6-thioguanine Nucleotides Research Articles

Usefulness of Measuring Thiopurine Metabolites in Children with Inflammatory Bowel Disease and Autoimmunological Hepatitis, Treated with Azathioprine.

Introduction Thiopurines, such as azathioprine (AZA) and 6-mercaptopurine (6-MP), are immunomodulatory agents, used for the maintenance of remission in children with inflammatory bowel disease (IBD)—Crohn's disease (CD) and ulcerative colitis (UC), as well as with autoimmunological hepatitis (AIH). Measurements of thiopurine metabolites may allow identifying patients at risk for toxicity and nonadherence. It can also provide an explanation for the ineffectiveness of the treatment, observed in some patients. Patients and Methods. A retrospective analysis was carried out of sixty-eight patients (thirty-six patients with CD, eighteen with UC, and fourteen with AIH), treated with AZA. Thiopurine metabolites, 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP), were assayed by high-performance liquid chromatography (HPLC), and the AZA dose was adjusted when 6-TGN concentration was known. Result Only twenty-five (41%) children had therapeutic 6-TGN concentrations, ten (16%) subjects had suboptimal 6-TGN concentrations, and twenty-six subjects (43%) had 6-TGN concentrations above the recommended therapeutic range. 6-MMP was not above the therapeutic range in any case. Seven subjects revealed undetectable 6-TGN and 6-MMP levels, indicating nonadherence. The mean AZA dose after the 6-TGN concentration-related adjustment did not differ, in comparison to the initial dose, either in IBD or AIH groups. The mean AZA dose was lower in AIH than in IBD. The subjects with an optimal 6-TGN level presented with a higher ratio of remission (88%) than the under- or overdosed patients (60% and 69%), respectively (Chi − square test = 3.87, p < 0.05). Conclusion Timely measurements of thiopurine metabolites can be a useful tool to identify nonadherent patients before a decision is taken to switch to another drug. We may also spot the patients who receive either too low or too high doses, compensating dose deviations in an appropriate way. The patients with optimal 6-TGN levels presented a higher percentage of remission than the under- or overdosed patients. In most patients, both initial and adjusted AZA doses, lower than suggested in guidelines, appeared to be sufficient to maintain remission.

Increments in DNA-thioguanine level during thiopurine-enhanced maintenance therapy of acute lymphoblastic leukemia.

Maintenance therapy containing methotrexate and 6-mercapto - purine is essential to cure acute lymphoblastic leukemia (ALL). Cytotoxicity is elicited by incorporation of thioguanine nucleotides into DNA (DNA-TG), and higher leukocyte DNA-TG is associated with increased relapse-free survival. As 6-thioguanine provides 6- fold higher cytosolic levels of thioguanine nucleotides than does 6- mercapto purine, we added low-dose 6-thioguanine to methotrexate/6- mercapto purine maintenance therapy to explore if this combination results in significantly higher DNA-TG. The target population of the “Thiopurine Enhanced ALL Maintenance therapy” (TEAM) study was 30 patients with non-high-risk ALL, aged 1-45 years on methotrexate/6-mercaptopurine maintenance therapy receiving no other systemic chemotherapy. Incremental doses of 6-thioguanine were added to methotrexate/6-mercaptopurine maintenance therapy (starting 6-thioguanine dose: 2.5 mg/m2/day, maximum: 12.5 mg/m2/day). The primary endpoint was DNA-TG increments. Thirty-four patients were included, and 30 patients completed maintenance therapy according to the TEAM strategy. Of these 30 patients, 26 (87%) tolerated 10.0-12.5 mg/m2/day as the maximum 6-thioguanine dose. TEAM resulted in significantly higher DNA-TG levels compared to those in both TEAM patients before their inclusion in TEAM (on average 251 fmol/mg DNA higher [95% confidence interval: 160-341; P<0.0001]), and with historical patients receiving standard methotrexate/6-mercapto - purine maintenance therapy (on average 272 fmol/mg DNA higher [95% confidence interval: 147-398; P<0.0001]). TEAM did not increase myelotoxicity or hepatotoxicity. In conclusion, TEAM is an innovative and feasible approach to improve maintenance therapy and results in higher DNA-TG levels without inducing additional toxicity. It may therefore be an effective strategy to reduce the risk of ALL relapse through increased DNA-TG. This will be tested in a randomized ALLTogether-1 substudy.

