thioinosine and 6-methylthiopurine-ribonucleoside-5--phosphate

Azathioprine and mercaptopurine have a pivotal role in the treatment of inflammatory bowel disease (IBD). However, because of their complex metabolism and potential toxicities, optimal use of biomarkers to predict adverse effects and therapeutic response is paramount.. To provide a comprehensive review focused on pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy in IBD.. A literature search up to July 2015 was performed in PubMed using a combination of relevant MeSH terms.. Pre-treatment thiopurine S-methyltransferase typing plus measurement of 6-tioguanine nucleotides and 6-methylmercaptopurine ribonucleotides levels during treatment have emerged with key roles in facilitating safe and effective thiopurine therapy. Optimal use of these tools has been shown to reduce the risk of adverse effects by 3-7%, and to improve efficacy by 15-30%. For the introduction of aldehyde oxidase (AOX) into clinical practice, the association between AOX activity and AZA dose requirements should be positively confirmed. Inosine triphosphatase assessment associated with adverse effects also shows promise. Nucleoside diphosphate-linked moiety X-type motif 15 variants have been shown to predict myelotoxicity on thiopurines in East Asian patients. However, the impact of assessments of xanthine oxidase, glutathione S-transferase, hypoxanthine guanine phosphoribosyltransferase and inosine monophosphate dehydrogenase appears too low to favour incorporation into clinical practice.. Measurement of thiopurine-related enzymes and metabolites reduces the risk of adverse effects and improves efficacy, and should be considered part of standard management. However, this approach will not predict or avoid all adverse effects, and careful clinical and laboratory monitoring of patients receiving thiopurines remains essential.

Topics: Azathioprine; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Methyltransferases; Pharmacogenetics; Thioinosine; Thionucleotides

Patients' beliefs about medicine may either reflect the necessity for treatment or concerns regarding the treatment. We explored the extent to which these beliefs have an effect on thiopurine metabolite levels and premature discontinuation in patients with inflammatory bowel disease (IBD).. Patients enrolled in the 'Thiopurine response Optimization by Pharmacogenetic testing in Inflammatory Bowel Disease Clinics' (TOPIC) trial were asked to complete the Beliefs about Medicine Questionnaire (BMQ) 4 weeks after thiopurine initiation. The BMQ measures perceptions about treatment necessity and concerns. On the basis of the necessity and concern scores, patients can be categorized as accepting, ambivalent, indifferent, or skeptical. The thiopurine discontinuation rates for these belief subgroups were compared by Kaplan-Meier curves. Furthermore, clinical response and metabolite levels were compared between the belief subgroups.. A total of 767 patients with IBD started thiopurine treatment, of whom 576 (75%) completed the BMQ. Patients could be classified as accepting (34%), indifferent (17%), ambivalent (34%), or skeptical (15%). Compared with patients in the accepting group (discontinuation rate 22%), patients with an indifferent (35%; P=0.02), ambivalent (37%; P<0.01), or skeptical belief (54%; P<0.01) had higher thiopurine discontinuation rates. No differences were observed in the steady-state thiopurine metabolite levels between the different belief subgroups.. Patients with a low perceived treatment necessity or high concerns toward IBD treatment were more likely to discontinue thiopurine treatment prematurely. Extra attention toward these patients might prevent premature discontinuation.

Topics: Adult; Female; Guanine Nucleotides; Health Knowledge, Attitudes, Practice; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Medication Adherence; Mercaptopurine; Middle Aged; Surveys and Questionnaires; Thioinosine; Thionucleotides; Treatment Outcome; Young Adult

Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related to high steady-state 6-methylmercaptopurine ribonucleotide (6-MMPR) metabolite concentrations.. To determine the predictive value of 6-MMPR concentrations 1 week after treatment initiation (T1) for the development of these adverse reactions, especially hepatotoxicity, during the first 20 weeks of treatment.. The cohort study consisted of the first 270 IBD patients starting thiopurine treatment as part of the Dutch randomised-controlled trial evaluating pre-treatment thiopurine S-methyltransferase genotype testing (ClinicalTrials.gov NCT00521950). Blood samples for metabolite assessment were collected at T1. Hepatotoxicity was defined by alanine aminotransaminase elevations >2 times the upper normal limit or a ratio of alanine aminotransaminase/alkaline phosphatase ≥5.. Forty-seven patients (17%) presented hepatotoxicity during the first 20 weeks of thiopurine treatment. A T1 6-MMPR threshold of 3615 pmol/8 × 10. In more than 80% of patients, thiopurine-induced hepatotoxicity could be explained by elevated T1 6-MMPR concentrations and the independent risk factors age, gender and BMI, allowing personalised thiopurine treatment in IBD to prevent early failure.

