Influence of TPMT and NUDT15 Genetic Polymorphisms on Mercaptopurine Pharmacokinetics in Healthy Volunteers.

Aims: This study aimed to investigate the impact of genetic polymorphisms of thiopurine methyltransferase (TPMT) and NUDT15 on pharmacokinetics profile of mercaptopurine in healthy adults in China. Methods: Blood samples were obtained from 45 healthy adult volunteers who were administered azathioprine. Genomic DNA was extracted and sequenced for TPMT and NUDT15. The plasma concentrations of 6-mercaptopurine (6-MP) were determined by ultra-performance liquid chromatography-tandem mass spectrometry. Finally, pharmacokinetic parameters were calculated based on the time-concentration curve. Results: Among the 45 healthy adult volunteers enrolled in the study, two TPMT allelic variants and three NUDT15 allelic variants were detected. In total, six genotypes were identified, including TPMT*1/*1&NUDT15*1/*1, TPMT*1/*1&NUDT15*1/*2, TPMT*1/*1&NUDT15*1/*9, TPMT*1/*1&NUDT15*2/*5, TPMT*1/*6&NUDT15*1/*2, and TPMT*1/*3&NUDT15*1/*2. The results indicated that Area Under Curve (AUC) of 6-MP in volunteers with TPMT*1/*3&NUDT15*1/*2 and TPMT*1/*6&NUDT15*1/*2 were 1.57-1.62-fold higher than in individuals carrying the wild type (TPMT*1/*1&NUDT15*1/*1). Compared with wild type, the half-life (T1/2) of TPMT*1/*6&NUDT15*1/*2 was extended by 1.98 times, whereas T1/2 of TPMT*1/*3&NUDT15*1/*2 decreased by 67%. The maximum concentration (Cmax) of TPMT*1/*3&NUDT15*1/*2 increased significantly by 2.15-fold, whereas the corresponding clearance (CL/F) decreased significantly by 58.75%. Conclusion: The findings of this study corroborate the notion that various genotypes of TPMT and NUDT15 can impact the pharmacokinetics of mercaptopurine, potentially offering foundational insights for personalized mercaptopurine therapy.

Innovating Thiopurine Therapeutic Drug Monitoring: A Systematic Review and Meta-Analysis on DNA-Thioguanine Nucleotides (DNA-TG) as an Inclusive Biomarker in Thiopurine Therapy.

