Metabolite 6-mercaptopurine Research Articles

Erythrocyte-incorporated 6-mercaptopurine metabolite levels are not affected by recent drug administration during maintenance therapy for childhood acute lymphoblastic leukemia

BackgroundChildhood acute lymphoblastic leukemia (ALL) maintenance therapy with oral 6-mercaptopurine (6-MP) and methotrexate prevents leukemic relapse by acting against residual lymphoblasts. Non-adherence to oral maintenance therapy significantly increases ALL relapse risk. The levels of intracellular 6-MP metabolites erythrocyte incorporated (ery-) thioguanine nucleotides (ery-TGN) and methylated 6-MP metabolites (ery-MeMP) can be used to evaluate adherence to 6-MP, however, little is known on their short-term pharmacokinetics. The aim of this study was to assess the changes between trough levels and the levels at 2-hours after 6-MP intake when the 6-MP levels in plasma is expected to have peaked. Materials and methodsTen ALL patients with stable 6-MP dose were prospectively included. Two blood samples were collected for ery-TGN and ery-MeMP analysis (before 6-MP intake and 2 h after). ResultsThe median trough ery-TGN was 173 (range 126–299) nmol/mmol hemoglobin (HGB), and 2 h after 6-MP intake the median level was 175 (range 120–293) nmol/mmol HGB. For ery-MeMP, the median trough and 2 h-concentrations were 9 239 (range 1 278–19 645) nmol/mmol HGB and 9 216 (range 1 215–19 519) nmol/mmol HGB, respectively. The median absolute percentual change for both ery-TGN and ery-MeMP were not statistically different from 0, p = 0.28 and p = 0.06, respectively. ConclusionsIntracellular 6-MP metabolites, ery-MeMP and ery-TGN, remain stable 2 h after oral 6-MP intake in patients with a stable 6-MP dose. This data support that blood samples may be used to assess patient adherence irrespective of the timing of 6-MP intake.

Impact of Mercaptopurine Metabolites on Disease Outcome in the AIEOP-BFM ALL 2009 Protocol for Acute Lymphoblastic Leukemia.

In the maintenance phase of Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP)- Berlin-Frankfurt-Muenster (BFM) acute lymphoblastic leukemia (ALL) 2009 protocol, mercaptopurine (MP) is given at the planned dose of 50 mg/m2 /day; however, dose adjustments are routinely performed to target patients' white blood cells to the optimal range of 2,000-3,000 cells/μL. Pediatric patients with ALL (n = 290, age: median (1st-3rd quartile): 4.8 (3.0-8.1) years; boys: 56.9%) were enrolled mainly in 4 medium-large Italian pediatric hospitals; 14.1% of patients relapsed after a median (1st-3rd quartile) follow-up time of 4.43 (3.82-5.46) years from maintenance beginning. MP metabolites (thionucleotide (TGN) and methyl-derivatives (MMPN)) were measured in the erythrocytes of 387 blood samples of 200 patients by high performance liquid chromatography with ultraviolet detection. Single-nucleotide polymorphisms (SNPs; (rs1800462, rs1800460, and rs1142345 in TPMT gene, rs116855232 in NUDT15, rs1127354, rs7270101, rs6051702 in ITPA, and rs2413739 in PACSIN2) were characterized by Taqman SNP genotyping assays. Cox proportional hazard models did not show an impact of TGN levels and variability on relapse. In contrast, after multivariate analysis, relapse hazard ratio (HR) increased in children with ALL of the intermediate risk arm compared with those in standard risk arm (3.44, 95% confidence interval (CI), 1.31-9.05, P = 0.012), and in carriers of the PACSIN2 rs2413739 T allele compared with those with the CC genotype (heterozygotes CT: HR, 2.32, 95% CI, 0.90-5.97, P = 0.081; and homozygous TT: HR, 4.14, 95% CI, 1.54-11.11, P = 0.005). Future studies are needed to confirm the lack of impact of TGN levels and variability on relapse in the AIEOP-BFM ALL trials, and to clarify the mechanism of PACSIN2 rs2413739 on outcome.

DNA polymerase gamma variants and hepatotoxicity during maintenance therapy of childhood acute lymphoblastic leukemia: is there a causal relationship?

