TG and 6-MMP from blood samples of patients with AIH to monitor the level of these bioactive metabolites. The detection and quantification of the analytes was carried out by Selected Reaction Monitoring (SRM)-based targeted mass spectrometry. The method was validated according to the EMA guidelines. Blood samples from patients with AIH treated with AZA were analysed with the developed method. The method was successfully validated with appropriate accuracy and precision for the target biomolecules and their concentration in the samples from patients with AIH was determined. The developed and validated UHPLC-MS method enables the fast and precise analysis of AZA metabolites.

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 (Ag core Au shell NP) suspension. The Au/Ag ratio of the Ag core Au shell 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 R 2 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., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2020 The Authors. Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

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., (Copyright © 2019. Publicado por Elsevier España, S.L.U.)

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 × 10 8 Ery for 6-TG and from 260 to 5300 pmol/8 × 10 8 Ery for 6-MMP. Range can be increased to 8000 pmol/8 × 10 8 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2018 Elsevier España, S.L.U. All rights reserved.)

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.

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 (Fe 3 O 4 @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 Fe 3 O 4 @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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., Competing Interests: No potential conflicts of interest relevant to this article were reported., (© The Korean Society for Laboratory Medicine.)

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(F 0 /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., (Copyright © 2017. Published by Elsevier B.V.)

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.

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., (© 2017 British Society for Immunology.)

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., (Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com)

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 ± 5.1 years) with a mean follow-up period of 2.9 ± 3.2 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 ± 9.2 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.

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.

Purpose Children with acute lymphoblastic leukemia (ALL) are generally instructed to take mercaptopurine (6-MP) in the evening and without food or dairy products. This study examines the association between 6-MP ingestion habits and 6-MP adherence, red cell thioguanine nucleotide (TGN) levels, and risk of relapse in children with TMPT wild-type genotype. Methods Participants included 441 children with ALL receiving oral 6-MP for maintenance. Adherence was monitored over 48,086 patient-days using the Medication Event Monitoring System; nonadherence was defined as adherence rate < 95%. 6-MP ingestion habits examined included: takes 6-MP with versus never with food, takes 6-MP with versus never with dairy, and takes 6-MP in the evening versus morning versus varying times. Results Median age at study was 6 years (range, 2 to 20 years); 43.8% were nonadherent. Certain 6-MP ingestion habits were associated with nonadherence (taking 6-MP with dairy [odds ratio (OR), 1.9; 95% CI, 1.3 to 2.9; P = .003] and at varying times [OR, 3.4; 95% CI, 1.8 to 6.3; P = .0001]). After adjusting for adherence and other prognosticators, there was no association between 6-MP ingestion habits and relapse risk (6-MP with food: hazard ratio [HR], 0.7; 95% CI, 0.3 to 1.9; P = .5; with dairy: HR, 0.3; 95% CI, 0.07 to 1.5; P = .2; taken in evening/night: HR, 1.1; 95% CI, 0.2 to 7.8; P = .9; at varying times: HR, 0.3; 95% CI, 0.04 to 2.7; P = .3). Among adherent patients, there was no association between red cell TGN levels and taking 6-MP with food versus without (206.1 ± 107.1 v 220.6 ± 121.6; P = .5), with dairy versus without (220.1 ± 87.8 v 216.3 ± 121.3; P =.7), or in the evening/night versus morning/midday versus varying times (218.8 ± 119.7 v 195.5 ± 82.3 v 174.8 ± 93.4; P = .6). Conclusion Commonly practiced restrictions surrounding 6-MP ingestion might not influence outcome but may hinder adherence. Future recommendations regarding 6-MP intake during maintenance therapy for childhood ALL should aim to simplify administration.

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/m 2 once per day and methotrexate 20 mg/m 2 once per week, targeted to a leucocyte count of 1·5-3·0 × 10 9 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Early-onset inflammatory bowel disease (IBD) is generally aggressive, with a high probability of complications and need of surgery. Despite the introduction of highly effective biological drugs, treatment with azathioprine continues to be important even for early-onset IBD; however, in these patients azathioprine response seems to be reduced. This study evaluated azathioprine doses, metabolite concentrations, and their associations with patients' age in children with IBD treated at 6 tertiary pediatric referral centers., Methods: Azathioprine doses, metabolites, and clinical effects were assessed after at least 3 months of therapy in 17 early-onset (age < 6 yr, cases) and 51 nonearly-onset (aged > 12 and <18 yrs, controls) patients with IBD. Azathioprine dose was titrated on therapeutic efficacy (response and adverse effects). Azathioprine metabolites and thiopurine methyltransferase activity were determined by high-performance liquid chromatography with ultra violet-vis detection (HPLC-UV) methods., Results: Frequency of patients in remission was similar among early-onset and control groups, respectively (82% and 84%, P value = 0.72). Early-onset patients required higher doses of azathioprine (median 2.7 versus 2.0 mg·kg·d, P value = 1.1 × 10). Different doses resulted in comparable azathioprine active thioguanine nucleotide metabolite concentrations (median 263 versus 366 pmol/8 × 10 erythrocytes, P value = 0.41) and methylmercaptopurine nucleotide concentrations (median 1455 versus 1532 pmol/8 × 10 erythrocytes, P value = 0.60). Lower ratios between thioguanine nucleotide metabolites and azathioprine doses were found in early-onset patients (median 98 versus 184 pmol/8 × 10 erythrocytes·mg·kg·d, P value = 0.017). Interestingly, early-onset patients presented also higher thiopurine methyltransferase activity (median 476 versus 350 nmol methylmercaptopurine/mg hemoglobin/h, P-value = 0.046)., Conclusions: This study demonstrated that patients with early-onset IBD present increased inactivating azathioprine metabolism, likely because of elevated activity of the enzyme thiopurine methyltransferase.