BACKGROUND AND OBJECTIVE: Thioguanine (TG), azathioprine (AZA), and mercaptopurine (MP) are thiopurine prodrugs commonly used to treat diseases, such as leukemia and inflammatory bowel disease (IBD). 6-thioguanine nucleotides (6-TGNs) have been commonly used for monitoring treatment. High levels of 6-TGNs in red blood cells (RBCs) have been associated with leukopenia, the cutoff levels that predict this side effect remain uncertain. Thiopurines are metabolized and incorporated into leukocyte DNA. Measuring levels of DNA-incorporated thioguanine (DNA-TG) may be a more suitable method for predicting clinical response and toxicities such as leukopenia. Unfortunately, most methodologies to assay 6-TGNs are unable to identify the impact of NUDT15 variants, effecting mostly ethnic populations (e.g., Chinese, Indian, Malay, Japanese, and Hispanics). DNA-TG tackles this problem by directly measuring thioguanine in the DNA, which can be influenced by both TPMT and NUDT15 variants. While RBC 6-TGN concentrations have traditionally been used to optimize thiopurine therapy due to their ease and affordability of measurement, recent developments in liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques have made measuring DNA-TG concentrations in lymphocytes accurate, reproducible, and affordable. The objective of this systematic review was to assess the current evidence of DNA-TG levels as marker for thiopurine therapy, especially with regards to NUDT15 variants. METHODS: A systematic review and meta-analysis were performed on the current evidence for DNA-TG as a marker for monitoring thiopurine therapy, including methods for measurement and the illustrative relationship between DNA-TG and various gene variants (such as TPMT, NUDT15, ITPA, NT5C2, and MRP4). PubMed and Embase were systematically searched up to April 2024 for published studies, using the keyword "DNA-TG" with MeSH terms and synonyms. The electronic search strategy was augmented by a manual examination of references cited in articles, recent reviews, editorials, and meta-analyses. A meta-analysis was performed using R studio 4.1.3. to investigate the difference between the coefficients (Fisher's z-transformed correlation coefficient) of DNA-TG and 6-TGNs levels. A meta-analysis was performed using RevMan version 5.4 to investigate the difference in DNA-TG levels between patients with or without leukopenia using randomized effect size model. The risk of bias was assessed using the Newcastle-Ottowa quality assessment scale. RESULTS: In this systematic review, 21 studies were included that measured DNA-TG levels in white blood cells for either patients with ALL (n = 16) or IBD (n = 5). In our meta-analysis, the overall mean difference between patients with leukopenia (ALL + IBD) versus no leukopenia was 134.15 fmol TG/µg DNA [95% confidence interval (CI) (83.78-184.35), P < 0.00001; heterogeneity chi squared of 5.62, I2 of 47%]. There was a significant difference in DNA-TG levels for patients with IBD with and without leukopenia [161.76 fmol TG/µg DNA; 95% CI (126.23-197.29), P < 0.00001; heterogeneity chi squared of 0.20, I2 of 0%]. No significant difference was found in DNA-TG level between patients with ALL with or without leukopenia (57.71 fmol TG/µg DNA [95% CI (- 22.93 to 138.35), P < 0.80]). DNA-TG monitoring was found to be a promising method for predicting relapse rates in patients with ALL, and DNA-TG levels are likely a better predictor for leukopenia in patients with IBD than RBC 6-TGNs levels. DNA-TG levels have been shown to correlate with various gene variants (TPMT, NUDT15, ITPA, and MRP4) in various studies, points to its potential as a more informative marker for guiding thiopurine therapy across diverse genetic backgrounds. CONCLUSIONS: This systematic review strongly supports the further investigation of DNA-TG as a marker for monitoring thiopurine therapy. Its correlation with treatment outcomes, such as relapse-free survival in ALL and the risk of leukopenia in IBD, underscores its role in enhancing personalized treatment approaches. DNA-TG effectively identifies NUDT15 variants and predicts late leukopenia in patients with IBD, regardless of their NUDT15 variant status. The recommended threshold for late leukopenia prediction in patients with IBD with DNA-TG is suggested to be between 320 and 340 fmol/µg DNA. More clinical research on DNA-TG implementation is mandatory to improve patient care and to improve inclusivity in thiopurine treatment.

Modulation of mercaptopurine intestinal toxicity and pharmacokinetics by gut microbiota.

The interaction between the gut microbiota and mercaptopurine (6-MP), a crucial drug used in pediatric acute lymphoblastic leukemia (ALL) treatment, has not been extensively studied. Here we reveal the significant perturbation of gut microbiota after 2-week 6-MP treatment in beagles and mice followed by the functional prediction that showed impairment of SCFAs production and altered amino acid synthesis. And the targeted metabolomics in plasma also showed changes in amino acids. Additionally, targeted metabolomics analysis of feces showed changes in amino acids and SCFAs. Furthermore, ablating the intestinal microbiota by broad-spectrum antibiotics exacerbated the imbalance of amino acids, particularly leading to a significant decrease in the concentration of S-adenosylmethionine (SAM). Importantly, the depletion of gut microbiota worsened the damage of small intestine caused by 6-MP, resulting in increased intestinal permeability. Considering the relationship between toxicity and 6-MP metabolites, we conducted a pharmacokinetic study in pseudo germ-free rats to confirm that gut microbiota depletion altered the methylation metabolites of 6-MP. Specifically, the concentration of MeTINs, a secondary methylation metabolite, showed a negative correlation with SAM, the pivotal methyl donor. Additionally, we observed a strong correlation between Alistipes and SAM levels in both feces and plasma. In conclusion, our study demonstrates that 6-MP disrupts the gut microbiota, and depleting the gut microbiota exacerbates 6-MP-induced intestinal toxicity. Moreover, SAM derived from microbiota plays a crucial role in influencing plasma SAM and the methylation of 6-MP. These findings underscore the importance of comprehending the role of the gut microbiota in 6-MP metabolism and toxicity.