Topics: Adult; Aged; Aged, 80 and over; Azathioprine; Chemical and Drug Induced Liver Injury; Cohort Studies; Early Diagnosis; Female; Genotype; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Prognosis; Risk Factors; Thioinosine; Thionucleotides; Treatment Outcome; Young Adult

Therapeutic drug monitoring of active metabolites of thiopurines, azathioprine and 6-mercaptopurine, is relatively new. The proposed therapeutic threshold level of the active 6-thioguanine nucleotides (6-TGN) is ≥235 pmol/8×10(8) erythrocytes. The aim of this prospective cross-sectional study was to compare 6-TGN levels in adult thiopurine tolerant IBD patients with an exacerbation with those in remission, and to determine the therapeutic 6-TGN cut-off level.. Hundred IBD patients were included. Outcome measures were thiopurine metabolite levels, calculated therapeutic 6-TGN cut-off level, CDAI/CAI scores, thiopurine dose and TPMT enzyme activity.. Forty-one patients had an exacerbation, 59 patients were in remission. In 17% of all patients 6-TGN levels were compatible with non-compliance. The median 6-TGN levels were not significantly different between the exacerbation and remission group (227 versus 263 pmol/8×10(8) erythrocytes, p=0.29). The previous reported therapeutic 6-TGN cut-off level of 235 pmol/8×10(8) erythrocytes was confirmed in this study. Twenty-six of the 41 patients (63%) with active disease had 6-TGN levels below this threshold and 24 of 59 IBD patients (41%) in clinical remission (p=0.04).. Thiopurine non-compliance occurs frequently both in active and quiescent disease. 6-TGN levels below or above the therapeutic threshold are associated with a significant higher chance of IBD exacerbation and remission, respectively. These data support the role of therapeutic drug monitoring in thiopurine maintenance therapy in IBD to reveal non-compliance or underdosing, and can be used as a practical tool to optimize thiopurine therapy, especially in case of thiopurine non-response.

Topics: Adolescent; Adult; Aged; Azathioprine; Biomarkers, Pharmacological; Chromatography, High Pressure Liquid; Cross-Sectional Studies; Disease Progression; Dose-Response Relationship, Drug; Drug Monitoring; Female; Genetic Markers; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Maintenance Chemotherapy; Male; Medication Adherence; Mercaptopurine; Methyltransferases; Middle Aged; Prospective Studies; Severity of Illness Index; Thioinosine; Thionucleotides; Young Adult

This study examined the role of thiopurine methyltransferase (TPMT) polymorphism in the metabolism and clinical effects of azathioprine and 6-mercaptopurine in the treatment of inflammatory bowel disease and childhood leukemia. The current hypothesis is that the cytotoxic effects of thiopurines are caused by the incorporation of thioguanine nucleotides into DNA. In this context, S-methylation catalyzed by TPMT can be regarded as a competing metabolic pathway. The authors assayed the TPMT activity in red blood cells from 122 patients treated with azathioprine or 6-mercaptopurine (83 adults with inflammatory bowel disease and 39 children with acute lymphoblastic leukemia) and in 290 untreated controls (219 adult blood donors and 71 children). The concentrations of thioguanine nucleotides and methylthioinosine monophosphate were also assayed in red blood cells from the patients. The TPMT activity and the concentrations of methylthioinosine monophosphate and thioguanine nucleotides were higher in children than in adults. All children but no adult patient received concomitant methotrexate. Interaction between methotrexate and 6-mercaptopurine has been described, and may explain the results. Low TPMT activity in adult patients with inflammatory bowel disease correlated to an increased incidence of adverse drug reactions. However, there was no correlation between TPMT activity and the red blood cell concentrations of methylthioinosine monophosphate or thioguanine nucleotides, or between the concentrations of these metabolites and the occurrence of adverse effects. The results show that the role of thiopurine metabolism for drug effects is complex.