Individualized therapy is a recent approach aiming to specify dosage regimen for each patient according to its genetic state. Cancer chemotherapy requires continuous monitoring of the plasma concentration levels of active forms of cytotoxic drugs and subsequent dose adjustment. In order to attain optimum therapeutic efficacy, correlation to pharmacogenetics data is crucial. In this study, a specific, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed for determination of methotrexate (MTX), 6-mercaptopurine (MP) and its metabolite 6-thioguanine nucleotide (TG) in human plasma. Based on the basic character of the studied compounds, solid phase extraction using a strong cation exchanger was found the optimum approach to achieve good extraction recovery. Chromatographic separation was carried out using RP-HPLC and isocratic elution by acetonitrile: 0.1% aqueous formic acid (85:15v/v) with a flow rate of 0.8mL/min at 40°C. The detection was performed by tandem mass spectrometry in MRM mode via electrospray ionization source in positive ionization mode. Analysis was carried out within 1.0min over a concentration range of 6.25-200.00ng/mL for the studied analytes. Validation was carried out according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the monitoring of the MTX, MP and TG and subsequent application to personalized therapy was demonstrated in a clinical study on children with acute lymphoblastic leukemia (ALL). Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Thiopurines are commonly used drugs in inflammatory bowel disease. Intracellular levels of thiopurine metabolites [i.e. 6-thioguaninenucleotides (6-TGN)] are associated with efficacy and toxicity. Because 6-TGN measurement is not globally available, the mean corpuscular volume (MCV) has been proposed as a surrogate marker for monitoring thiopurine therapy., Aims: To analyze the relationship between MCV and efficacy of thiopurines, defined as either response to therapy or 6-TGN levels., Methods: A systematic search on PubMed was performed., Results: Fifteen studies were included. In six studies, a positive association was found between ΔMCV and 6-TGN. In four studies, it was suggested that ΔMCV can be used to predict clinical remission. In five articles, no association was found., Conclusion: In the majority of articles, it was reported that ΔMCV is useful in guiding intracellular metabolite levels. However, there is insufficient evidence showing that ΔMCV can predict clinical remission.

Background: 6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are used in many autoimmune diseases and after solid-organ transplantation. Their properties are mediated by active metabolites, 6-thioguanine nucleotides (6-TGN), and 6-methylmercaptopurine (6-MMP). The most common adverse effects are myelo- and hepato-toxicity. The aim of the study was quantification of 6-TG and 6-MMP, with the use of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) in solid-organ transplant recipients., Methods: In 33 patients, kidney transplant recipient (n = 25) and liver transplant recipient (n = 8) intra-erythrocyte concentrations of 6-TG and 6-MMP were measured with the use of LC/MS/MS., Results: The mean concentration of 6-TG was 205.35 ± 157.62 pmol/8 × 10(8) red blood cells (RBC); median concentration of 6-MMP was 1064.1 (35.78-11,552.9) pmol/8 × 10(8) RBC. There were no correlations between 6-TG levels and peripheral blood parameters (white blood cell count, WBC; hemoglobin, Hb concentration; PLT, blood platelet count) or alanine aminotransferase activity (AlAT) activity. Relationships between 6-MMP concentrations and peripheral blood parameters (WBC, Hb, PLT) or AlAT activity have not been found. Subgroups with leukopenia, anemia, thrombocytopenia, and liver dysfunction did not differ in concentrations of 6-TG or 6-MMP. We have observed a negative correlation between daily azathioprine dose and WBC count (r = -0.37, P = .04)., Conclusions: Relationships between concentrations of azathioprine metabolites and myelotoxicity or hepatotoxicity have not been confirmed. Further studies on larger groups of patients would be helpful in a more accurate understanding of the impact of azathioprine metabolites on parameters of bone marrow and liver function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: The thiopurine drugs, 6-mercaptopurine and azathioprine, remain the mainstay of immunomodulator therapy for inflammatory bowel disease (IBD). Optimal management depends on achieving therapeutic levels of 6-thioguanine (6-TGN), but measuring thiopurine metabolites is associated with significant cost. Thiopurines cause lymphopenia and an increase in mean corpuscular volume (MCV). It is unclear whether any clinically useful correlation exists between 6-TGN levels and lymphocyte count or MCV., Aims: The aim of this study is to investigate the correlation between 6-TGN levels and lymphocyte count and MCV in thiopurine-treated patients with IBD., Methods: We analysed a prospectively acquired database of 67 patients with IBD treated with thiopurine therapy. The data were analysed looking at the relationship between 6-TGN levels and both lymphocyte count and MCV by using the Spearman's rank correlation coefficient., Results: Twenty-seven (40%) patients had therapeutic 6-TGN levels. Thirty-three (49%) patients had sub-therapeutic 6-TGN levels. A weak positive correlation between 6-TGN levels and lymphocyte count was demonstrated, but this was not statistically significant (Spearman's R = 0.14, P = 0.23). Spearman's rank correlation coefficient between 6-TGN levels and MCV was statistically significant (R = 0.42, P = 0.0005). MCV >101 fL excluded a subtherapeutic 6-TGN level with positive predictive value of 92%., Conclusions: There is no specific lymphopenia that can be assumed to indicate a therapeutic 6-TGN level. The relationship between 6-TGN levels and MCV is likely to be clinically relevant. If MCV is elevated, 6-TGN is unlikely to be sub-therapeutic. MCV is a potential surrogate marker which can rule out sub-therapeutic thiopurine metabolites in patients with IBD treated with azathioprine or 6-mercaptopurine., (© 2016 Royal Australasian College of Physicians.)