Bioequivalence of Two 6-Mercaptopurine Tablet Formulations in Healthy Fasting Chinese Volunteers.

The supply of branded 6-mercaptopurine (6-MP) is limited in China, necessitating the local production and clinical evaluation of generic alternatives. We evaluated the in vivo bioequivalence (BE) of a new generic mercaptopurine tablet (50 mg) formulation by comparing peak plasma concentration and area under the concentration-time curve (AUC) with a branded 6-MP formulation as the reference in 36 healthy fasting Chinese adults. The in vivo BE was evaluated by the average BE test. The safety parameters of the test and reference formulations were also evaluated. The geometric mean ratios for AUC over the dosing interval and AUC from time zero to infinity were 104% and 104%, respectively, of the reference values, while the point estimate of the geometric mean ratio for peak plasma concentration was 104% of the reference value. The test and reference formulations in this study were both deemed safe as only 23 Grade 1 adverse events were observed in 13 of 36 subjects. The test and reference formulations of 6-MP tablets meet the regulatory criteria for BE in healthy fasting Chinese adults.

Adverse Events of Thiopurine Therapy in Pediatric Inflammatory Bowel Disease and Correlations with Metabolites: A Cohort Study.

BACKGROUND: In the recent era of growing availability of biological agents, the role of thiopurines needs to be reassessed with the focus on toxicity. AIMS: We assessed the incidence and predictive factors of thiopurine-induced adverse events (AE) resulting in therapy cessation in pediatric inflammatory bowel disease (IBD), related to thiopurine metabolites and biochemical abnormalities, and determined overall drug survival. METHODS: We performed a retrospective, single-center study of children diagnosed with IBD between 2000 and 2019 and treated with thiopurine therapy. The incidence of AE and overall drug survival of thiopurines were evaluated using the Kaplan-Meier method. Correlations between thiopurine metabolites and biochemical tests were computed using Spearman's correlation coefficient. RESULTS: Of 391 patients with IBD, 233 patients (162 Crohn's disease, 62 ulcerative colitis, and 9 IBD-unclassified) were prescribed thiopurines (230 azathioprine and 3 mercaptopurine), of whom 50 patients (22%) discontinued treatment, at least temporary, due to thiopurine-induced AE (median follow-up 20.7 months). Twenty-six patients (52%) were rechallenged and 18 of them (70%) tolerated this. Sixteen patients (6%) switched to a second thiopurine agent after azathioprine intolerance and 10 of them (63%) tolerated this. No predictive factors for development of AE could be identified. Concentrations of 6-thioguanine nucleotides (6-TGN) were significantly correlated with white blood cell and neutrophil count, 6-methylmercaptopurine (6-MMP) concentrations with alanine aminotransferase and gamma-glutamyltranspeptidase. CONCLUSIONS: Approximately 20% of pediatric patients with IBD discontinued thiopurine treatment due to AE. A rechallenge or switch to mercaptopurine is an effective strategy after development of AE. Concentrations of 6-TGN and 6-MMP are associated with biochemical abnormalities.

Understanding thiopurine methyltransferase polymorphisms for the targeted treatment of hematologic malignancies.

INTRODUCTION: Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines (mercaptopurine (MP) and tioguanine (TG)), chemotherapeutic agents used in the treatment of acute lymphoblastic leukemia (ALL). Polymorphisms in TPMT gene encode diminished activity enzyme, enhancing accumulation of active metabolites, and partially explaining the inter-individual differences in patients' clinical response. AREAS COVERED: This review gives an overview on TPMT gene and function, and discusses the pharmacogenomic implications of TPMT variants in the prevention of severe thiopurine-induced hematological toxicities and the less known implication on TG-induced sinusoidal obstruction syndrome. Additional genetic and non-genetic factors impairing TPMT activity are considered. Literature search was done in PubMed for English articles published since1990, and on PharmGKB. EXPERT OPINION: To titrate thiopurines safely and effectively, achieve the right degree of lymphotoxic effect and avoid excessive myelosuppression, the optimal management will combine a preemptive TPMT genotyping to establish a safe initial dose with a close phenotypic monitoring of TPMT activity and/or of active metabolites during long-term treatment. Compared to current ALL protocols, replacement of TG by MP during reinduction phase in TPMT heterozygotes and novel individualized TG regimens in maintenance for TPMT wild-type subjects could be investigated to improve outcomes while avoiding risk of severe hepatotoxicity.