Towards personalised medicine in autoimmune hepatitis: Measurement of thiopurine metabolites results in higher biochemical response rates

Patients with autoimmune hepatitis (AIH) usually receive maintenance therapy with thiopurines, such as azathioprine (AZA) or mercaptopurine. Genetic polymorphisms in AZA metabolism can lead to variations in thioguanine nucleotide (TGN) and 6-methylmercaptopurine, both of which can cause adverse drug reactions (ADRs). In inflammatory bowel disease, a therapeutic TGN range (225-450 pmol/8x108 erythrocytes) has been identified to optimise effectiveness. We evaluated the benefits of a personalised medicine approach to thiopurine dosing, in comparison to standard weight-based dosing. A retrospective matched cohort study of 214 patients with AIH who were seen at King's College between 1999-2019 was performed. Metabolite levels were measured in 109 patients. The control group included 105 patients on weight-based thiopurine dosing with no metabolite monitoring. Biochemical response (BR) occurred more frequently at 6-month follow-up in patients with metabolite monitoring compared to those on a weight-based regimen (77% vs. 60%, p= 0.008). This remained true with data analysis based on clinicians who measure metabolites and those who do not (BR at 6 months: 84% vs. 64%, p= 0.016). Patients with BR had TGN levels within the therapeutic range of 225-450 pmol/8x108 erythrocytes significantly more often than those who failed to achieve or lost BR (40% vs. 13%, p <0.0001). Moreover, TGN levels within the pre-defined therapeutic range predicted more stable disease within 6 months of testing compared to levels outside the range (p <0.0001). A high proportion of patients with sub-therapeutic TGN levels (75-225 pmol/8x108 erythrocytes) remained in BR (75% vs. 81%, p= 0.589) with fewer ADRs (44% vs. 86%, p= 0.0002) when compared to patients with therapeutic TGN levels. A strategy of personalised medicine using metabolite levels can optimise treatment regimens in AIH, resulting in fewer ADRs whilst maintaining BR. This study looked to see if measuring the breakdown products of a medication used in autoimmune hepatitis increases the chances of gaining good control of the disease, when compared to a group of patients who were on a dose of this medication based on their weight. A group of 214 patients with autoimmune hepatitis were split into 2 groups: roughly half had their medication dose adjusted according to measurements of breakdown products in the blood, whilst the other half received their weight-based dose as normal. The results confirmed that using a personalised approach and checking drug breakdown products resulted in fewer side effects and potentially improved control of disease.

Association of genetic variants in TPMT, ITPA, and NUDT15 with azathioprine-induced myelosuppression in southwest china patients with autoimmune hepatitis

This study aimed to investigate the influence of TPMT*3C, ITPA, NUDT15, and 6-thioguanine nucleotides (6-TGN) on azathioprine (AZA)-induced myelosuppression in Southwest China patients with autoimmune hepatitis (AIH). A total of 113 Chinese patients with AIH receiving AZA maintenance treatment were evaluated. The relevant clinical data of the patients were collected from the hospital information system. Genotyping of TPMT*3C(rs1142345), ITPA (rs1127354) and NUDT15(rs116855232) was conducted using a TaqMan double fluorescent probe. The concentration of 6-TGN was determined using UPLC-MS/MS. Among AIH patients treated with AZA, 40 (35.4%) exhibited different degrees of myelosuppression. The NUDT15 variant was associated with leukopenia (P = 8.26 × 10–7; OR = 7.5; 95% CI 3.08–18.3) and neutropenia (P = 3.54 × 10–6; OR = 8.05; 95% CI 2.96–21.9); however, no significant association with myelosuppression was observed for TPMT*3C and ITPA variants (P > 0.05). There was no significant difference in 6-TGN concentration between AIH patients with or without myelosuppression (P = 0.556), nor was there a significant difference between patients with variant alleles of TPMT*3C, ITPA, or NUDT15 and wild-type patients (P > 0.05). Interestingly, it was found that patients with a lower BMI had higher adjusted 6-TGN levels and a higher incidence of myelosuppression (P = 0.026 and 0.003). This study confirmed that NUDT15 variants are a potential independent risk predictor for AZA-induced leukopenia and neutropenia. BMI may be a crucial non-genetic factor that affects the concentration of AZA metabolites and myelosuppression. In addition, the 6-TGN concentration in red blood cells does not reflect the toxicity of AZA treatment, and new biomarkers for AZA therapeutic drug monitoring need further research.