Topics: Adult; Age Factors; Antimetabolites, Antineoplastic; Azathioprine; Child; Drug Therapy, Combination; Erythrocytes; Female; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methotrexate; Methyltransferases; Polymorphism, Genetic; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thioinosine; Thionucleotides

Inhibition of pyrimidine and purine synthesis has been demonstrated to potentiate 5-fluorouracil (5-FU) activity in preclinical models. Low-dose phosphonacetyl-L-aspartate (PALA) potentiates the incorporation of 5-FU into RNA, without detectably increasing its toxicity. 6-Methylmercaptopurine riboside (MMPR) results in inhibition of purine biosynthesis with elevation of phosphoribosyl pyrophosphate (PRPP), which in turn is believed to increase the phosphorylation and intracellular retention of 5-FU. We conducted a phase I clinical trial to determine the maximum tolerated dose of 5-FU in combination with low-dose PALA and a biochemically-optimized dose of MMPR. The regimen consisted of PALA 250 mg/m2 given on day 1, followed 24 h later by MMPR 150 mg/m2, and escalating doses of 5-FU from 1625 to 2600 mg/m2 by 24 h continuous infusion. This regimen was repeated weekly. A group of 29 patients with a diagnosis of malignant solid tumor were entered; their median performance status was 1. The dose-limiting toxicity was mucositis, while other gastrointestinal toxicity was minimal. Two patients also experienced ischemic chest pain during the 5-FU infusion. The maximum tolerated dose of 5-FU in this combination was 2600 mg/m2. Several responses were observed including a complete remission in a previously treated breast cancer patient and two partial responses in breast and colon cancer. MMPR pharmacokinetics were obtained from urine analyses in 21 patients on this trial; there was no correlation between the pharmacokinetics of MMPR and the toxicity observed. This regimen was well tolerated and phase II trials are warranted using PALA 250 mg/m2, MMPR 150 mg/m2, and 5-FU 2300 mg/m2 by continuous infusion over 24 h.

Topics: Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Antineoplastic Agents; Aspartic Acid; Female; Fluorouracil; Humans; Infusions, Intravenous; Male; Middle Aged; Neoplasms; Phosphonoacetic Acid; RNA, Neoplasm; Thioinosine; Thionucleotides

Topics: Chromatography, High Pressure Liquid; Guanine Nucleotides; Laboratories, Hospital; Limit of Detection; Quality Control; Reference Standards; Thioinosine; Thionucleotides

The implication of inosine triphosphate pyrophosphatase (ITPA) on thiopurine drug response variability has been investigated but little data are available on its role on thiopurine metabolites. The ability of ITPA to modify the thiopurine metabolite levels is currently used to optimize azathioprine (AZA) therapy in relation to thiopurine S-methyltransferase (TPMT) activity, the aim of this study is to investigate ITPA phenotype in a large population and to evaluate the relation between ITPA and TPMT activities and thiopurine metabolites.. ITPA activity was determined in 183 adults and 138 children with or without AZA therapy. 6-thioguanine nucleotides (6-TGN), 6-methylmercaptopurine nucleotides (6-MeMPN) levels, and ITPA as well as TPMT activities were measured in red blood cells. Using the Gaussian mixture model, distribution of ITPA activity was evaluated. Intraindividual variability and influence of age, sex, AZA treatment and associated co-medications on ITPA activity were also assessed.. This retrospective study shows a quadrimodal distribution in ITPA activity. No influence of age, sex, AZA therapy, and co-medications was found. In adults, ITPA activity was not significantly associated with 6-TGN or 6-MeMPN concentrations, whereas a weak negative correlation was observed with 6-MeMPN levels in pediatric populations (rs = -0.261; P = 0.024). A weak positive correlation was observed between ITPA and TPMT activities in children (rs = 0.289; P = 0.001).. ITPA activity was poorly influenced by nongenetic parameters and has no influence on 6-TGN and 6-MeMPN concentrations in adults and only a weak correlation with 6-MeMPN and TPMT activity in children. These results demonstrate that ITPA is not a rate-limiting enzyme in the formation of 6-TGN but suggest that a decrease in ITPA activity in children may be a risk factor for accumulation of 6-MeMPN in cells.