Maintenance therapy and risk of osteonecrosis in children and young adults with acute lymphoblastic leukemia: a NOPHO ALL2008 sub-study.

PURPOSE: Osteonecrosis is a burdensome treatment-related toxicity that is mostly diagnosed during or soon after 6-mercaptopurine (6MP)/methotrexate (MTX) maintenance therapy for acute lymphoblastic leukemia (ALL), possibly indicating a pathogenic role of these drugs. METHODS: We prospectively registered symptomatic osteonecrosis during treatment of 1234 patients aged 1.0-45.9 years treated according to the Nordic Society of Hematology and Oncology (NOPHO) ALL2008 protocol. MTX/6MP metabolites were measured as part of the NOPHO ALL2008 maintenance therapy study. RESULTS: After a median follow-up of 5.6 years [interquartile range (IQR) 3.6-7.5], 68 patients had been diagnosed with symptomatic osteonecrosis. The cumulative incidence was 2.7% [95% confidence interval (CI) 1.6-3.8%] for patients aged < 10 years, 14.9% (95% CI 9.7-20.2%) for patients aged 10.0-17.9 years, and 14.4% (95% CI 8.0-20.8%) for patients aged ≥ 18 years. The median time from diagnosis of ALL to diagnosis of osteonecrosis in these age groups was 1.0 year (IQR 0.7-2.0), 2.0 years (IQR 1.1-2.4), and 2.2 years (IQR 1.8-2.8), respectively (p = 0.001). With 17,854 blood samples available for MTX and 6MP metabolite analysis, neither erythrocyte levels of 6-thioguanine (TG) nucleotides (p > 0.99), methylated 6MP metabolites (p = 0.37), MTX polyglutamates (p = 0.98) nor DNA-TG (p = 0.53) were significantly associated with the hazard of osteonecrosis in Cox models stratified by the three age groups and adjusted for sex. CONCLUSION: Maintenance therapy intensity determined by 6MP and MTX metabolites was not associated with the risk of developing osteonecrosis in the NOPHO ALL2008 cohort.

Preparation, Characterization, Pharmacokinetic, and Therapeutic Potential of Novel 6-Mercaptopurine-Loaded Oral Nanomedicines for Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children. It requires a long and rigorous course of chemotherapy treatments. 6-Mercaptopurine (6-MP) is one of the primary drugs used in chemotherapy. Unfortunately, its efficacy has been limited due to its insolubility, poor bioavailability and serious adverse effects. To overcome these drawbacks, we constructed 6-mercaptopurine (6-MP)-loaded nanomedicines (6-MPNs) with biodegradable poly(lactide-co-glycolide) (PLGA) to enhance the anticancer efficacy of 6-MP. METHODS: We prepared the 6-MPNs using a double-emulsion solvent evaporation method, characterizing them for the physicochemical properties. We then investigated the plasma, intestinal region and other organs in Sprague Dawley (SD) rats for pharmacokinetics. Additionally, we evaluated its anticancer efficacy in vitro on the human T leukemia cell line Jurkat and in vivo on the ALL model mice. RESULTS: The 6-MPNs were spherical in shape with uniform particle size and high encapsulation efficiency. The in vitro release profile showed that 6-MPNs exhibited a burst release that a sustained release phase then followed. The apoptosis assay demonstrated that 6-MPNs could improve the in vitro cytotoxicity in Jurkat cells. Pharmacokinetics profiles revealed that 6-MPNs had improved oral bioavailability. Tissue distribution experiments indicated that 6-MPNs increased the duodenum absorption of 6-MP, at the same time having a low accumulation of the toxic metabolites of 6-MP. The in vivo pharmacodynamics study revealed that 6-MPNs could prolong the survival time of the ALL model mice. The prepared 6-MPNs, therefore, have superior properties in terms of anticancer efficacy against ALL with reduced systemic toxicity. CONCLUSION: Our nanomedicines provide a promising delivery strategy for 6-MP; they offer a simple preparation method and high significance for clinical translation.