Dynamics of leucocyte DNA thioguanine nucleotide levels during maintenance therapy of childhood acute lymphoblastic leukemia.

Methotrexate (MTX)/6-Mercaptopurine (6MP)-based maintenance therapy is crucial to cure childhood acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated by incorporation of thioguanine nucleotides (TGN) into DNA (DNA-TG) with higher levels in leucocytes being associated with reduced relapse risk. To further understand the dynamics of DNA-TG formation, we measured DNA-TG levels in leucocyte subsets during maintenance therapy and in the months following its discontinuation. DNA-TG levels were measured in leucocytes (DNA-TGTotal), polymorph nucleated granulocytes (neutrophils, eosinophils, basophils [DNA-TGPMN]) and mononucleated cells (lymphocytes, monocytes [DNA-TGMNC]) in 1013 samples from 52 patients on ALL maintenance therapy (951 samples during therapy and 62 samples after therapy discontinuation, respectively). Median DNA-TGTotal, DNA-TGPMN and DNA-TGMNC during maintenance therapy were 539, 563 and 384 fmol/µg DNA, respectively. DNA-TGPMN displayed more pronounced fluctuation than DNA-TGMNC (range 0-3084 [interquartile range IQR 271-881] versus 30-1411 [IQR 270-509] fmol/µg DNA). DNA-TGTotal was more strongly correlated with DNA-TGPMN (rS = 0.95, p < 0.0001) than DNA-TGMNC (rS = 0.73, p < 0.0001). DNA-TGPMN correlated less with DNA-TGMNC (rS = 0.64, p < 0.0001) and to a much lesser extent with absolute neutrophil count (rS = 0.35, p < 0.0001). Following discontinuation of therapy, DNA-TGPMN was rapidly eliminated, and not measurable beyond day 22 after discontinuation, whereas DNA-TGMNC was slowly eliminated, and five patients demonstrated a measurable DNA-TGMNC more than 365days after therapy discontinuation. Fluctuations in DNA-TGTotal are predominantly caused by corresponding fluctuations in DNA-TGPMN, thus DNA-TGTotal measures recent TGN incorporation in these short-lived cells. Measurement of DNA-TGTotal at 2-4weeks intervals provides a reliable profile of DNA-TG levels.

Allopurinol to Prevent Mercaptopurine Adverse Effects in Children and Young Adults With Acute Lymphoblastic Leukemia.