Background: Impact of first-line induction therapy on medium-term outcomes in the setting of early thiopurine (TP) use in children with Crohn's disease has not been evaluated, in particular whether choice of exclusive enteral nutrition (EEN) over corticosteroids (CS) for induction impacts clinical outcomes at 12 and 24 months., Aims and Methods: In this retrospective study, 89 children from our database with new diagnosis CD and follow-up of at least 2 years following induction with exclusive course of CS or EEN and early, dose-optimized TP (within 6 months from diagnosis) were evaluated. We compared steroid dependency (relapse <3 months of tapering first course CS or inability to wean <10 mg prednisolone), need for IFX, linear growth, and surgical resections over the first 2 years., Results: Choice of EEN over CS induction was associated with reduced linear growth failure (7 vs. 26%, p = 0.02), CS dependency (7 vs. 43%, p = 0.002), and improved primary sustained response to IFX (86 vs. 68%, p = 0.02). Combined CS/IFX-free remission and surgical resection rates were similar., Conclusion: In the setting of early TP commencement, EEN induction is superior to CS induction for reducing growth failure, CS dependency, and loss of response to IFX over the first 2 years.

Background and Aims: The thiopurines are widely used in the treatment of inflammatory bowel disease, but are limited by poor dose-effect relationship. The objective was to assess the ability of a novel assay, determining the mono-, di-, and triphosphates, of thioguanine as well as methylthioinosine as individual metabolites in erythrocytes, to predict clinical outcome compared to a routine assay, determining metabolites as sums., Methods: Samples from 79 patients with Crohn's disease or ulcerative colitis treated with azathioprine or mercaptopurine were analysed by both assays. Clinical status was determined by the Harvey-Bradshaw and Walmsley indices. The genotypes of thiopurine methyltransferase (TPMT) and inosine triphosphatase were determined., Results: TPMT wild-type patients with thioguanine nucleotide (TGN) levels below the cut-off level were more likely to have active disease when TGN was measured by the novel assay (p=0.02), and when thioguanosine triphosphate (TGTP) was measured separately (p=0.01). When TGN was measured by the routine assay the correlation was not evident (p=0.12). Neither TGN levels nor TGTP correlated to disease activity in TPMT deficient patients. Patients with methyl thioinosine nucleotide (meTIN) levels above 1500 pmol/8×10^8 RBCs were more likely to have active disease (p=0.07). We observed good correlations between the mono-, di-, and triphosphates and their respective sums (R(2)>0.88)., Conclusions: The novel TGN assay was better in predicting clinical outcome compared to the routine assay, while determination of TGTP had no clinical advantage and TGTP ratio was not correlated to disease activity., (Copyright © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Background: Azathioprine (AZA), a pro-drug metabolised to the active metabolites 6-tioguanine nucleotides (6TGN), is a steroid-sparing therapy for Crohn's disease (CD)., Aim: To investigate whether AZA therapy is optimised by individualised dosing based on thiopurine methyltransferase (TPMT) activity and 6TGN concentrations., Methods: This multicentre, double-blind, randomised controlled trial compared the efficacy and safety of weight-based vs. individualised AZA dosing in inducing and maintaining remission in adults and children with steroid-treated CD. The primary outcome was clinical remission (CR) at 16 weeks. In the weight-based arm, subjects received 2.5 mg/kg/day. In the individualised dosing arm, the initial AZA dose was 1.0 mg/kg/day (if intermediate TPMT) or 2.5 mg/kg/day (if normal TPMT). Starting at week 5, the dose was adjusted to target 6TGN concentrations of 250-400 pmol/8 × 10(8) red blood cells (RBC), or to a maximal dose of 4 mg/kg/day., Results: After randomising 50 subjects, the trial was stopped prematurely due to insufficient enrolment. In intention-to-treat analysis, CR rates at week 16 were 40% in the individualised arm vs. 16% in the weight-based arm (P = 0.11). In per-protocol (PP) analysis, week 16 CR rates were 60% in the individualised arm and 25% in the weight-based arm (P = 0.12). At week 16, median 6TGN concentrations in PP remitters and nonremitters were 216 and 149 pmol/8 × 10(8) RBC respectively (P = 0.07)., Conclusions: Despite trends favouring individualised over weight-based AZA dosing, there were no statistically significant differences in efficacy, likely due to low statistical power and inability to achieve the target 6TGN concentrations in the individualised arm. [Clinicaltrials.Gov Identifier Nct00113503]., (© 2013 John Wiley & Sons Ltd.)