Hybrid modelling of biological systems using fuzzy continuous Petri nets.

Integrated modelling of biological systems is challenged by composing components with sufficient kinetic data and components with insufficient kinetic data or components built only using experts' experience and knowledge. Fuzzy continuous Petri nets (FCPNs) combine continuous Petri nets with fuzzy inference systems, and thus offer an hybrid uncertain/certain approach to integrated modelling of such biological systems with uncertainties. In this paper, we give a formal definition and a corresponding simulation algorithm of FCPNs, and briefly introduce the FCPN tool that we have developed for implementing FCPNs. We then present a methodology and workflow utilizing FCPNs to achieve hybrid (uncertain/certain) modelling of biological systems illustrated with a case study of the Mercaptopurine metabolic pathway. We hope this research will promote the wider application of FCPNs and address the uncertain/certain integrated modelling challenge in the systems biology area.

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.

Systematic Review and Meta-Analysis of Thiopurine Metabolite Levels and Biochemical Remission in Autoimmune Hepatitis.

BACKGROUND: The utility of measuring thiopurine metabolites (TM) to individualize therapy in autoimmune hepatitis (AIH) has not been defined, and the evidence regarding its use in clinical practice is heterogeneous. This systematic review and meta-analysis aimed to compare the mean concentration of TM between patients in biochemical remission and those not in remission. METHODS: A systematic literature search was conducted using PubMed, Scopus, the Cochrane Library, and Google Scholar for keywords related to TM and AIH. Two reviewers independently searched and selected studies comparing the levels of 6-methyl mercaptopurine (6-MMP) and 6-thioguanine nucleotide (6-TGN) and their ratio in cases of AIH in remission and otherwise. Meta-analysis was performed by calculating the weighted mean difference using the inverse variance heterogeneity model. RESULTS: A total of 1066 records were identified through systematic search; of which, 7 (n = 3 pediatric, n = 4 adults) were considered for inclusion, and 442 TM measurements (n = 128 in children) were analyzed. Mean 6-TGN levels were significantly higher among patients in remission than in those who were not, with a pooled weighted mean difference (WMD) of 15.67 [95% confidence interval (CI), 6.68-24.66] pmol/8 × 108 red blood cells (RBC). The difference was higher in the pediatric age group (WMD, 56.11; 95% CI, 13.60-98.62) than in adults (WMD, 13.77; 95% CI, 4.58-22.97). There was no significant difference in the 6-MMP levels (WMD, -431.7; 95% CI, -1237.4 to 373.9 pmol/8 × 108 RBC; I2 = 82%; n = 3 studies) or 6-MMP/6-TGN ratio among the patients who were in biochemical remission and those who were not (WMD, -0.97; 95% CI, -5.77 to 3.84; I2 = 82%; n = 3 studies). CONCLUSIONS: This meta-analysis suggests a link between 6-TGN levels and biochemical remission in AIH. Further high-quality studies are required to determine the therapeutic cutoff of 6-TGN.

Pharmacokinetics of tablet and liquid formulations of oral 6-mercaptopurine in children with acute lymphoblastic leukemia.