Mercaptopurine (6MP) is used to treat acute lymphoblastic leukemia (ALL) and is metabolized by hypoxanthine guanine phosphoribosal transferase to form 6-thioguanine nucleotide (6TGN). It is also metabolized by thiopurine methyl-transferase to produce 6-methylmercaptopurine (6MMP). Elevated levels of 6MMP have been associated with toxic effects that may interfere with therapy. Allopurinol is known to inhibit thiopurine methyl-transferase which reduces red cell 6MMP and increases 6TGN. Allopurinol has been utilized successfully in adult and pediatric patients with inflammatory bowel disease who have experienced 6MMP related gastrointestinal toxicity. Between August 2015 and August 2018 we started 25 patients with ALL in maintenance on allopurinol in combination with a reduced dose of 6MP. They all had unacceptable side-effects from elevated 6MMP, including abdominal pain, nausea, vomiting, decreased appetite, hypoglycemia, fatigue, and liver toxicity. In addition many had a facial rash. All patients showed resolution of symptoms within a few weeks after starting allopurinol. The red cell levels of 6MMP rapidly declined in the first month. The red cell levels of 6TGN transiently increased in spite of the lower 6MP dose. There was no decrease in absolute neutrophil count or hemoglobin. Platelets decreased slightly not requiring therapy modification. Elevated bilirubin normalized, and alanine aminotransferase decreased significantly with most normalizing. All patients continued on allopurinol with reduced dose 6MP until completing therapy. Allopurinol, in conjunction with a reduced dose of 6MP, effectively resolves 6MMP related side-effects in ALL patients on maintenance chemotherapy. This approach may lead to increased adherence to oral 6MP during ALL maintenance in patients with 6MMP induced side-effects.

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

BackgroundIn the recent era of growing availability of biological agents, the role of thiopurines needs to be reassessed with the focus on toxicity.AimsWe 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.MethodsWe 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.ResultsOf 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.ConclusionsApproximately 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.

Relationship Between Thiopurine S-Methyltransferase Genotype/Phenotype and 6-Thioguanine Nucleotide Levels in 316 Patients With Inflammatory Bowel Disease on 6-Thioguanine.

Background:In inflammatory bowel disease (IBD), conventional thiopurine users cease treatment in 60% of cases within 5 years, mostly because of adverse events or nonresponse. In this study, the authors aimed to investigate the role of 6-thioguanine nucleotide (TGN) measurements, geno/phenotyping of thiopurine S-methyltransferase (TPMT), and their mutual relationship with TG therapy in IBD.Methods:An international retrospective, multicenter cohort study was performed at 4 centers in the Netherlands (Máxima Medical Centre) and the United Kingdom (Guy's and St. Thomas' Hospital, Queen Elizabeth Hospital, and East Surrey Hospital).Results:Overall, 526 6-TGN measurements were performed in 316 patients with IBD. The median daily dosage of TG was 20 mg/d (range 10–40 mg/d), and the median duration of TG use was 21.1 months (SD, 28.0). In total, 129 patients (40.8%) had a known TPMT status. In the variant-type and wild-type TPMT genotype metabolism groups, median 6-TGN values were 1126 [interquartile range (IQR) 948–1562] and 467.5 pmol/8 × 10E8 red blood cells (RBCs) (IQR 334–593). A significant difference was observed between the 2 groups (P = 0.0001, t test). For TPMT phenotypes, in the slow, fast, and normal metabolism groups, the median 6-TGN values were 772.0 (IQR 459–1724), 296.0 (IQR 200–705), and 774.5 pmol/8 × 10E8 RBCs (IQR 500.5–981.5), with a significant difference observed between groups (P < 0.001, analysis of variance).Conclusions:Our findings indicated that TPMT measurements at TG initiation can be useful but are not necessary for daily practice. TPMT genotypes and phenotypes are both associated with significant differences in 6-TGN levels between metabolic groups. However, the advantage of TG remains that RBC 6-TGN measurements are not crucial to monitor treatments in patients with IBD because these measurements did not correlate with laboratory result abnormalities. This presents as a major advantage in countries where patients cannot access these diagnostic tests.

Thiopurines: Recent Topics and Their Role in the Treatment of Inflammatory Bowel Diseases.