Background and Aims: A skewed thiopurine metabolism is a phenomenon associated with both poor treatment response and toxicity. Our aim was to evaluate the frequency of this phenomenon and the relationship to thiopurine methyltransferase (TPMT) function., Methods: All thiopurine metabolite measurements in adult patients (n=4033) between January 2006 and April 2012 were assessed to evaluate the occurrence of a skewed metabolism and the relationship to TPMT genotype and activity., Results: A skewed metabolism was observed in 14% of all patients. It only developed in patients with a normal TPMT genotype, but was observed at all TPMT activity levels within the normal range (9.1-24.2 U/ml RBC). Two cases that illustrate typical clinical scenarios of a skewed metabolism and the effect of combination treatment with low-dose azathioprine and allopurinol are presented., Conclusions: A skewed metabolism is a common clinical phenomenon in patients with a normal TPMT function, which can develop at all TPMT activity levels within the normal range. We suggest that metabolite measurements should be considered in patients not responding to treatment and in those with hepatotoxicity or myelotoxicity in order to detect a skewed metabolism, since this phenomenon can be successfully managed by a combination of low-dose azathioprine and allopurinol., (Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Gold nanoparticles exhibit the optical properties of localized surface plamon resonance (LSPR) and are widely applied to the biosensors. The application of gold nanoparticles to the determination of anticancer drug 6-thioguanine (6-TG) was discussed. The binding of 6-TG molecule to the surface of gold nanoparticles alters the local refractive index in the vicinity of the nanoparticles and results in a shift of the LSPR spectrum. The experimental conditions were examined and optimized. Under the optimal conditions, the ratios of absorbances at two wavelengths are directly proportional to the concentrations of 6-TG. The developed method is simple, rapid, and sensitive. In addition, this method is particularly attractive because organic cosolvents, light-sensitive dyes, and sophisticated instruments are not required. This method was successfully applied to the determination of 6-TG in real samples and the results were satisfactory., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes. The method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine). LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 × 10⁸ RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 °C for 8 h after sampling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The value of azathioprine metabolites (6-thioguanine nucleotides [6-TGN]) in monitoring clinical treatment response is still controversially discussed. Data regarding thiopurine metabolite levels and endoscopic improvement are lacking., Methods: Data were analyzed post hoc from a 1-year, multicenter, double-blind, double-dummy, randomized trial comparing azathioprine 2.0 to 2.5 mg/kg per day versus mesalamine 4 g/d in a subset of 23 postoperative patients with Crohn's disease (CD) treated with azathioprine and having moderate-to-severe endoscopic recurrence according to a modified 6-grade score. Red blood cell (RBC) concentrations of 6-TGN, 6-methyl-mercaptopurine ribonucleotides (6-MMPR), and 6-methyl-thioguanine nucleotides (6-MTGN) were indicated as follows: area under the concentration-time curve, average concentration (C av), and concentration at the final study visit., Results: Overall, 74% of patients showed an improvement in the modified endoscopic score (P = 0.022). Median endoscopic score reduced from 4 at the baseline to 2 at the final visit. Patients with a high C av for 6-TGN (≥ 193 pmol/8 × 10(8) RBC; P = 0.017) or 6-MTGN (≥ 79.2 pmol/8 × 10(8) RBC; P = 0.035) significantly improved in endoscopic score, and the improvement in endoscopic score correlated with C av for 6-TGN (r = -0.51; P = 0.013). For concentration at the final visit, higher values for 6-TGN (≥ 142 pmol/8 × 10(8) RBC; P = 0.017) were associated with a better postoperative score. Sensitivity analysis revealed a significant correlation between 6-TGN (area under the concentration-time curve) and postoperative endoscopic improvement., Conclusions: Our post hoc analysis from a double-blind, randomized trial suggests that higher RBC 6-TGN levels are associated with endoscopic improvement in patients with severe postoperative endoscopic recurrence of CD. Thus, our study provides first evidence on the utility of monitoring of thiopurine metabolites to achieve mucosal response in CD.

The United States Supreme Court recently issued an opinion regarding the patentability of claims directed to diagnostic methods in Mayo Collab. Service v. Prometheus Lab., Inc. In this opinion, the Supreme Court held that correlations between metabolite levels in the human body and either therapeutic efficacy or adverse effects are unpatentable laws of nature. It further found that a patent claim to a method including such a correlation is unpatentable if the remainder of the claim contains only conventional and well-known steps. The Prometheus decision creates uncertainty regarding the scope of patentable subject matter, particularly in the fields of diagnostic and personalized medicine, that will remain until future cases apply this new doctrine.

Therapeutic drug monitoring (TDM) of major metabolites of thiopurine drugs is a widely used tool for assessing treatment efficacy and toxicity in patients with inflammatory bowel disease (IBD). We report the laboratory and clinical validation of a simple and reliable high performance liquid chromatography (HPLC) method for the measurement of 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) on paediatric patients with IBD. The aim of this paper is to develop and validate a method for the measurement of 6-TGN and 6-MMP applicable to routine practice and to evaluate the usefulness of the TDM of thiopurine drugs in children with IBD attending our Gastroenterology Unit. The HPLC method was validated following international guidelines starting from red blood cells (RBC) and whole blood (WB). A comparison between RBC and WB was assessed. The usefulness of TDM was then evaluated using the new method from WB in 47 paediatric patients with IBD treated with thiopurine drugs. WB and RBC resulted in interchangeable matrices. The majority of patients had the metabolite levels inside the therapeutic ranges. A moderate correlation was found between 6-MMP concentration and the dose of thiopurines. A higher percentage of non responders was found among patients with lower levels of 6-TGN. Toxicity was found in eight patients and was evaluated in respect to the metabolite concentration. The described HPLC method is applicable to routine practice and it is suitable for its use in multicentric studies. Our results of TDM on paediatric IBD patients can contribute to clarify its role in their therapeutic management.