PURPOSE: Mercaptopurine (6MP) is essential to cure childhood acute lymphoblastic leukemia (ALL). A liquid 6MP formulation was recently introduced to facilitate oral 6MP administration, especially to children. Its approval and bioequivalence with 6MP tablet were based on comparative pharmacokinetics in 60 healthy adults. Due to potential pharmacokinetic differences between healthy adults and children with ALL, we compared pharmacokinetics of tablet and liquid 6MP formulations in children with ALL. METHODS: Pharmacokinetics of 50 mg 6MP tablet (Puri-Nethol®) and 20 mg/ml 6MP liquid suspension (Xaluprine®) were compared in a non-blinded, random order, single-dose, cross-over study in 16 children with ALL (eight males). 6MP was administered after a 12 h fast, and 6MP plasma concentrations measured consecutively over seven hours post-dose. Pharmacokinetic outcomes were as follows: Area under the curve (AUC), maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax), and terminal half-life (T½). RESULTS: Liquid 6MP formulation resulted in a 26% lower AUC (p = 0.02) compared with tablet (median 1215 vs. 1805 h × nmol/l). No significant differences were observed for Cmax,Tmax and T½ (p = 0.28, p = 0.09, p = 0.41, respectively). Based on criteria declared by the World Health Organization the results did not establish non-inferiority of liquid 6MP formulation compared with 6MP tablet. CONCLUSION: Non-inferiority of liquid 6MP formulation compared with 6MP tablet was not demonstrated. Yet, maintenance therapy doses are adjusted by degree of myelosuppression and not by 6MP dose. Thus, in spite of a lower bioavailability, a liquid 6MP formulation is still desirable in a clinical setting, especially for children. However, if shifting between 6MP formulation is indicated, dose adjustments should be anticipated to maintain equivalent treatment intensity in children with ALL. The study is registered on clinicaltrials.gov (NCT01906671). Date of registration: 24.07.13.

Outcome of concomitant treatment with thiopurines and allopurinol in patients with inflammatory bowel disease: A nationwide Danish cohort study.

BACKGROUND: Thiopurine and allopurinol in combination are associated with clinical remission in inflammatory bowel diseases but their influence on subsequent outcomes is unclear. We compared outcomes during exposure to both thiopurines and allopurinol versus thiopurines alone. METHODS: We established a nationwide cohort of patients with inflammatory bowel diseases exposed to thiopurines ± allopurinol during 1999-2014, using registry data. Patients were followed until hospitalization, surgery, anti-TNFα, or death (as a primary composite outcome). We used Poisson regression analyses to calculate incidence rate ratios overall and stratified by calendar period (assuming the combined exposure was unintended before 2009). RESULTS: A total of 10,367 patients with inflammatory bowel diseases (Crohn's disease, n = 5484; ulcerative colitis, n = 4883) received thiopurines. Of these, 217 (2.1%) also received allopurinol. During 24,714 person years of follow-up, we observed 40 outcomes among thiopurine-allopurinol-exposed patients, and 4745 outcomes among those who were thiopurine exposed; incidence rate ratio, 1.26 (95% confidence interval, 0.92-1.73). The incidence rate ratios decreased over time: 4.88 (95% confidence interval 2.53-9.45) for 1999-2003, 2.19 (95% confidence interval, 1.17-4.09) for 2004-2008 and 0.80 (95% confidence interval, 0.52-1.23) for 2009-2014. CONCLUSION: Our nationwide inflammatory bowel disease cohort study shows that concomitant thiopurine-allopurinol is as safe to use as thiopurines alone, with a tendency towards a positive effect on clinical outcomes in recent calendar periods when combined use was intended.

[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.

PACSIN2 rs2413739 influence on thiopurine pharmacokinetics: validation studies in pediatric patients.

The aim of the study was to validate the impact of the single-nucleotide polymorphism rs2413739 (T > C) in the PACSIN2 gene on thiopurines pharmacological parameters and clinical response in an Italian cohort of pediatric patients with acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD). In ALL, PACSIN2 rs2413739 T allele was associated with a significant reduction of TPMT activity in erythrocytes (p = 0.0094, linear mixed-effect model, multivariate analysis considering TPMT genotype) and increased severe gastrointestinal toxicity during consolidation therapy (p = 0.049). A similar trend was present also for severe hematological toxicity during maintenance. In IBD, no significant effect of rs2413739 could be found on TPMT activity, however azathioprine effectiveness was reduced in patients carrying the T allele (linear mixed effect, p = 0.0058). In PBMC from healthy donors, a positive correlation between PACSIN2 and TPMT protein concentration could be detected (linear mixed effect, p = 0.045). These results support the role of PACSIN2 polymorphism on TPMT activity and mercaptopurine adverse effects in patients with ALL. Further evidence on PBMC and pediatric patients with IBD supports an association between PACSIN2 variants, TPMT activity, and thiopurines effects, even if more studies are needed since some of these effects may be tissue specific.