Ulcerative colitis (UC) and Crohn's disease (CD) are chronic inflammatory bowel diseases (IBD) of unknown etiology, characterized by repeated relapse and remission. The efficacy of thiopurine in IBD was first reported in the late 1960s. Thiopurines are used to alleviate the symptoms of IBD, especially UC. These drugs have a steroid-sparing potential and are widely used for the purpose of maintaining long-term remission in steroid-dependent cases. Therefore, thiopurines tend to be used long-term, but adverse events that accompany long-term use, such as lymphoproliferative disorders, must be monitored with care. In contrast, thiopurine plays a critical role in controlling the immunogenicity of biologics. Furthermore, although thiopurine is an old drug, new findings, including the prediction of serious adverse events such as severe alopecia and acute advanced leukopenia, by nudix hydrolase 15 gene polymorphism analysis, as well as the possibility of appropriate drug monitoring by detailed analysis of 6-thioguanine nucleotides have been clarified. However, the consequences of thiopurine withdrawal have not been determined and further studies, including randomized controlled trials, are necessary to answer the clinical question regarding the scenarios in which thiopurine withdrawal is possible.

Differential effects of mesalazine formulations on thiopurine metabolism through thiopurine S-methyltransferase inhibition.

Thiopurines are often used in combination with mesalazine for the treatment of ulcerative colitis (UC). Mesalazine formulations are delivered to the digestive tract by various delivery systems and absorbed as 5-aminosalicylic acid (5-ASA). 5-ASA is known to inhibit thiopurine S-methyltransferase (TPMT) activity and to affect thiopurine metabolism. There have been no studies comparing TPMT inhibition by multimatrix mesalazine (MMX) with other formulations. We investigated the difference in TPMT inhibition by different mesalazine formulations and prospectively confirmed the clinical relevance. Plasma concentrations of 5-ASA, N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA), and TPMT activities were measured in UC patients receiving various mesalazine formulations (time-dependent or pH-dependent mesalazine or MMX) as monotherapy. Patients already on both time-dependent or pH-dependent mesalazine and thiopurines switched their mesalazine to MMX, examining 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP) 0 and 8weeks after switching. Clinical relapse after switching was also monitored for 24weeks. Plasma 5-ASA and N-Ac-5-ASA levels were significantly higher in patients receiving time-dependent mesalazine (n=12) compared with pH-dependent mesalazine (n=12) and MMX (n=15), accompanied by greater TPMT inhibition. Prospective switching from time-dependent mesalazine to MMX decreased 6-TGN levels, increased those of 6-MMP, and increased 6-MMP/6-TGN ratios. Furthermore, this resulted in significantly more relapses than switching from pH-dependent mesalazine to MMX. Time-dependent mesalazine has higher plasma 5-ASA and N-Ac-5-ASA levels and greater TPMT inhibition than MMX. Therefore, switching from time-dependent mesalazine to MMX may lead to an increase of 6-MMP/6-TGN, which may reduce the clinical effectiveness of thiopurines, warranting close monitoring after switch.

Long-term efficacy and tolerability of dose-adjusted thiopurine treatment in maintaining remission in inflammatory bowel disease patients with NUDT15 heterozygosity.

Background/AimsThiopurines are key drugs for inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD). Recently, NUDT15 polymorphism (R139C, c.415C > T) has been shown to be associated with thiopurine-induced adverse events in Asian populations. In patients with the C/T genotype, low-dose thiopurine treatment is recommended, but its long-term efficacy and tolerability remain unclear. This study aimed to uncover the long-term efficacy and appropriate dosage of thiopurine for IBD patients with the C/T genotype.MethodsA total of 210 patients with IBD (103 UC and 107 CD) determined to have NUDT15 R139C variants were enrolled. Clinical data were retrospectively reviewed from medical records.ResultsOf 46 patients (21.9%) with the C/T genotype, 30 patients (65.2%) were treated with thiopurines. Three of whom (10.0%) discontinued thiopurine treatment due to adverse events and 27 of whom continued. The median maintenance dosage of 6-mercaptopurine was 0.25 mg/kg/day (range, 0.19–0.36 mg/kg/day), and 6-thioguanine nucleotides level was 230 (104–298) pmol/8 × 108 red blood cells. Cumulative thiopurine continuation rates for 120 months for patients with the C/C and C/T genotypes were not significantly different (P = 0.895). Cumulative non-relapse rates in the patients with UC treated with thiopurine monotherapy and surgery-free rates in CD patients treated with combination therapy (thiopurines and anti-tumor necrosis factor-α agents) for maintenance remission were not significantly different at 60 months (C/C vs. C/T, P = 0.339 and P = 0.422, respectively).ConclusionsLow-dose thiopurine treatment is an effective and acceptable treatment for patients with C/T genotype.