Background: Thiopurine drugs are widely used in the treatment of inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and adverse events occurrence. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the effects of TPMT and hypoxanthine guanine phosphoribosyltransferase (HPRT) activities on 6-thioguanine nucleotides (6-TGNs) concentrations and thiopurine-induced leukopenia in patients with IBD., Methods: Clinical data and blood samples were collected from 120 IBD patients who were receiving azathioprine (AZA)/6-mercaptopurine (6-MP) therapy. Erythrocyte TPMT, HPRT activities and 6-TGNs concentrations were determined. HPRT activity and its correlation with TPMT activity, 6-TGNs level, and leukopenia were evaluated., Results: The HPRT activity of all patients ranged from 1.63-3.33 (2.31 ± 0.36) μmol/min per g Hb. HPRT activity was significantly higher in patients with leukopenia (27, 22.5%) than without (P < 0.001). A positive correlation between HPRT activity and 6-TGNs concentration was found in patients with leukopenia (r = 0.526, P = 0.005). Patients with HPRT activity > 2.70 μmol/min per g Hb could have an increased risk of developing leukopenia (odds ratio = 7.47, P < 0.001). No correlation was observed between TPMT activity and HPRT activity, 6-TGNs concentration, or leukopenia., Conclusions: High levels of HPRT activity could be a predictor of leukopenia and unsafe 6-TGN concentrations in patients undergoing AZA/6-MP therapy. This could partly explain the therapeutic response or toxicity that could not be adequately explained by the polymorphisms of TPMT., (Copyright © 2011 Crohn's & Crohn's & Colitis Foundation of America, Inc.)

Background: Circulating concentrations of 6-thioguanine nucleotide (6-TGN) and 6-methyl mercaptopurine (6-MMP) are associated with thiopurine efficacy and may predict toxicity. This study aimed to examine retrospectively the utility of measuring metabolite concentrations in patients with inflammatory bowel disease (IBD) who had continuing symptoms despite stable thiopurine treatment., Methods: Concentrations of 6-TGN and 6-MMP were measured in lysates of washed red cells by high-performance liquid chromatography in peripheral blood drawn from 63 symptomatic patients with IBD (63% men, mean age 37, range 14-74 years, 67% Crohn's disease, 33% ulcerative colitis) treated with azathioprine or 6-mercaptopurine. Short-term clinical outcomes were examined., Results: 6-TGN concentrations weakly correlated with the thiopurine dose (r = 0.28, P = 0.08). On weight-based criteria, 50% of patients were underdosed. However, metabolite patterns suggested 7 (11%) patients were noncompliant, 18 (29%) were being underdosed, 33 (52%) were refractory to treatment with either appropriate (41%) or elevated (11%) metabolite concentrations, and 6 (10%) had a raised 6-MMP:6-TGN ratio consistent with aberrant thiopurine metabolism. The clinical outcome improved in 40 of 46 (87%) of patients in whom the course of action taken was as recommended by a metabolite-directed algorithm, while 3 of 17 patients (18%) improved where discordant actions were taken (P = 0.0001; Fisher's exact test). Fifteen patients (24%) avoided inappropriate escalation of therapy., Conclusions: Dose-optimization or toxicity-avoidance strategies frequently result from metabolite testing in patients with inadequate efficacy from thiopurines, with evidence of better outcomes. Thiopurine metabolite testing is a potentially powerful tool for optimizing thiopurine usage in IBD., (Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.)

Azathioprine is commonly used in the treatment of autoimmune hepatitis (AIH). Few data are available on drug monitoring of azathioprine metabolites in patients with AIH, especially in pediatric patients. The purpose of this study was to investigate intracellular thiopurine metabolites in children with AIH and to assess the relevance of drug monitoring compared with the efficacy and toxicity. Data from 28 patients with AIH treated by azathioprine for at least 3 months were recorded. 6-Thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine nucleotides (6-MeMPN) concentrations and TPMT activity were determined by high-performance liquid chromatography. Blood cell counts and liver function tests were also collected and the clinical outcome was documented. A wide interindividual variability in 6-TGN and 6-MeMPN concentrations was observed with values ranging from 51 to 1966 pmol/8 x 10(8) red blood cells (RBCs) for 6-TGN and from 42 to 8189 pmol/8 x 10(8) RBCs for 6-MeMPN. A total of 61.4% of the patients achieved remission and only 32.6% of these children had 6-TGN values within the target range proposed for inflammatory bowel disease (250-450 pmol/8 x 10(8) red blood cells). No difference in metabolite concentrations was observed between children in remission and those with active disease. Azathioprine dosage was significantly related to 6-TGN and 6-MeMPN levels (r = 0.308, P < 0.001 and r = 0.405, P < 0.001, respectively). A significant negative correlation was observed between 6-TGN concentrations and erythrocyte and lymphocyte counts, whereas 6-MeMPN was not related to blood cell counts. Although leukocyte counts were not related to 6-TGN concentrations, patients with leucopenia exhibited higher 6-TGN values than those without leucopenia (median values 438 pmol/8 x 10(8) RBCs versus 405 pmol/8 x 10(8) RBCs, respectively). Thiopurine metabolite monitoring appears useful in identifying the myelotoxicity and the hepatotoxicity as a result of azathioprine with disease recurrence and to assess adherence to therapy. A further larger study will be required to confirm the optimal recommended target for thiopurine metabolites to achieve remission in patients with AIH.