Hepatotoxicity is a frequent complication during maintenance therapy of acute lymphoblastic leukemia (ALL) with 6-mercaptopurine and methotrexate. Elevated levels of methylated 6-mercaptopurine metabolites (MeMP) are associated with hepatotoxicity. However, not all mechanisms are known that lead to liver failure in patients with ALL. Variants in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma (POLG1), have been related to drug-induced hepatotoxicity, for example, by sodium valproate. The association of common POLG variants with hepatotoxicity during maintenance therapy was studied in 34 patients with childhood ALL. Of the screened POLG variants, four different variants were detected in 12 patients. One patient developed severe hepatotoxicity without elevated MeMP levels and harbored a heterozygous POLG p.G517V variant, which was not found in the other patients.

Influence of the Photodegradation of Azathioprine on DNA and Cells.

Azathioprine (AZA) is a pharmacologic immunosuppressive agent administrated in various conditions such as autoimmune disease or to prevent the rejection of organ transplantation. The mechanism of action is based on its biologically active metabolite 6-mercaptopurine (6-MP), which is converted, among others, into thioguanine nucleotides capable of incorporating into replicating DNA, which may act as a strong UV chromophore and trigger DNA oxidation. The interaction between azathioprine and DNA, before and after exposure to solar simulator radiation, was investigated using UV-vis spectrometry and differential pulse voltammetry at a glassy carbon electrode. The results indicated that the interaction of AZA with UV radiation was pH-dependent and occurred with the formation of several metabolites, which induced oxidative damage in DNA, and the formation of DNA-metabolite adducts. Moreover, the viability assays obtained for the L929 cell culture showed that both azathioprine and degraded azathioprine induced a decrease in cell proliferation.

Improved HPLC Quantification of 6-Mercaptopurine Metabolites in Red Blood Cells: Monitoring Data and Literature Analysis.

Thiopurine drugs azathioprine (AZA) and 6-mercaptopurine (6-MP) are used extensively in pediatric and adult patients with inflammatory and neoplastic diseases. They are metabolized to 6-thioguanine nucleotides (6-TGN) or to 6-methyl-mercaptopurine nucleotides (6-MMPN). The balance between 6-TGN and 6-MMPN is highly variable and monitoring is recommended, but its benefit in outcome gives rise to conflicting results, potentially increased by differences in quantifying 6-MP metabolism. Our aim was to report (1) the HPLC-UV procedure used in our laboratory to quantify red blood cells (RBCs) with 6-TGN and 6-MMPN (as its derivate: 6-MMP(d)) in patients treated with thiopurines and (2) additional tests, sometimes confirmatory, to improve method standardization. The comparison of two methods to count RBCs shows that metabolite concentrations were slightly lower in the washed and resuspended RBCs than in whole blood. Perchloric acid (0.7 M), dithiothreitol (DTT, final 0.013 M sample concentration) and 60 min hydrolysis were selected for acid hydrolysis. (3) Monitoring data from 83 patients receiving AZA or 6-MP showed that at steady state, only 53/183 (29%) had 6-TGN and 6-MMPN in the recommended therapeutic range. Our method is discussed in light of the technical conditions and sample stability data from 17 publications identified since the first analytical report in 1987. Monitoring data demonstrate, if required, that inter-patient variability in 6-TGN and 6-MMPN concentrations is high in samples from treated patients.

Socioeconomic position and maintenance therapy in children with acute lymphoblastic leukemia: A national cohort study.