Combination therapy with anti-TNFs and thiopurines does affect drug metabolite levels but it is not associated with body composition in inflammatory bowel disease patients: A cross-sectional study

In this cross-sectional, real-life study we have investigated the potential association between 6-thioguanine nucleotide (6-TGN) and anti-TNF [infliximab (IFX), adalimumab (ADA)], anti-drug antibody levels and body composition parameters. Based on our results thiopurine and anti-TNF combination therapy resulted in decreased antibody formation in IFX-treated patients. AZA-ADA-treated patients showed increased anti-TNF drug concentrations, regardless of antibody formation. Drug metabolites did not correlate with body composition parameters.

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

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. 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. 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). 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.

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.

Population pharmacokinetics of azathioprine active metabolite in patients with inflammatory bowel disease and dosage regimens optimisation.

Azathioprine is a first-line drug used to maintain the remission of inflammatory bowel disease (IBD). As a prodrug, azathioprine is metabolised to produce active 6-thioguanine nucleotides (6-TGN). There are large individual variations in the pharmacokinetics/pharmacodynamics of 6-TGN in patients with IBD. Here, we aimed to develop a model to quantitatively investigate factors that affect 6-TGN pharmacokinetics to formulate a dosage guideline for azathioprine. Data were collected prospectively from 100 adult patients with IBD who were receiving azathioprine. Patients were genotyped for two single-nucleotide polymorphisms (TPMT*3C c.719A>G and NUDT15 c.415C>T). Using high-performance liquid chromatography, we measured 156 steady-state trough concentrations of 6-TGN within the range 0.09 to 1.16mg/L (ie 133-1733pmol per 8×108 RBC). The covariates analysed included sex, age, body-weight, laboratory tests and concomitant medications. A population pharmacokinetic model was established using "non-linear mixed-effects modelling" software and the "first-order conditional estimation method with interaction." Body-weight, TPMT*3C polymorphisms and co-therapy with mesalazine were found to be important factors influencing the clearance of 6-TGN. A dosage guideline for azathioprine was developed based on the PPK model that enables individualised azathioprine dosing in adult patients with different body-weights, TPMT*3C genotypes and co-administration with mesalazine.

Thalidomide Combined With Azathioprine as Induction and Maintenance Therapy for Azathioprine-Refractory Crohn's Disease Patients.

The combination therapy of thalidomide and azathioprine (AZA) offers an alternative in clinical practice for Crohn's disease (CD) patients experiencing a loss of response to AZA monotherapy. However, little is known about the efficacy and safety of this combination therapy for patients with CD. This was a retrospective study of 122 consecutive CD patients who lost response to AZA therapy and had switched to a combination therapy of thalidomide and AZA. The primary outcomes were clinical response and clinical remission rates at week 24. Patients who had an initial response to combination therapy were continued on the treatment for remission maintenance. The secondary outcomes were the proportion of clinical relapse throughout maintenance. The Kaplan–Meier method was used to calculate cumulative rates, and Cox regression analysis was used for multivariate analysis. During induction, 80.3% (98/122) patients achieved clinical response within a median duration of 6.5 weeks, (interquartile range, 4.3–8.1 weeks). The rate of clinical remission at 24 weeks was 70.5%. During follow-up, 22.4% (22/98) of the patients that were maintained on combination therapy experienced clinical relapse. The proportions of patients in remission status at 12, 24, and 36 months were 85.1, 78.3, and 70.1%, respectively. Multivariate analysis revealed C-reactive protein >10 mg/L at disease relapse on AZA monotherapy [adjusted hazard ratio (HR), 4.72; 95% CI, 1.19–18.75, P = 0.027] and 6-thioguanine nucleotides level ≥235 pmol/8 × 108 erythrocytes at AZA monotherapy (adjusted HR, 5.32; 95% CI, 1.40–20.14, P = 0.014) were associated with disease relapse on combination therapy. The endoscopic remission rate was 63.6%. Mucosal healing was achieved in 23.6% of the patients. Both Crohn's Disease Endoscopic Index of Severity (13.4 ± 4.92 vs. 6.12 ± 5.24, P < 0.001) and Rutgeerts scores (3.23 ± 0.73 vs. 1.77 ± 1.59, P = 0.003) were significantly decreased with the use of combination therapy. Adverse events occurred in 62 (50.8%) patients, but only 13 (10.7%) necessitated therapy discontinuation. Thalidomide combined with AZA was effective in inducing clinical remission and sustaining long-term steroid-free remission in CD patients who lost response to AZA monotherapy.