Topics: Adolescent; Adult; Aged; Azathioprine; Child; Child, Preschool; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Infant; Male; Methyltransferases; Middle Aged; Phenotype; Pyrophosphatases; Retrospective Studies; Thioinosine; Thionucleotides; Young Adult

The conventional thiopurines azathioprine and mercaptopurine are considered maintenance immunosuppressive drugs of choice in the treatment of inflammatory bowel disease (IBD). Unfortunately, treatment is often discontinued because of adverse events (AEs) or refractoriness, retrospectively associated with the high levels of the thiopurine metabolites 6-methylmercaptopurine ribonucleotides (6-MMPR). Patients with a clinically "skewed" thiopurine metabolism may be particularly at risk for therapy failure. We determined the predictive value of this pharmacological phenomenon in patients with IBD during regular thiopurine therapy.. Clinical effectiveness and tolerability of weight-based thiopurine therapy were determined in all patients with IBD displaying a skewed metabolism [ratio 6-MMPR/6-thioguanine nucleotide (6-TGN) >20]. All samples were routinely assessed between 2008 and 2012, as part of standard clinical follow-up after initiation of conventional thiopurine therapy.. Forty-one (84%) of 49 included patients with IBD discontinued thiopurines (55% female, 53% with Crohn disease) with a median duration of 14 weeks (range, 7-155). The majority of patients with a skewed metabolism discontinued thiopurines because of adverse events (55%) or refractoriness (12%). The most commonly observed adverse event was hepatotoxicity (18 patients, 37%). Median 6-TGN level was 159 pmol/8 × 10 RBC (range, 46-419), median 6-MMPR level was 11,020 pmol/8 × 10 RBC (range, 3610-43,670), and the median 6-MMPR/6-TGN ratio was 72 (range, 29-367). Thiopurine therapy failure was associated with a ratio above 50 (P < 0.03). Hepatotoxicity occurred more frequently in patients with an extremely skewed metabolism (6-MMPR/6-TGN ratio >100) (P < 0.01).. This study demonstrates that a routinely established skewed metabolism is a major risk factor for future thiopurine failure in patients with IBD. These observations imply that routine thiopurine metabolite measurements may be used as a prognostic tool to identify those patients with an aberrant-skewed metabolism at an early stage, possibly benefitting from therapy adjustments.

Topics: Adolescent; Adult; Aged; Azathioprine; Cohort Studies; Colitis, Ulcerative; Crohn Disease; Female; Follow-Up Studies; Guanine Nucleotides; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Middle Aged; Retrospective Studies; Risk Factors; Thioinosine; Thionucleotides; Treatment Failure; Young Adult

A drug interaction between infliximab and azathioprine has previously been reported in Crohn's disease patients: the concentration of the main active thiopurine metabolites, the 6-thioguanine nucleotides (6-TGN), increased 1-3 weeks after the first infliximab infusion by 50% compared to baseline. The aim of this prospective study was to determine the effect of adalimumab on thiopurine metabolism in Crohn's disease patients, evaluated by 6-TGN and 6-methylmercaptopurine ribonucleotides (6-MMPR) concentration measurement.. Crohn's disease patients on azathioprine or mercaptopurine maintenance therapy starting with concomitant adalimumab treatment were included. 6-TGN and 6-MMPR concentrations were determined before initiation of adalimumab and after 2, 4, 6 and 12 weeks of combination therapy. The activity of three essential enzymes involving thiopurine metabolism, thiopurine S-methyltransferase (TPMT), hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and inosine-triphosphate pyrophosphatase (ITPase), was evaluated at baseline and week 4. Clinical outcome was evaluated by the Crohn's disease activity index and C-reactive protein concentrations at baseline, week 4 and week 12.. Twelve Crohn's disease patients were analyzed. During the follow-up period of 12 weeks the median 6-TGN and 6-MMPR concentrations did not significantly change compared to baseline. TPMT, ITPase and HGPRT enzyme activity did not change either after 4 weeks. In two patients (17%) myelotoxicity was observed within 2-4 weeks, in whom both low therapeutic 6-TGN and 6-MMPR concentrations were found.. In this study in Crohn's disease patients no pharmacokinetic interaction was shown between adalimumab and the conventional thiopurines, azathioprine and mercaptopurine.