Socioeconomic differences in survival among children with acute lymphoblastic leukemia (ALL) have been reported in high-income countries and there is an unmet need for strategies to identify vulnerable patient subgroups. Reported differences in survival for children from families with different socioeconomic positions seem to arise when starting maintenance therapy. This could reflect reduced physician's compliance or family adherence to maintenance therapy. This nationwide cohort study with extensive monitoring of systemic methotrexate (MTX)/6-mercaptopurine (6MP) dosing and metabolite levels, retrospectively investigated 173 Danish children treated according to The Nordic Society for Pediatric Hematology and Oncology ALL2008 protocol from 2008 to 2016. Significantly lower prescribed doses of MTX and 6MP were seen in the children in families with short parental education (short vs. medium vs. higher education: mMTX: 13.8, 16.2, and 18.6mg/m2 /week; p<.01; m6MP: 47.4, 64.9, and 66.1mg/m2 /day; p=.03) or parents unemployed/not in workforce (unemployed/not in workforce vs. mixed vs. at work: mMTX: 15.0, 19.9, and 17.2mg/m2 /week; p<.01; m6MP: 54.8, 72.0, and 65.1mg/m2 /day; p<.01). When assessing family adherence by analyzing MTX and 6MP metabolite levels, including per prescribed dose of MTX and 6MP, we found no significant differences by levels of parental education, affiliation to work market, or income (p>.05 for all comparisons). These results indicate that inferior physician compliance to protocol recommendations on drug dosage rather than families' adherence to therapy may contribute to the association between socioeconomic position and cure rates in childhood ALL, although precise mechanisms remain to be explored.

PACSIN2 rs2413739 Polymorphism and Thiopurine Pharmacokinetics: Validation Studies in Pediatric Patients

Thiopurines such as thioguanine (TG), mercaptopurine (MP) and its prodrug azathioprine (AZA) are key antileukemic and immunosuppressive drugs in the treatment of several pediatric diseases such as acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD): their therapeutic action is affected by the enzyme thiopurine-S-methyltransferase (TPMT) whose encoding gene is polymorphic and whose enzymatic activity is also trans -regulated by the intronic single nucleotide polymorphism (SNP) rs2413739 (T>C) in the PACSIN2 gene.This study was aimed at validating the impact of PACSIN2 rs2413739 on thiopurine pharmacokinetic and pharmacodynamic parameters in two Italian pediatric cohorts: the first cohort was multicentric, consisted of 297 pediatric patients newly diagnosed with ALL (median (IQ) age: 4.8 (3.0-9.2) years; male: 54.9%) treated according to the AIEOP-BFM ALL 2009 protocol; the second consisted of 119 pediatric IBD patients (median age (interquartile range (IQ)): 15.1 (12.3-16.9) years; male: 52.1%) treated continuously with daily weight-based AZA therapy for at least three months before enrollment at the Gastroenterology Unit of the Pediatric Hospital IRCCS “Burlo Garofolo” in Trieste. Moreover, the role of rs2413739 polymorphism on TPMT and PACSIN2 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) of adult healthy donors.The presence of the PACSIN2 rs2413739 T allele significantly reduced the TPMT activity only in the ALL cohort (p=0.026, linear mixed-effect model, multivariate analysis considering the TPMT SNP rs1142345 and rs1800460), without affecting MP metabolites levels in patients' erythrocytes: the PACSIN2 rs2413739 genetic effect on TPMT activity was particularly evident within TPMT heterozygotes (linear mixed-effect model, p=0.041). Interestingly, TPMT enzymatic function was significantly higher in ALL patients undergoing a long-term thiopurine-based therapy compared to IBD patients (p=0.032, linear mixed-effect model). In IBD patients, treatment response to AZA was significantly affected by PACSIN2 genotype: AZA effectiveness was reduced in patients carrying the T allele of the SNP rs2413739 (linear mixed effects, p=0.018; genotyping results: CC in 19, CT in 29 and TT in 20 patients): indeed, a clinically active disease was observed in ~40% of TT patients, in 30% of those with the CT genotype and in 20% of the CC subjects. For ALL patients, clinical data are still under collection and are not available at the time of preparation of the abstract.To evaluate the role of PACSIN2 SNP rs2413739 on TPMT and PACSIN2 protein expression, 20 adult healthy donors were genotyped for the SNP of interest, and Western-blot analysis against human TPMT and human PACSIN2 were performed on their PBMCs lysates. No significant contribution of the polymorphism was found on TPMT and PACSIN2 protein expression in this small population. In contrast, a significant direct correlation between the TPMT and PACSIN2 levels was observed (linear mixed effect, p = 0.045), suggesting a putative coordination in their protein expression in PBMCs.These results support the role of PACSIN2 polymorphism on TPMT activity, although this effect may be tissue-specific or dependent on the patients' clinical condition. DisclosuresNo relevant conflicts of interest to declare.

When Multiple Objective Measures of Medication Adherence Indicate Incongruent Adherence Results: An Example with Pediatric Cancer.