Prediction of Thiopurine Metabolite Levels Based on Haematological and Biochemical Parameters.

OBJECTIVES: Therapeutic drug monitoring of thiopurine erythrocyte levels is not available in all centers and it usually requires quite a long time to obtain the results. The aims of this study were to build a model predicting low levels of 6-thioguanine and 6-methylmercaptopurine in pediatric inflammatory bowel disease (IBD) patients and to build a model to predict nonadherence in patients treated with azathioprine (AZA). METHODS: The study consisted of 332 observations in 88 pediatric IBD patients. Low AZA dosing was defined as 6-thioguanine levels <125 pmol/8 × 10 erythrocytes and 6-methylmercaptopurine levels <5700 pmol/8 × 10 erythrocytes. Nonadherence was defined as undetectable levels of 6-thioguanine and 6-methylmercaptopurine <240 pmol/8 × 10 erythrocytes. Data were divided into training and testing part. To construct the model predicting low 6-thioguanine levels, nonadherence, and the level of 6-thioguanine, the modification of random forest method with cross-validation and resampling was used. RESULTS: The final models predicting low 6-thioguanine levels and nonadherence had area under the curve, 0.87 and 0.94; sensitivity, 0.81 and 0.82; specificity, 0.80 and 86; and distance, 0.31 and 0.21, respectively, when applied on the testing part of the dataset. When the final model for prediction of 6-thioguanine values was applied on testing dataset, a root-mean-square error of 110 was obtained. CONCLUSIONS: Using easily obtained laboratory parameters, we constructed a model with sufficient accuracy to predict patients with low 6-thioguanine levels and a model for prediction of AZA treatment nonadherence (web applications: https://hradskyo.shinyapps.io/6TG_prediction/ and https://hradskyo.shinyapps.io/Non_adherence/).

Systematic review with meta-analysis: risk factors for thiopurine-induced leukopenia in IBD.

BACKGROUND: Thiopurine-induced leukopenia, a frequently observed and potentially life-threatening adverse event, complicates the clinical management of IBD patients. AIM: To assess risk factors for thiopurine-induced leukopenia in IBD. METHODS: MEDLINE, EMBASE, BIOSIS and Cochrane library were searched for studies reporting at least one risk factor for thiopurine-induced leukopenia. Pooled odds ratio (OR) was calculated for each potential risk factor using a random effects model. Studies that were not eligible for meta-analysis were described qualitatively. RESULTS: Seventy articles were included, 34 (11 229 patients) were included in meta-analyses. A significantly higher thiopurine-induced leukopenia risk was found for TPMT (OR 3.9, 95% [CI] 2.5-6.1) and for NUDT15 R139C (OR 6.9, 95% CI 5.2-9.1), G52A (OR 3.2, 95% CI 1.3-7.9) and 36_37ins/delGGAGTC variant carriers (OR 5.6, 95% CI 2.8-11.4). A potential association between high 6-thioguanine nucleotides (6-TGN) or 6-methylmercaptopurine (6-MMP) levels and leukopenia was observed, since most studies reported higher metabolite levels in leukopenic patients (6-TGN: 204-308 (Lennard method) and 397 (Dervieux method), 6-MMP: 4020-10 450 pmol/8 x 108 RBC) compared to controls (6-TGN: 170-212 (Lennard method) and 269 (Dervieux method), 6-MMP: 1025-4550 pmol/8 x 108 RBC). CONCLUSIONS: TPMT and NUDT15 variants predict thiopurine-induced leukopenia. High 6-TGN and 6-MMP levels might induce leukopenia, although exact cut-off values remain unclear. Potential preventive measures to reduce the risk of thiopurine-induced leukopenia include pre-treatment TPMT and NUDT15 genotyping. Routine thiopurine metabolite measurement might be efficient, yet cut-off levels must be validated in advance.

Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter.