Background: 6-Mercaptopurine (6-MP) is an effective maintenance medication in patients with ulcerative colitis (UC), but toxic effects like myelosuppression limit its clinical benefit. In the blood, 6-thioguanine (6-TGN) is formed from 6-MP and mediates the therapeutic efficacy and most of the toxicities of 6-MP. The level of 6-TGN depends on the activity of thiopurine methyltransferase (TPMT), inherited as 1 of its 3 polymorphic forms with low, moderate, or normal/high activity. Accordingly, the 6-MP dose needs to be pharmacogenetically guided., Methods: Patients with quiescent UC received 6-MP as maintenance therapy and 6-TGN was assayed as its concentrations in red blood cells (RBCs) done by high-performance liquid chromatography. In a preliminary investigation, 30 mg/day 6-MP (n = 50) was given orally over 12 weeks to determine the time course of blood 6-TGN level. Then 257 patients were given 6-MP at 15-80 mg/day in a stepwise manner based on RBC 6-TGN, white blood cell count, and body weight to monitor 6-MP efficacy and safety profiles., Results: At 30 mg/day 6-MP, RBC 6-TGN peaked over 4-8 weeks. In the main dosing study, the mean RBC 6-TGN level in patients who remained in remission during the 1-year observation time (n = 151) was 322.3 +/- 119.5 pmole/8 x 10(8) RBC versus 204.8 +/- 78.7 pmole/8 x 10(8) RBC in patients (n = 19) who relapsed (P < 0.001). Bone marrow suppression was seen almost exclusively at high 6-TGN concentration ranges. Further, a regression plot showed an inverse relationship between 6-TGN levels in RBC and TPMT enzyme activity., Conclusions: By regularly measuring RBC 6-TGN in patients with quiescent UC receiving 6-MP as maintenance therapy, we could monitor bone marrow suppression as well as other toxic side effects. Potentially, this strategy should enable physicians to avoid thiopurine-related adverse effects and identify individuals who may benefit most from 6-MP maintenance therapy.

Background: Monitoring of thiopurine metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) is used to assess compliance and explain adverse reactions in IBD-patients. Correlations between dosage, metabolite concentrations and therapeutic efficacy or toxicity are contradictive. Research is complicated by analytical problems as matrices analyzed and analytical procedures vary widely. Moreover, stability of thiopurine metabolites is not well documented, yet pivotal for interpretation of analytical outcomes. Therefore, we prospectively investigated metabolite stability in blood samples under standard storage conditions., Methods: Stability at room temperature and refrigeration (22 degrees C, 4 degrees C) was investigated during 1 week and frozen samples (-20 degrees C, -80 degrees C) were analyzed during 6 months storage. Ten patient samples were analyzed for each study period., Results: Median 6-TGN concentrations on day 7 decreased significantly to 53% and 90% during storage at ambient temperature or refrigeration. Median 6-MMP concentrations on day 7 decreased significantly to 55% and 86%, respectively. Samples stored at -20 degrees C also showed significant decreases in both 6-TGN and 6-MMP in comparison with baseline values. At -80 degrees C, only 6-MMP showed a significant decrease in values compared to baseline., Conclusion: The stability of thiopurine metabolites is clearly a limiting factor in studies investigating utilisation of TDM and correlations with therapeutic outcome in IBD-patients. This has to be accounted for in clinical practice and (multi-center) trials investigating thiopurine drugs., (Copyright 2010. Published by Elsevier B.V.)

Background: The monitoring of 6-thioguanine nucleotides (6-TGN) levels is warranted during thiopurine therapy for patients with inflammatory bowel diseases., Aims: The aim of this study was to elucidate the parameters that can predict the 6-TGN levels among Japanese patients with inflammatory bowel diseases undergoing thiopurine therapy., Material and Methods: The 6-TGN levels were measured in 54 patients with inflammatory bowel diseases (32 with ulcerative colitis and 22 with Crohn's disease), who had been administered azathioprine or 6-mercaptopurine for more than 90 days. Possible correlations between the hematologic parameters and 6-TGN levels were investigated. The clinical and hematologic variables were evaluated to determine the 6-TGN levels of less than or over 235 pmol/8 x 10(8) RBCs., Results: The 6-TGN levels correlated significantly with changes in the mean corpuscular volume (R = 0.423, p = 0.001) and the lymphocyte counts (R = -0.280, p = 0.04). A multivariate analysis revealed changes in the mean corpuscular volume (OR: 1.22, 95% CI: 1.07-1.40) and hemoglobin levels (OR: 0.59, 95% CI: 0.35-0.99) to be factors predictive of the 6-TGN levels. An increase in the mean corpuscular volume of 3.5 fl was determined to be the most preferable cut-off value to distinguish patients with 6-TGN >or= 235 pmol/8 x 10(8) RBCs from those with a lower concentration., Conclusions: Changes in the mean corpuscular volume are considered to be predictive of the 6-TGN levels in patients with inflammatory bowel diseases receiving thiopurine therapy.