Previous research suggests that children and adolescents with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) often have difficulty adhering to complex treatment regimens during the maintenance phase of therapy. Measurement of treatment adherence can be done via objective (e.g., electronic monitoring (EM), pharmacological assays) or subjective methods (patient, parent, or physician reports). This paper provides an illustration of recommended strategies for comparing discrepancies between two objective measures of medication adherence (e.g., behavioral adherence using electronic monitoring versus pharmacological adherence using 6-mercaptopurine (6MP) metabolite data) within a relatively large cohort of pediatric patients with ALL or LBL (N = 139) who had longitudinal data for both measures of medication adherence over a 15-month period. Additionally, individual- and family-level factors such as gender, socioeconomic status, household environment, and dose intensity will be examined to identify possible sources of discrepancies between adherence measures. This information will provide practical advice for physicians, healthcare providers, and psychologists in identifying nonadherence and the caveats therein so patients achieve the best possible health outcomes.

Adherence to Azathioprine/6-Mercaptopurine in Children and Adolescents with Inflammatory Bowel Diseases: A Multimethod Study

Background Measurement of the degree of adherence is a key element for the evaluation of treatment efficacy and safety; thus, adherence plays an important role in clinical research and practice. The aim of this study was to investigate medication adherence in children with inflammatory bowel disease (IBD) utilizing a multimethod assessment approach. A further aim was to examine factors that can influence adherence within this population. Methods Medication adherence in 47 children (age range 3 to 17 years) with IBD in three centers in Northern Ireland and Jordan was assessed via subjective (parent and child versions of the Medication Adherence Report Scale (MARS) specific questionnaire) and objective methods, that is, high-performance liquid chromatography (HPLC) determination of the 6-mercaptopurine (6-MP) and azathioprine (AZA) metabolites in packed red blood cell samples taken during a clinic visit. Beliefs about prescribed medicines were also assessed in parents/guardians using the Beliefs about Medicines Questionnaire (BMQ). Results An overall nonadherence to AZA/6-MP therapy in children with IBD was found to be 36.17% (17 out of 47 patients were classified as nonadherent using at least one of the assessment methods). A total of 41 patients (91.1%) were classified as adherent to AZA or 6-MP using the blood sampling, while adherence rates using the MARS questionnaire completed by children and parents/guardians were 60.6% and 72.7%, respectively. The latter provides a more longitudinal measure of adherence. Child self-reported nonadherence rates were significantly higher than parent/guardian reported rates (p=0.013). Binary logistic regression analysis identified age to be independently predictive of adherence, with adolescents (children aged ≥ 13 years old) more likely to be classified as nonadherent. Regarding the BMQ, when parental/guardian necessity beliefs outweighed concerns, that is, higher scores in the necessity-concern differential (NCD), adolescents were more likely to be classified as adherent. Conclusion Results provide evidence for ongoing adherence challenges in the paediatric population with IBD. It is recommended that parents/guardians (particularly of older children) and older children themselves, should receive enhanced counselling and education about their prescribed medicines.

Mechanisms of NT5C2-Mediated Thiopurine Resistance in Acute Lymphoblastic Leukemia.

Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5'-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.

Effects of azathioprine and its metabolites on inflammatory cytokines in human nasal polyp organ cultures.

Oral steroids are recommended for the treatment of nasal polyps (NPs), but prolonged use is avoided because of side effects. Topical steroids can also control NPs without significant complications; however, the response to this is partially successful, and additional therapies are needed to treat glucocorticoid-resistant NPs. Azathioprine (AZA) and its first metabolite 6-mercaptopurine (6-MP) are important immunosuppressants used for the therapy of various diseases. The aim of this study was to investigate the effects of AZA and 6-MP on inflammatory cytokines in organ-cultured NPs. NP explants were cultured using an air-liquid interface method. Cultures were maintained in the absence and presence of steroid, AZA, and 6-MP for 72 hours. Elaboration of cytokines tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, IL-4, IL-5, and IL-13 into the supernatant was quantitated using the enzyme-linked immunosorbent assay (ELISA). The messenger RNA (mRNA) expression levels of TNF-α, IL-2, IL-4, IL-5, and IL-13 in cultured mucosa were measured using real-time polymerase chain reaction. Hematoxylin and eosin staining of cultured mucosa was performed to observe inflammatory cells. Immunohistochemistry was done to evaluate the distribution pattern of inflammatory cytokines in NP explants. On histologic examination, less inflammatory cell infiltration was found in NPs treated by steroid, AZA, and 6-MP than in control, but there was no statistical significance (p=0.218). On immunohistochemistry, IL-13 showed a steady falling tendency in submucosal glands by steroid, AZA, and 6-MP. Expression of TNF-α, IL-2, IL-4, IL-5, and IL-13 mRNA in the NPs treated by steroid, AZA, and 6-MP were significantly lower than those of the control (p<0.001 for all). By ELISA, IL-2 and IL-13 were significantly lower with topical steroid, AZA, and 6-MP treatment (p=0.012 and p<0.001). Topical AZT decreases inflammatory cytokines on human NP explants and this could have future therapeutic implications for NPs.

