6-thioguanine Nucleotides Research Articles

Mercaptopurine metabolite levels are predictors of bone marrow toxicity following high-dose methotrexate therapy of childhood acute lymphoblastic leukaemia.

High-dose methotrexate (HD-MTX) courses with concurrent oral low-dose MTX/6-mercaptopurine (6MP) for childhood acute lymphoblastic leukaemia (ALL) are often followed by neutro- and thrombocytopenia necessitating treatment interruptions. Plasma MTX during HD-MTX therapy guides folinic acid rescue to prevent toxicities, but myelosuppression can also be prevented by pre-HD-MTX 6MP dose reductions. Accordingly, we monitored pre-HD-MTX erythrocyte levels of methylated 6MP metabolites (Ery-MeMP) and of thioguanine nucleotides (Ery-6TGN) as well as DNA-incorporated thioguanine nucleotides (DNA-TGN) in circulating leucocytes to identify patients at highest risk of post-HD-MTX myelosuppression. In multiple linear regression analyses of neutrophil and thrombocyte nadir values (adjusted for gender, age, risk group and 6MP dose) after 48 HD-MTX courses in 17 childhood ALL patients on MTX/6MP maintenance therapy, the pre-HD-MTX DNA-TGN levels in neutrophils (P<0.0001), Ery-MeMP (P<0.0001) and Ery-6TGN (P=0.01) levels were significant predictors of post-HD-MTX neutrophil nadirs, whereas Ery-MeMP (P<0.0001) was the only predictor of post-HD-MTX thrombocyte nadir. In conclusion, pre-HD-MTX 6MP metabolite levels may be applicable for 6MP dose adjustments to prevent HD-MTX-induced myelosuppression.

A Rapid and Robust HPLC- DAD Method for the Monitoring of Thiopurine Metabolites in Whole Blood: Application to Paediatric Patients with Inflammatory Bowel Disease

Mercaptopurine (MP) and azathioprine (AZA) are thiopurine drugs that are used in paediatrics for the treatment of inflammatory bowel disease (IBD), Therapeutic drug monitoring (TDM) of thiopurine active metabolites, 6-methylmercaptopurine (6MMP) and 6-thioguanine nucleotides (6-TGN) is a common practice for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new High Performance Liquid Chromatography coupled to Diode Array Detection (HPLC-DAD) method for the simultaneous quantification of 6-TGN and 6-MMP in whole blood with a total runtime of 7.5 minutes over a wide range of concentrations (50 – 2000 pmol/8x10^8 RBC for 6-TG and 20 – 15000 pmol/8x10^8 RBC for 6-MMP respectively). The new method has been validated following international guidelines on bioanalytical method validation. Within- and between-run precision and accuracy results for quality control samples were always ≤ 15% of the nominal concentrations. The correlation with our reference method was excellent. The new HPLCDAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses.

P588. A prospective evaluation of adalimumab drug levels and anti-drug antibodies using two commercial ELISA and the influence of 6-thioguanine nucleotides amongst patients with Crohn's disease

P588. A prospective evaluation of adalimumab drug levels and anti-drug antibodies using two commercial ELISA and the influence of 6-thioguanine nucleotides amongst patients with Crohn's disease

Change in erythrocyte mean corpuscular volume during combination therapy with azathioprine and infliximab is associated with mucosal healing: a post hoc analysis from SONIC.

The adequacy of exposure of purine analogs as measured by 6-thioguanine nucleotides concentrations in the setting of combination therapy remains poorly understood. The aim of this study was to investigate the relationship between the mean corpuscular volume (MCV) value (as a surrogate marker of 6-thioguanine nucleotides concentration) and Crohn's disease outcomes in the setting of combination therapy with infliximab. The SONIC trial was a randomized controlled trial comparing infliximab to azathioprine and to infliximab plus azathioprine in 508 Crohn's disease patients. An increase of at least 7 femtoliter (fL) of the MCV (ΔMCV) was used for statistical analysis. At week 26, the mean increase of MCV was similar among patients treated with azathioprine alone (mean of 7.9 fL) or in combination with infliximab (mean of 8.5 fL). In the azathioprine group, 63.6% of patients with ΔMCV >7 were in steroid-free clinical remission at week 26 as compared with 33.3% of patients without ΔMCV >7 (P = 0.0046). In the combination therapy group, ΔMCV above 7 was associated with mucosal healing (75.0% for ΔMCV >7 versus 47.1% for ΔMCV <7, P = 0.0172) but not with steroid-free clinical remission. Patients with a ΔMCV above 7 were more likely to have infliximab trough level above 3 μg/mL at week 30 (68.4% versus 38.8% for ΔMCV <7, P = 0.0032). These results suggest that ΔMCV above 7 (which is a surrogate for a higher 6-thioguanine nucleotides concentration) leads to improved Crohn's disease outcomes, even when combined with infliximab. It also suggests the possibility that a lower azathioprine exposure might be less effective in combination therapy.