Allopurinol use during pediatric acute lymphoblastic leukemia maintenance therapy safely corrects skewed 6-mercaptopurine metabolism, improving inadequate myelosuppression and reducing gastrointestinal toxicity.

Background:Inadequate myelosuppression during maintenance therapy for acute lymphoblastic leukemia (ALL) is associated with an increased risk of relapse. One mechanism is skewed metabolism of 6-mercaptopurine (6MP), a major component of maintenance therapy, which results in preferential formation of the hepatotoxic metabolite (6-methyl mercaptopurine [6MMP]) with low levels of the antileukemic metabolite, 6-thioguanine nucleotides (6TGN). Allopurinol can modify 6MP metabolism to favor 6TGN production and reduce 6MMP.Methods:Patients in maintenance were considered for allopurinol treatment who had the following features: (a) Grade ≥3 hepatotoxicity; (b) Grade ≥2 nonhepatic gastrointestinal (GI) toxicity; or (c) persistently elevated absolute neutrophil count (ANC) despite >150% protocol dosing of oral chemotherapy.Results:From 2013 to 2017, 13 ALL patients received allopurinol: nine for hepatotoxicity, five for inadequate myelosuppression, and three for nonhepatic GI toxicity (four met multiple criteria). Allopurinol was well tolerated, without significant adverse events. Allopurinol resulted in a significant decrease in the average 6MMP/6TGN ratio (mean reduction 89.1, P = .0001), with a significant increase in 6TGN (mean 550.4, P = .0008) and a significant decrease in 6MMP (mean 13 755, P = .0013). Patients with hepatotoxicity had a significant decrease in transaminase elevation after starting allopurinol (alanine transaminase [ALT] mean decrease 22.1%, P = .02), and all with nonhepatic GI toxicity had improved symptoms. Those with inadequate myelosuppression had a significant increase in the time with ANC in goal (mean increase 26.4%, P = .0004).Conclusions:Allopurinol during ALL maintenance chemotherapy is a safe, feasible, and effective intervention for those who have altered metabolism of 6MPcausing toxicity or inadequate myelosuppression.

Thiopurines and their optimization during infliximab induction and maintenance: A retrospective study in Crohn's disease.

Combining therapy with a thiopurine is favored when commencing infliximab in Crohn's disease; however, the optimal 6-thioguanine nucleotide (TGN) level and how long to continue thiopurines after induction are uncertain. We aimed to compare outcomes after induction and during maintenance in combination therapy versus infliximab monotherapy in Crohn's and to examine whether TGN levels were associated with outcomes. Crohn's patients induced with infliximab with or without concomitant thiopurines were retrospectively identified. Response to induction and clinical outcomes in subsequent 6-month maintenance semesters were analyzed. A TGN level ≥235pmol/8×108 red blood cells was considered therapeutic. In 89 patients, response to induction was higher in combination therapy than monotherapy (74 vs 47%, P=0.04). This benefit was only seen in patients with a therapeutic TGN (odds ratio 3.72, confidence interval 1.07-13.0, P=0.04). Combination therapy during induction yielded a three times longer time to subsequent need for treatment escalation or treatment failure compared with monotherapy (29 vs 9months, P=0.01), with both therapeutic and subtherapeutic TGNs independent predictors on multivariate analysis. Among 370 semesters, there was no difference in outcomes between combination therapy and monotherapy (P=0.42), nor when combination semesters were stratified by therapeutic versus subtherapeutic TGN (P=0.56). In semester 1 only, a significantly higher remission rate was observed with therapeutic compared with subtherapeutic TGN (76% vs 33%, P=0.02). Combination therapy dosed with an optimized thiopurine was superior to infliximab monotherapy for induction of response, durability of response, and clinical outcomes in the first 6months following induction. Thereafter, combination therapy yielded no clinical advantage, supporting consideration of thiopurine withdrawal on a case-by-case basis.