Three Faces of Mercaptopurine Cytotoxicity In Vitro: Methylation, Nucleotide Homeostasis, and Deoxythioguanosine in DNA.

Mercaptopurine (MP) is a cytotoxic thiopurine important for the treatment of cancer and autoimmune diseases. MP and other thiopurine drugs undergo extensive intracellular metabolism, but the mechanisms of action are poorly characterized. In particular, it is unknown how different metabolites contribute to cytotoxicity and incorporation of thiopurine bases into DNA. The aim of this study was to ask whether cytotoxicity results from the incorporation of thioguanosine nucleotides into DNA, an alternative thiopurine metabolite, or a combination of factors. Therefore, we measured the cytotoxicity, metabolism, and incorporation of thioguanosine into DNA in response to MP or MP metabolites. Thiopurine metabolites varied in cytotoxicity, with methyl-thioinosine-mono-phosphate and thioguanosine-tri-phosphate the most toxic, and the methyl-thioguanosine nucleotides the least. We show, using liquid chromatography-tandem mass spectrometry, how different metabolites may perturb biochemical pathways, particularly disrupting guanosine nucleotide homeostasis, that may contribute to the mechanism of action of thiopurines. Although there was no correlation between metabolite cytotoxicity and the levels of 6-methylthioinosine-mono-phosphate or thioguanosine incorporation into DNA as individual factors, a combined analysis suggested that these factors together had a major influence on cytotoxicity. This study emphasizes the importance of enzymes of nucleotide homeostasis, methylation, and demethylation in thiopurine effects. These results will facilitate the development of dynamic biochemical models of thiopurine biochemistry that will improve our understanding of mechanisms of action in relevant target tissues.

A Role for Thiopurine Metabolites in the Synergism Between Thiopurines and Infliximab in Inflammatory Bowel Disease.

Interactions between principal cytotoxic thiopurine metabolites, that is 6-thioguanine nucleotides [6-TGN], and infliximab [IFX] and anti-IFX antibodies [Abs] may contribute to higher effectiveness of IFX-thiopurine combination therapy than monotherapies in inflammatory bowel disease. To examine if thiopurine metabolites influenced trough IFX and anti-IFX Abs, 89 patients previously assessed for anti-IFX Abs were included. To assess if IFX influenced thiopurine metabolites, eight patients who had responded to 12 weeks of intensified IFX at a constant thiopurine dosing were included. In the first cohort, IFX-thiopurine combination therapy reduced anti-IFX Ab detection [8/40; 20%] as compared with IFX monotherapy [22/49; 45%], odds ratio [OR] 0.31 [0.12-0.80], p < 0.05. 6-TGN was significantly lower in anti-IFX Ab-positive patients (50 pmol/8 × 108 red blood cells [RBC] vs 105, p < 0.01). All anti-IFX Ab-positive patients had 6-TGN < 117 pmol/8 × 108 RBC (sensitivity 100% [63-100], specificity 47% [29-65], area under the curveROC = 0.82, p < 0.01). Trough IFX was similar between anti-IFX Ab-negative patients in IFX monotherapy and IFX-thiopurine combination therapy [5.1 μg/mL vs 4.9, p = 0.76]. 6-TGN and IFX did not correlate [rP = 0.04, p = 0.83; rS = 0.02, p = 0.89, respectively]. In the second cohort, trough IFX increased during IFX intensification [ΔIFX median 6.5 μg/mL, p = 0.02], but 6-TGN was stable [6-TGN at Weeks 0, 4, 8, 12: 90 pmol/8 × 108 RBC, 93, 101, 90; p > 0.05]. Methylated mercaptopurine metabolite associations were consistently negative. Superior effect of IFX-thiopurine combination therapy over monotherapies partly relates to decrease in anti-IFX Abs, which associates with 6-TGN levels and has a lower therapeutic threshold than during thiopurine monotherapy. Additional benefit likely ascribes to synergy between different anti-inflammatory modes of action rather than direct drug interactions.