Background & Aims: Corticosteroids alone or in conjunction with azathioprine (AZA) is the standard treatment in autoimmune hepatitis (AiH). Individual variations in thiopurine (TP) metabolism may affect both drug efficacy and toxicity. Our aim was to investigate the utility of thiopurine methyltransferase (TPMT) as well as thioguanine nucleotide (TGN) and methylthioinosine monophosphate (meTIMP) metabolite measurements with regard to clinical outcome., Methods: Two hundred thirty-eight patients with AiH were included in this cross-sectional study. TPMT status was assessed in all patients, while TGN and meTIMP were measured in patients with ongoing TP medication. Clinical outcome was evaluated by liver tests and the ability to withdraw steroids., Results: TPMT genotyping (n=229) revealed 207 (90.4%) wild-type and 22 heterozygous patients. One hundred forty-three patients had ongoing TP therapy with AZA (n=134) or mercaptopurine (MP; n=9); response was judged as complete response (CR) in 113 patients and partial response (PR) in 30 patients. Both TP dose (1.64 vs 1.19 mg/kg; p=0.012) and TPMT activity (14.3 vs 13.5; p=0.05) were higher in PR, resulting in similar TGN levels (PR: 121 pmol/8 x 10(8) red blood cells [RBC]; CR: 113 pmol/8 x 10(8) RBC; p=0.33) but higher meTIMP levels in PR (1350 vs 400 pmol/8 x 10(8) RBC; p=0.004). Patients able to withdraw steroids or who were using 5 mg prednisolone daily were treated with lower TP doses than patients on higher steroid doses (1.15 vs 1.18 vs 1.82 mg/kg; p<0.001)., Conclusions: TP metabolite measurements are of clinical value in AiH patients who do not respond to standard TP treatment and for the identification of a shifted metabolism, which may demand an alternative treatment strategy.

Aims: To develop a method that prospectively assesses adherence rates in paediatric patients with acute lymphoblastic leukaemia (ALL) who are receiving the oral thiopurine treatment 6-mercaptopurine (6-MP)., Methods: A total of 19 paediatric patients with ALL who were receiving 6-MP therapy were enrolled in this study. A new objective tool (hierarchical cluster analysis of drug metabolite concentrations) was explored as a novel approach to assess non-adherence to oral thiopurines, in combination with other objective measures (the pattern of variability in 6-thioguanine nucleotide erythrocyte concentrations and 6-thiouric acid plasma levels) and the subjective measure of self-reported adherence questionnaire., Results: Parents of five ALL patients (26.3%) reported at least one aspect of non-adherence, with the majority (80%) citing "carelessness at times about taking medication" as the primary reason for non-adherence followed by "forgetting to take the medication" (60%). Of these patients, three (15.8%) were considered non-adherent to medication according to the self-reported adherence questionnaire (scored > or = 2). Four ALL patients (21.1%) had metabolite profiles indicative of non-adherence (persistently low levels of metabolites and/or metabolite levels clustered variably with time). Out of these four patients, two (50%) admitted non-adherence to therapy. Overall, when both methods were combined, five patients (26.3%) were considered non-adherent to medication, with higher age representing a risk factor for non-adherence (P < 0.05)., Conclusions: The present study explored various ways to assess adherence rates to thiopurine medication in ALL patients and highlighted the importance of combining both objective and subjective measures as a better way to assess adherence to oral thiopurines.

We studied the pharmacokinetics of 6-thioguanine (6TG) in 50 children treated for newly diagnosed acute myeloid leukemia, four of them with Down syndrome (DS). They received oral 6TG 100 mg/m2 body surface area twice daily for 4 days. Etoposide, 100 mg/m2/24 h, and cytarabine, 200 mg/m2/24 h, were administered concomitantly by intravenous infusion. On day 5, doxorubicin 75 mg/m2 was given as an 8-h infusion. The concentration of thioguanine nucleotides (TGN) in erythrocytes, the active metabolites of 6TG, was determined by high-performance liquid chromatography. The mean TGN concentration from 72, 95, and 106-h samples was used as a measure of drug exposure for each individual. The median TGN concentration in non-DS children above 2 years of age was 2.30 micromol/mmol Hb (range 0.57-25.3). The TGN concentrations varied widely (30-fold) also after dose normalization. We found no correlation with demographic, clinical, or biochemical parameters, and differences in bioavailability might be the most important explanation to interpatient variability. Children with high TGN concentration tended to have longer treatment interval to the next course, but we found no correlation with our predefined parameters for clinical response, that is, remission and relapse rate. Therefore, 6TG does not seem to be a candidate for therapeutic drug monitoring by TGN measurement, at least not in the setting of short multidrug treatment courses. Children with DS had significantly higher TGN concentrations, indicating that dose reduction might be considered to reach the same drug exposure as in non-DS children.

Objective: Individualized therapy based on genetic background and monitoring of metabolites can optimize drug safety and efficacy. Such an approach is available for azathioprine (AZA), the thiopurine antimetabolite. AZA exerts therapeutic effects when metabolized to the active thiopurine nucleotide, 6-thioguanine (6-TGN). In inflammatory bowel disease (IBD), 6-TGN levels in the target range of 235-400 pmol/8x10(8) red blood cells (RBC) are associated with a high likelihood of response. Our objective was to evaluate whether drug escalation based on metabolite levels improves efficacy and maintains safety in patients with systemic lupus erythematosus (SLE)., Methods: We conducted a 6-month open-label dose-escalation clinical study of patients with active SLE treated with azathioprine dosed by body weight and metabolite levels. The primary endpoint was >or=50% improvement in any one parameter of disease activity, or 50% decrease in glucocorticoid dose., Results: Of 50 patients enrolled in the study, 21 achieved clinical responses, 13 of whom had 6-TGN<235 pmol/8 x10(8) RBC. Ten patients had no clinical response at 6 months, yet achieved either therapeutic IBD 6-TGN levels (>235, n=4) or received maximum AZA dose>or=3.5 mg/kg (n=6). In 19 patients the drug was discontinued prematurely due to side effects or SLE activity. For those patients in whom either liver function test or white blood cell count abnormalities were encountered, metabolites guided attribution to drug or disease activity., Conclusion: Clinical responses in SLE can occur at levels of 6-TGN lower than the target range established for IBD. During followup, measurements of AZA metabolites may provide a rational approach to safety.