Topics: Adalimumab; Adolescent; Adult; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal, Humanized; Azathioprine; C-Reactive Protein; Crohn Disease; Drug Interactions; Drug Therapy, Combination; Erythrocytes; Female; Guanine Nucleotides; Humans; Hypoxanthine Phosphoribosyltransferase; Immunosuppressive Agents; Inosine Triphosphatase; Male; Methyltransferases; Middle Aged; Prospective Studies; Pyrophosphatases; Severity of Illness Index; Thioinosine; Thionucleotides; Young Adult

TPMT activity and metabolite determination (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) remain controversial during thiopurine management. This study assessed associations between patient characteristics and TPMT activity, and their impact on metabolite levels.. A retrospective review of the laboratory database from a French university hospital identified 7360 patients referred for TPMT phenotype/genotype determination, and/or for 6-TGN/6-MMPN monitoring.. Four TPMT phenotypes were identified according to TPMT activity distribution: low, intermediate, normal/high and very high. Based on 6775 assays, 6-TGN concentrations were 1.6-fold higher in TPMT-deficient patients compared with TPMT-normal patients. Azathioprine dose and TPMT genotype were significant predictors of metabolite levels. Furthermore, 6-MMPN and 6-MMPN: 6-TGN ratios were, respectively, 1.6- and 2.2-fold higher in females than in males, despite similar TPMT, 6-TGN and azathioprine doses. An unfavorable ratio (≥20) was associated with a slightly higher TPMT activity.. These results illustrate the usefulness of pharmacogenomics and metabolite measurement to improve the identification of noncompliance and patients at high risk for toxicity or therapeutic resistance. Original submitted 13 November 2013; Revision submitted 30 January 2014.

Topics: Adult; Azathioprine; Databases, Factual; Drug Monitoring; Female; Genotype; Guanine Nucleotides; Humans; Individuality; Male; Methyltransferases; Middle Aged; Pharmacogenetics; Phenotype; Retrospective Studies; Thioguanine; Thioinosine; Thionucleotides; Young Adult

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

Topics: Adolescent; Adult; Aged; Azathioprine; Cross-Sectional Studies; Drug Monitoring; Erythrocytes; Female; GTP Phosphohydrolases; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methylthioinosine; Middle Aged; Thioguanine; Thioinosine; Thionucleotides; Young Adult

Thiopurine therapy is often discontinued in inflammatory bowel disease (IBD) patients. The xanthine oxidase (XO) inhibitor allopurinol has previously shown to enhance thiopurine efficacy and to prevent adverse reactions, the mechanism of this beneficial interaction is not completely clarified. The aim of this study is to observe possible effects of allopurinol and low-dose thiopurine combination therapy on the activity of three pivotal thiopurine metabolizing enzymes.. A prospective study of IBD patients failing thiopurine therapy due to a skewed thiopurine metabolism was performed. Patients were treated with allopurinol and azathioprine or mercaptopurine. Xanthine oxidase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and thiopurine S-methyl transferase (TPMT) activities, and thiopurine metabolites concentrations were measured during thiopurine monotherapy, and after 4 and 12 weeks of combination therapy.. Of fifteen IBD patients, XO activity decreased from 0.18 (IQR 0.08-0.3) during thiopurine monotherapy to 0.14 (IQR 0.06-0.2) and 0.11 (IQR 0.06-0.2; p=0.008) mU/hour/ml at 4 and 12 weeks, respectively. HGPRT activity increased from 150 (IQR 114-176) to 180 (IQR 135-213) and 204 nmol/(h×mg protein) (IQR 173-213; p=0.013). TPMT activity seemed not to be affected. 6-Thioguanine nucleotide concentrations increased from 138 (IQR 119-188) to 235 (223-304) and to 265 pmol/8×10^8 (IQR 188-344), whereas 6-methyl mercaptopurine ribonucleotides concentrations decreased from 13230 (IQR 7130-17420) to 690 (IQR 378-1325) and 540 (IQR 240-790) pmol/8×10^8 at 4 and 12 weeks of combination therapy (both p<0.001).. Allopurinol and thiopurine combination-therapy seems to increase HGPRT and decrease XO activity in IBD patients, which at least in part may explain the observed changes in thiopurine metabolite concentrations.