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

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

Hepatotoxicity During Maintenance Therapy and Prognosis in Children With Acute Lymphoblastic Leukemia.

Hepatotoxicity is a known toxicity to treatment of childhood acute lymphoblastic leukemia. Hepatotoxicity occurs during maintenance therapy and is caused by metabolites of 6-Mercaptopurine (6 MP) and Methotrexate (MTX). Our objective was to investigate the association between alanine aminotransferases (ALAT) levels and relapse rate. We included 385 patients enrolled in the NOPHO ALL-92 protocol. Data on ALAT levels, 6 MP and MTX doses, cytotoxic MTX/6 MP metabolites, and thiopurine methyltransferase (TPMT) activity were prospectively registered. In total, 91% of the patients had a mean ALAT (mALAT) level above upper normal limit (40 IU/L) and ALAT levels were positively correlated to 6 MP doses (rs=0.31; P<0.001). In total, 47 patients suffered a relapse, no difference in mALAT levels were found in these compared with nonrelapse patients (median, 107 vs. 98 IU/L; P=0.39). mALAT levels in patients classified as TPMT high activity (TPMT) were higher than in TPMT low-activity patients (median, 103 vs. 82 IU/L; P=0.03). In a Cox regression model risk of relapse was not associated with ALAT levels (P=0.56). ALAT levels increased 2.7%/month during the last year of maintenance therapy (P<0.001). In conclusion, elevated ALAT levels are associated with TPMT and may indicate treatment adherence in these patients. If liver function is normal, elevated ALAT levels should not indicate treatment adaptation.

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

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. In this substudy of the NOPHO ALL2008 phase 3 trial done in 23 hospitals in seven European countries (Denmark, Estonia, Finland, Iceland, Lithuania, Norway, and Sweden), we analysed data from centralised and blinded analyses of 6-mercaptopurine and methotrexate metabolites in blood samples from patients with non-high-risk childhood acute lymphoblastic leukaemia. Eligible patients were aged 1·0-17·9 years; had been diagnosed with non-high-risk precursor B-cell or T-cell leukaemia; had been treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol; and had reached maintenance therapy in first remission. Maintenance therapy was (mercaptopurine 75 mg/m2 once per day and methotrexate 20 mg/m2 once per week, targeted to a leucocyte count of 1·5-3·0 × 109 cells per L). We measured DNA-TGN and erythrocyte concentrations of TGN nucleotides, methylated mercaptopurine metabolites, and methotrexate polyglutamates. The primary objective was the association of DNA-TGN concentrations and 6-mercaptopurine and methotrexate metabolites with relapse-free survival. The secondary endpoint was the assessment of DNA-TGN concentration and 6-mercaptopurine and methotrexate metabolites during maintenance therapy phase 2. 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). 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. Danish Cancer Society, Childhood Cancer Foundation (Denmark), Childhood Cancer Foundation (Sweden), Nordic Cancer Union, Otto Christensen Foundation, University Hospital Rigshospitalet, and Novo Nordic Foundation.

Metabolite monitoring to guide thiopurine therapy in systemic autoimmune diseases.