Background and Aim: There have been no reports on 6-thioguanine nucleotide (6-TGN) concentrations in Japanese patients with inflammatory bowel disease (IBD) undergoing azathioprine (AZA) or 6-mercaptopurine (6-MP) therapy. The aim of this study was to assess 6-TGN concentrations in Japanese IBD patients., Methods: Eighty-three patients with Crohn's disease (n = 42) and ulcerative colitis (n = 41) were enrolled. In 69 patients, AZA was prescribed at 50 mg/day, and seven patients were given 75 (n = 5) or 100 mg/day (n = 2). 6-MP was administered at 30 mg/day (n = 7). The 6-TGN concentrations were then assayed by high-performance liquid chromatography., Results: The mean 6-TGN concentrations of the entire study population (n = 83) were 277.9 +/- 179.8 pmol/8 x 10(8) red blood cells (RBC). The mean 6-TGN concentrations in those patients with active disease (n = 38) and those in remission (n = 45) were 232.9 +/- 159.7(mean +/- SD) and 342.8 +/- 184.6 pmol/8 x 10(8) RBC, respectively (P < 0.05). The odds ratio of being in remission and having a 6-TGN value >235 pmol/8 x 10(8) RBC was 2.6 (95% CI 1.05-6.2). A significant inverse correlation was found between the white blood cell (WBC) counts and 6-TGN concentrations (r = -0.301, P < 0.05, n = 83); the mean WBC counts of the active patients (6780 +/- 2412) were significantly higher than the patients in clinical remission (5468 +/- 1920, P < 0.05). Three patients with severe leukopenia and 10 patients with high 6-TGN concentrations had no thiopurine S-methyl transferase mutations., Conclusion: The 6-TGN concentrations in Japanese patients with IBD on low-dose AZA and 6-MP therapy were comparable to those reported from Western countries. The monitoring of 6-TGN concentrations may be helpful for developing a therapeutic strategy for Japanese IBD patients.

Background: 6-Thioguanine nucleotides (6-TGN) are active metabolites of azathioprine (AZA) and 6-mercaptopurine (6MP). Higher remission rates have been observed in patients with higher 6-TGN levels. However, many physicians prescribe AZA/6MP using milligrams per kilogram (mg/kg) dosing regimens without measuring 6-TGN levels. The aim of this study was to examine the association between 6MP dose and 6-TGN levels., Methods: We conducted a cross-sectional study of patients treated for inflammatory bowel disease (IBD) with AZA or 6MP, whose 6-TGN levels were measured. Patients with low or intermediate thiopurine methyl transferase (TPMT) activity were excluded. AZA dose was converted to 6MP equivalents. The relationship between dose and 6-TGN levels was assessed with the Spearman correlation coefficient. We used logistic regression to assess the relationship between dose and 6-TGN levels of >230 pmol/8 x 10(8)., Results: In this study, 155 patients met our inclusion criteria (median dose, 1.01 +/- 0.40; range, 0.61-1.41 mg/kg). There was a weak correlation between 6-TGN levels and the absolute dose (rho = 0.18, P = 0.04) and the dose in mg/kg (rho = 0.19, P = 0.03). The correlation between mg/kg dosage and 6-TGN levels was slightly stronger in those using concomitant 5-aminosalicylate (5-ASA) medications (rho = 0.24, P = 0.02). Compared with <1.0 mg/kg per day, doses of > or =1.5 mg/kg per day were strongly associated with 6-TGN levels of >230 pmol/8 x 10(8) RBC (adjusted odds ratio [OR] = 3.7, 95% confidence interval [CI] = 1.1-11.8). However, only 37% of patients receiving > or =1.0 mg/kg per day had 6-TGN levels of >230 pmol/8 x 10(8) RBC., Conclusions: 6MP dose is weakly associated with 6-TGN levels. The use of standard mg/kg dosing regimens will result in low 6-TGN levels in most patients.

Aims: To determine infant exposure to 6-thioguanine and 6-methylmercaptopurine nucleotides (6-TGN and 6-MMPN, respectively) during maternal use of azathioprine in breastfeeding., Methods: Mother-infant pairs provided blood for determination of 6-TGN and 6-MMPN concentrations, and TPMT genotype., Results: Four women taking azathioprine 1.2-2.1 mg kg(-1) day(-1) and their infants were studied. All had the wild-type TPMT genotype. Maternal 6-TGN and 6-MMPN concentrations ranged from 234 to 291 and 284 to 1178 pmol per 8 x 10(8) red blood cells, respectively, and were consistent with those associated with improved therapeutic outcomes. Neither 6-TGN nor 6-MMPN was detected in any of the infants, despite a sensitive assay., Conclusions: The data suggest that azathioprine may be 'safe' during breastfeeding in patients with the wild-type TPMT genotype ( approximately 90% of caucasian patients) taking 'normal' doses.