Topics: Adult; Allopurinol; Azathioprine; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Fatigue; Female; Guanine Nucleotides; Humans; Hypoxanthine Phosphoribosyltransferase; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Nausea; Prospective Studies; Thioinosine; Thionucleotides; Xanthine Oxidase

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

Topics: Adolescent; Adult; Aged; Azathioprine; Biomarkers; Crohn Disease; Drug Administration Schedule; Drug Monitoring; Endoscopy, Gastrointestinal; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Intestinal Mucosa; Male; Middle Aged; Recurrence; ROC Curve; Severity of Illness Index; Thioguanine; Thioinosine; Thionucleotides; Treatment Outcome; Young Adult

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) into the DNA of thiopurine-treated cells causes cell death, but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Because the growth of MTAP-deleted tumor cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP(-)) transfected to express MTAP constitutively (A549-MTAP(+)). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP(-) conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dG(s) incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.

Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Deletion; Humans; Immunoblotting; Mercaptopurine; Neoplasms; Purine-Nucleoside Phosphorylase; Purines; Thioguanine; Thioinosine; Thionucleotides

There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5'-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5'-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated.. IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤ 20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities.. Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤ 20 (P < 0.001). Metabolite ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001).. These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

Topics: Adolescent; Adrenal Cortex Hormones; Adult; Aged; Aminosalicylic Acids; Child; Female; Guanine Nucleotides; HEK293 Cells; Humans; IMP Dehydrogenase; Inflammatory Bowel Diseases; Male; Methyltransferases; Middle Aged; Phenotype; Phosphorylation; RNA, Messenger; Thioinosine; Thionucleotides; Treatment Outcome; Young Adult

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

Topics: Adrenal Cortex Hormones; Adult; Aged; Azathioprine; Biomarkers; Cross-Sectional Studies; Female; Follow-Up Studies; Hepatitis, Autoimmune; Humans; Male; Methyltransferases; Middle Aged; Prognosis; Thioguanine; Thioinosine; Thionucleotides; Treatment Outcome

In a 23-year-old female with colonic Crohn's disease 6-mercaptopurine 100 mg daily (1.7 mg/kg) was added to mesalamine and prednisolone therapy because of ongoing disease activity. One month later she had fever and a pancytopenia. 6-methylmercaptopurine ribonucleotides levels were extremely elevated (57,000 pmol/8x10(8) red blood cells) and 6-thioguanine nucleotides levels were subtherapeutically (126 pmol/8x10(8) red blood cells). Genotyping showed a wildtype thiopurine S-methyltransferase TPMT(H/H) (*1/*1) genotype and a wildtype inosine triphosphate pyrophosphatase gene. TPMT and inosine triphosphate pyrophosphatase activity were normal. The pancytopenia recovered spontaneously within a few weeks, parallel with decreasing 6-methylmercaptopurine ribonucleotides levels after interrupting 6-mercaptopurine treatment. Epstein-Barrvirus, Cytomegalovirus and Herpesvirus infections were excluded by serology. This is the first report of pancytopenia due to extremely high 6-methylmercaptopurine ribonucleotides levels. No relation was found with the genotype of TPMT and inosine triphosphate pyrophosphatase enzymes, which play key roles in the thiopurine metabolic pathway. Apparently, 6-methylmercaptopurine ribonucleotides metabolites can cause pancytopenia, as was already known for 6-thioguanine nucleotides.

Topics: Adult; Crohn Disease; Female; Humans; Mercaptopurine; Methyltransferases; Pancytopenia; Thioinosine; Thionucleotides

The studies described indicate that me-t-IMP formation is an important pathway, contributing to cytotoxicity in Molt F4 cells, which exhibit a highly active de novo purine synthesis. On three levels cytotoxicity is induced during methylation of thiopurines. 1. Purine synthesis de novo is inhibited during formation of me-t-IMP. Inhibition of PDNS results in depletion of purine nucleotides, with subsequently diminishing DNA and RNA synthesis. 2. The increased PRPP levels, induced by me-T-IMP, induce increased pyrimidine biosynthesis and cause an imbalance in purine nucleotides. This imbalance may lead to inhibition of cell growth and after prolonged exposure, to cell death. 3. The observed depletion of SAM and the decrease of the SAM/SAH ratio may be an additional mechanism by which 6MP and me-MPR exert their effects on cell growth and cell viability. Changes in SAM/SAH ratio may directly influence methylation reactions. The significant decrease of DNA methylation by 6MP and me-t-IMP may influence gene regulation and tumor progression. Administration of SAM leads to chemoprevention of rat liver carcinogenesis, indicating a role of DNA methylation in tumor progression. Besides the effects on methylation of DNA, a decrease of SAM/SAH ratio may also affect other processes, such as methylation of RNA, proteins and phospholipids, thereby disturbing their functionality. In conclusion, decrease of the SAM/SAH ratio resulting from treatment with 6MP and me-MPR may exert many effects in these cells. This may open a new field of research, possibly contributing to a deeper understanding of the complex mechanisms by which 6MP provokes cytotoxicity.