6-Thioguanine nucleotide (6-TGN) is the active metabolite of thiopurine drugs azathioprine and 6-mercaptopurine. 6-Methylmercaptopurine (6-MMP) is an inactive and potentially hepatotoxic metabolite. A subgroup of patients (shunters) preferentially produce 6-MMP instead of 6-TGN, therefore displaying thiopurine resistance and risk for hepatotoxicity. Outside inflammatory bowel disease literature, few data exist regarding individualized thiopurine therapy based on metabolite monitoring. This study sought to describe metabolite monitoring in patients receiving weight-based thiopurine for systemic autoimmune diseases. Patients were enrolled using a laboratory database, and data were retrospectively collected. The correlation between the highest thiopurine dose (mg/kg) and the 6-TGN concentration (pmol/8×108 erythrocytes) was estimated with Pearson's correlation coefficient. Seventy-one patients with various systemic autoimmune conditions were enrolled. The correlation between the thiopurine dose and the 6-TGN level was weak for the overall patient sample (r=0.201, p=0.092) and for the subgroup of non-shunters (r=0.278, p=0.053). Subjects with 6-MMP levels >5700pmol/8×108 erythrocytes had more hepatic cytolysis compared to subjects with 6-MMP <5700, OR=4.36 (CI 95% 1.18-16.13, p=0.027). Twenty-two patients (31%) were identified as shunters. Six shunters developed hepatotoxicity, five of which had 6-MMP concentration >5700. Eleven non-shunters had hepatotoxicity, one of which had 6-MMP >5700. Thiopurine metabolite monitoring shows wide variability in 6-TGN levels among patients treated with weight-based thiopurine for systemic autoimmune diseases. Thirty-one percent of the patients in our series fulfilled the shunter definition. Thiopurine metabolite monitoring and dose adjustment to improve maintenance of remission and avoid hepatotoxicity should be studied prospectively.

HPLC Determination of the Levels of 6-Mercaptopurine Metabolites Suitable for the Clinical Risk Assessment of its Toxicity among Egyptian Children with Acute Lymphocytic Leukemia

The need of a robust, sensitive HPLC method for the quantitation of 6-thioguaninenucleotides (6-TG) and 6-methylmercaptopurine (6-MMP) is indispensable to relate levels of these metabolites with emergence of signs of toxicity in patients undergoing treatment with 6-mercaptopurine (6-MP), paving the road to accurate dose calculations and thus providing a cost-effective treatment approach. Previously reported methods were either laborious, required special types of C18 columns, or had long run times. A Design of Experiments (DoE) approach targeting the shortest run time with greatest selectivity was adopted using a user friendly HPLC method development simulation software (DryLab®). Analytes eluted within 10 min, at 3.8, 4.2, 5.6 and 7.5 min for 6-TG, 6-MP, 6-MMP and Dithiothreitol (DTT) respectively. Excellent recovery percentages of 90.9 ± 14.4, 87.8 ± 6.7 and 92.1 ± 9.08, respectively were obtained. The method proved its validity and robustness according to the International Conference on Harmonization (ICH) guidelines. The LOD of 6-MP, 6-TG and 6-MMP were 6, 9 and 24 pmol/8 × 108 RBCs, respectively. Twenty-Two Acute Lymphocytic Leukemia (ALL) children recruited from 57357 Cancer Hospital (Cairo, Egypt) had their 6-MP metabolites measured using the developed method. A strong negative correlation was manifested between TG and RBCs count and hemoglobin (p=0.009 and 0.002 respectively). WBC and neutrophils showed a negative correlation to TG at Continuation 1 phase of treatment, confirming the association of TG with myelotoxicity. The significant correlation between MMP and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (p=0.030, 0.004) explained its potential hepatotoxicity.

262 - Potential Involvement of Peroxymonocarbonate and SOD in the Oxidation of the Anticancer Drug Metabolite, 6-Mercaptopurine