6-Mercaptopurine (6-MP) is a nucleobase analog used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disorders. However, the mechanisms underlying its transport into target cells have remained elusive. The protein encoded by SLC43A3_1 [equilibrative nucleobase transporter 1 (ENBT1)] has recently been shown to transport endogenous nucleobases. A splice variant (SLC43A3_2), encoding a protein with 13 additional amino acids in the first extracellular loop, is also expressed but its function is unknown. We hypothesized that 6-MP is a substrate for both variants of ENBT1. Human embryonic kidney 293 (HEK293) cells (lacking endogenous ENBT1 activity) were transfected with each of the coding region variants of SLC43A3. ENBT1 function was assessed via the rate of flux of [3H]adenine and [14C]6-MP across the plasma membrane. Both SLC43A3 variants encoded proteins with similar functional properties. [14C]6-MP and [3H]adenine had K m values (±S.D.) of 163 ± 126 and 37 ± 26 µM, respectively, for this system. Decynium-22, 6-thioguanine, and 6-methylmercaptopurine inhibited 6-MP uptake with K i values of 1.0 ± 0.4, 67 ± 30, and 73 ± 20 µM, respectively. ENBT1 also mediated adenine-sensitive efflux of 6-MP from the SLC43A3-HEK293 cells. MRP4 also contributed to the efflux of 6-MP in this model, but was less efficient than ENBT1 in this regard. Furthermore, transfection of HEK293 cells with SLC43A3 increased the sensitivity of the cells to the cytotoxic effects of 6-MP by more than 7-fold. Thus, both variants of ENBT1 are key players in the transfer of 6-MP into and out of cells, and changes in SLC43A3 expression impact 6-MP cytotoxicity.

pH-Dependent 5-Aminosalicylates Releasing Preparations Do Not Affect Thiopurine Metabolism.

BACKGROUND/AIMS: Thiopurines are key drugs in maintenance therapy for treating inflammatory bowel disease (IBD). Time-dependent 5-aminosalicylates (5-ASA) releasing preparations (time-dependent 5-ASA) increase 6-thioguanine nucleotide (6-TGN), an active metabolite of thiopurines. However, the effects of pH-dependent 5-ASA releasing preparations (pH-dependent 5-ASA) on thiopurine metabolism were not reported. METHODS: We conducted a retrospective study of 134 IBD patients who received thiopurine treatment. The 6-methylmercaptopurine (6-MMP)/6-TGN values after taking the same dose of thiopurine preparations for at least 28 days were included. RESULTS: There was a significant decrease in the 6-MMP/6-TGN ratio in time-dependent 5-ASA compared with group without 5-ASA preparations and the pH-dependent 5-ASA group (p = 0.008 and < 0.001 respectively). Spearman's rank correlation coefficient indicated a negative relationship between the daily oral dose of time-dependent 5-ASA and the 6-MMP/6-TGN ratio (r = -0.362, p = 0.003). Multivariate logistic regression analysis was performed in the groups with 6-MMP/6-TGN ratios of 1 or more and less than 1. The use of time-dependent 5-ASA and concomitant allopurinol negatively affected the independent 6-MMP/6-TGN ratio (p = 0.006 and 0.007 respectively). CONCLUSION: Our study revealed that time-dependent but not pH-dependent 5-ASA decreases the 6-MMP/6-TGN ratio. We also confirmed that concomitant allopurinol results in a low 6-MMP/6TGN ratio.

A mathematical model of white blood cell dynamics during maintenance therapy of childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the most common malignancy in childhood and requires prolonged oral maintenance chemotherapy to prevent disease relapse after remission induction with intensive intravenous chemotherapy. In maintenance therapy, drug doses of 6-mercaptopurine (6-MP) and methotrexate (MTX) are adjusted to achieve sustained antileukemic activity without excessive myelosuppression. However, uncertainty exists regarding timing and extent of drug dose responses and optimal dose adaptation strategies. We propose a novel comprehensive mathematical model for 6-MP and MTX pharmacokinetics, pharmacodynamics and myelosuppression in acute lymphoblastic maintenance therapy. We personalize and cross-validate the mathematical model using clinical data and propose a real-time algorithm to predict chemotherapy responses with a clinical decision support system as a potential future application.