Topics: Antimetabolites, Antineoplastic; Cell Death; DNA, Neoplasm; Humans; Mercaptopurine; Methylation; S-Adenosylhomocysteine; S-Adenosylmethionine; Thioinosine; Thionucleotides; Tumor Cells, Cultured

6-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.

Topics: Adenine; Adenosine; Antineoplastic Agents; Cell Division; Cell Survival; Cells, Cultured; Guanosine; Humans; Inosine; Mercaptopurine; Methylthioinosine; Nucleosides; Nucleotides; Purine Nucleosides; Ribonucleosides; T-Lymphocytes; Thioinosine; Thionucleotides

The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 microM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP from Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.

Topics: Cell Division; Cell Survival; Humans; Inosine Monophosphate; Mercaptopurine; Methylthioinosine; Phosphoribosyl Pyrophosphate; Purine Nucleotides; T-Lymphocytes; Thioinosine; Thionucleosides; Thionucleotides; Tumor Cells, Cultured

Cytotoxicity of 6-mercaptopurine (6MP) and 6-methylmercaptopurine ribonucleoside (Me-MPR) was studied in Molt F4 human malignant lymphoblasts. Both drugs are converted into methylthioIMP (Me-tIMP), which inhibits purine de novo synthesis. Addition of amidoimidazole carboxamide ribonucleoside (AICAR) circumvented inhibition of purine de novo synthesis, and thus partly prevented 6MP and Me-MPR cytotoxicity. Purine nucleotides, and especially adenine nucleotides, were recovered by addition of AICAR. Under these conditions, Me-tIMP formation decreased. The results of this study indicate that formation of Me-tIMP may be important for 6MP cytotoxicity in Molt F4 cells. These data suggest that depletion of adenine nucleotides is the main cause for Me-tIMP cytotoxicity.

Topics: Adenine Nucleotides; Aminoimidazole Carboxamide; Cell Count; Cell Death; Drug Interactions; Guanine Nucleotides; Humans; Mercaptopurine; Methylthioinosine; Ribonucleosides; Thioinosine; Thionucleosides; Thionucleotides; Time Factors; Tumor Cells, Cultured

Methotrexate pretreatment of L1210 cells had been shown previously by us to cause an enhancement of the intracellular accumulation of 5-fluorouracil and of the formation of 5-fluorouracil nucleotides which was correlated with synergistic cytotoxicity. This effect of methotrexate was associated with increases in 5-phosphoribosyl-1-pyrophosphate, the cofactor required for the conversion of 5-fluorouracil to 5-fluorouridine-5'-monophosphate (FUMP). Because these influences on 5-fluorouracil metabolism were most likely mediated by the activity of methotrexate as an inhibitor of purine synthesis, the effects of other agents that inhibit purine synthesis were examined. An inhibitor of amidophosphoribosyltransferase, 6-methylmercaptopurine ribonucleoside, the glutamine antagonists, azaserine and 6-diazo-5-oxo-L-norleucine (DON), and the L-aspartate analogue inhibitor of adenylsuccinate synthetase, L-alanosine, all reduced the incorporation of [1-14C]glycine into adenine and guanine bases isolated from nucleic acids. Each drug also resulted in intracellular elevations of 5-phosphoribosyl-1-pyrophosphate that were 15- to 25-fold greater than control levels. These alterations in de novo purine nucleotide synthesis were associated with enhanced intracellular 5-fluorouracil accumulation and synergistic cytotoxicity.

Topics: Alanine; Animals; Azaserine; Cell Line, Tumor; Cell Survival; Diazooxonorleucine; Dose-Response Relationship, Drug; Fluorouracil; Methotrexate; Nucleic Acid Synthesis Inhibitors; Purines; Thioinosine; Thionucleotides; Uracil Nucleotides