Thiopurines' Metabolites and Drug Toxicity: A Meta-Analysis.

Many questions remain unanswered regarding therapeutic drug monitoring (TDM) utility with thiopurines. This study aims to establish a relationship between thiopurines’ metabolites and drug toxicity. We performed a systematic review with inclusion of studies evaluating the relationship between thiopurines’ metabolites and drug toxicity. Meta-analysis of mean difference (MD), correlations and odds ratio (OR) was performed. We identified 21,240 records, 72 of which were eligible for meta-analysis. Levels of 6-thioguanine nucleotides (6-TGN) were higher in patients with leukopenia (MD 127.06 pmol/8 × 108 RBC) and gastrointestinal intolerance (MD 201.46 pmol/8 × 108 RBC), and lower in patients with hepatotoxicity (MD −40.6 pmol × 108 RBC). We established a significant correlation between 6-TGN and leukocytes (r = −0.21), neutrophils (r = −0.24) and alanine aminotransferase levels (r = −0.24). OR for leukopenia in patients with elevated 6-TGN was 4.63 (95% CI 2.24; 9.57). An optimal cut-off of 135 pmol/8 × 108 RBC for leukopenia was calculated (sensitivity 75.4%; specificity 46.4%). 6-methylmercaptopurine ribonucleotides (6-MMPR) were significantly associated with hepatotoxicity (MD 3241.2 pmol/8 × 108 RBC; OR 4.28; 95% CI 3.20; 5.71). Levels of 6-MMPR measured in the first 8 weeks of treatment were associated with leukopenia. We conclude that TDM could be used to prevent thiopurines’ toxicity. As optimal metabolites level may vary according to indication, physicians may adapt posology to decrease toxicity without compromising efficacy.

Gut microbiota-driven drug metabolism in inflammatory bowel disease.

Background and AimsThe gut microbiota plays an important role in the metabolization and modulation of several types of drugs. With this study we aimed to review the literature relating to microbial drug metabolism of medication prescribed in inflammatory bowel disease [IBD] practice.MethodsA systematic literature search was performed in Embase and PubMed from inception to October 2019. The search was conducted with predefined MeSH/Emtree and text terms. All studies regarding drug metabolism by microbiota of medication prescribed in IBD practice were eligible. A total of 1018 records were encountered and 89 articles were selected for full text reading.ResultsIntestinal bacterial metabolism or modulation is of influence in four specific drugs used in IBD (mesalazines, methotrexate, glucocorticoids and thioguanine). The gut microbiota cleaves the azo-bond of sulfasalazine, balsalazide and olsalazine and releases the active moiety 5-aminosalicylic acid. It has an impact on the metabolization and potentially on the response of methotrexate therapy. In particular, thioguanine can be converted by intestinal bacteria into the pharmacologically active 6-thioguanine nucleotides without the requirement of host metabolism. Glucocorticoid compounds can be prone to bacterial degradation.ConclusionThe human intestinal microbiota can have a major impact on drug metabolism and efficacy of medication prescribed in IBD practice. A better understanding of these interactions between microbiota and drugs is needed and should be an integral part of the drug development pathway of new IBD medication.