Introduction 6-mercaptopurine (6MP), an immunosuppressive and cytotoxic metabolite of azathioprine, is used therapeutically in the treatment of many inflammatory and immunological diseases. 6MP is known to undergo metabolism through oxidation by xanthine oxidase, S-methylation via thiopurine methyltransferase, and can be metabolized to form 6-thioguanine, which is believed to be the active metabolite. At sites of inflammation, reactive oxygen species (ROS) are generated, including H2O2 that inactivates superoxide dismutase. In the presence of HCO3-, peroxymonocarbonate (HCO4–) can potentially be produced, which enhances the oxidation potential of H2O2. H2O2 can also produce CO3●– through SOD peroxidase activity. Thiols have been shown to be oxidized by HCO4– or CO3●– but no studies have investigated the effect on 6MP. Hypothesis HCO4– and CO3●– will oxidize 6-mercaptopurine at the sulfur atom, and SOD peroxidase activity will act as a catalyst. Methods and Results UV-Vis spectrometry was used at room temperature to monitor the spectral changes (200 – 500 nm) using 20 µM 6MP and other purine analogs including its parent drug, azathioprine, and related metabolites including 6-methyl-MP, 6-thioguanine, 6-thioxanthine, and 6-thiouric acid. Reactions were run in 0.1 M KPO4 buffer pH 7.4 containing 250 µM DTPA, and were initiated by the addition of 25 mM HCO3– and 1 mM H2O2. The most prominent changes were observed with 6MP and 6-thioguanine; less changes were observed with 6-thioxanthine and 6-thiouric acid. Oxidation of 6MP by H2O2 was observed in the absence of HCO3– but was significantly enhanced using 12.5 and 25 mM HCO3–. The reactions were analyzed by high resolution LC/MS and were compared with reactions that contained SOD to catalyze SOD peroxidase activity. 6MP was detected at its expected m/z (151.0086, C5H4N4S) and the major product peak found corresponded to an m/z of 182.9981 (C5H4N4O2S), representing a sulfoxide product. SOD inclusion in these reactions produced the same products but with only 30 min incubation time vs. 24 h without SOD. The reaction of CO3●– with 6-MP was confirmed by EPR and spectrometric measurements. SOD activity was significantly enhanced by 6-MP but not azathioprine and the CO3●– radicals were scavenged by only 6-MP. 6MP was oxidized by H2O2/HCO3– and treated with HEK293 and HepG2 cells. 6MP-sulfoxide was significantly less cytotoxicity to both cell lines compared to unadulterated 6MP. Conclusions Exposure of 6MP to H2O2/HCO3– with and without SOD significantly enhanced its oxidation, and led to the formation of a 6MP-sulfoxide, which loses its cytotoxic activity and consequently ameliorates SOD activity.

PTH-081 Allopurinol Abrogates Thiopurine Induced Liver Injury Without The Need for Metabolite Measurments

Introduction The thiopurines azathioprine and mercaptopurine are widely used as long term immunomodulators in inflammatory bowel diseases (IBD). Reported side effects vary between 10–40% hindering the efficacy and safety of these useful treatments. Hepatotoxicity is one of the commoner side effects (up to 10%) and some investigators have made an association to thiopurine methyltransferase (TMPT) levels as well as hypermethylation. Aim Report on the use of allopurinol in conjuction to azathioprine (Low Dose Azathioprine Allopurinol- LDAA) in patients who developed thiopurine induced liver injury. Methods This is a retrospective cohort study. The patients were drawn from a large database of patients at a single District General Hospital by the authors. The outcomes were assessed by review of patient records. Results We identified 15 (10%) patients who developed thiopurine induced liver injury [median (range) ALT: 74 iu/L (49,630), ALP: 35 iu/L (20, 120), bilirubin: 8μmol/L (5, 45), time of onset from drug commencement: 12 weeks (6, 30)] in the last 5 years from a prospective cohort of 150 patients who required azathioprine for IBD (Crohn’s disease: 8, Ulcerative colitis: 7, 1 with concomitant psoriatic arthropathy and 1 with Takayasu’s arteritis). There were 7 (47%) patients with wild type TPMT and 8 (53%) heterozygotes. Seven (78%)of the 9 patients receiving LDAA had a normalisation of aminotransferases after an increase in allopurinol dose from 100 mg to 200 mg while two were eventually diagnosed with primary sclerosing cholangitis. Addition of allopurinol (100 mg) for the 6 patients on azathioprine monotherapy lead to normalisation of aminotransferases. Conclusion Observations form this data suggest i) Patients who cannot produce high methylated mercaptopurine metabolites (MMP) can develop hepatotoxicity which responds well to allopurinol. ii) Patients may still develop hepatotoxicity on co-therapy with 100 mg of allopurinol, which responds to a dose increase to 200 mg, suggesting an independent hepatocyte protective effect of allopurinol. We have previously suggested a role for reactive oxygen species. iii) Thiopurine metabolites profiles should not be used to guide allopurinol use, as the protective effect is not predicted by high MMP levels. Disclosure of Interest None Declared