Concentrations of 6-Thioguanine Nucleotide Correlate With Trough Levels of Infliximab in Patients With Inflammatory Bowel Disease on Combination Therapy

In patients with inflammatory bowel diseases, the combination of infliximab and thiopurines (such as 6-thioguanine) is more effective treatment than monotherapy. We assessed the correlation between serum levels of 6-thioguanine (6-TGN) and infliximab levels or antibodies to infliximab (ATI). We performed a cross-sectional study of 72 patients receiving maintenance therapy with infliximab and a thiopurine for inflammatory bowel disease at the Crohn's and Colitis Center of the University of Miami, FL. We collected clinical, endoscopic, and biochemical data, and levels of thiopurine metabolites. The primary outcomes were trough level of infliximab and the presence of ATI. Levels of 6-TGN correlated with those of infliximab (ρ, 0.53; P < .0001). The cut-off point of 6-TGN that best predicted a higher level of infliximab was 125 pmol/8 × 10(8) red blood cells (RBCs) (area under receiver operating characteristic, 0.86; P < .001). Patients in the lowest quartile of 6-TGN had infliximab levels that were similar to patients on no thiopurines (4.3 vs. 4.8 mcg/mL, respectively; P = .8). An infliximab level of 8.3 mcg/mL or greater was associated with mucosal healing. Only 8 patients (11%) had detectable ATI. Patients with 6-TGN levels less than 125 pmol/8 × 10(8) RBCs were significantly more likely to have ATI (odds ratio, 1.3; 95% confidence interval, 2.3-72.5; P < .01). Although 6-TGN levels of greater than 230 pmol/8 × 10(8) RBCs have been associated with improved outcomes in patients on monotherapy, a level of 6-thioguanine of 125 pmol/8 × 10(8) RBCs or greater may be adequate to achieve therapeutic levels of infliximab. In the long term, this may minimize the toxicity for patients on combination therapy.

Thiopurine metabolites variations during co-treatment with aminosalicylates for inflammatory bowel disease: effect of N-acetyl transferase polymorphisms.

To evaluate variation of the concentration of thiopurine metabolites after 5-aminosalicylate (5-ASA) interruption and the role of genetic polymorphisms of N-acetyl transferase (NAT) 1 and 2. Concentrations of thioguanine nucleotides (TGN) and methymercaptopurine nucleotides (MMPN), metabolites of thiopurines, were measured by high performance liquid chromatography in 12 young patients (3 females and 9 males, median age 16 years) with inflammatory bowel disease (6 Crohn's disease and 6 ulcerative colitis) treated with thiopurines (7 mercaptopurine and 5 azathioprine) and 5-ASA. Blood samples were collected one month before and one month after the interruption of 5-ASA. DNA was extracted and genotyping of NAT1, NAT2, inosine triphosphate pyrophosphatase (ITPA) and thiopurine methyl transferase (TPMT) genes was performed using PCR assays. Median TGN concentration before 5-ASA interruption was 270 pmol/8 x 10(8) erythrocytes (range: 145-750); after the interruption of the aminosalicylate, a 35% reduction in TGN mean concentrations (absolute mean reduction 109 pmol/8 × 10(8) erythrocytes) was observed (median 221 pmol/8 × 10(8) erythrocytes, range: 96-427, P value linear mixed effects model 0.0011). Demographic and clinical covariates were not related to thiopurine metabolites concentrations. All patients were wild-type for the most relevant ITPA and TPMT variants. For NAT1 genotyping, 7 subjects presented an allele combination corresponding to fast enzymatic activity and 5 to slow activity. NAT1 genotypes corresponding to fast enzymatic activity were associated with reduced TGN concentration (P value linear mixed effects model 0.033), putatively because of increased 5-ASA inactivation and consequent reduced inhibition of thiopurine metabolism. The effect of NAT1 status on TGN seems to be persistent even after one month since the interruption of the aminosalicylate. No effect of NAT1 genotypes was shown on MMPN concentrations. NAT2 genotyping revealed that 6 patients presented a genotype corresponding to fast enzymatic activity and 6 to slow activity; NAT2 genotypes were not related to thiopurine metabolites concentration in this study. NAT1 genotype affects TGN levels in patients treated with thiopurines and aminosalicylates and could therefore influence the toxicity and efficacy of these drugs; however the number of patients evaluated is limited and this has to be considered a pilot study.

Impact of 6 Mercaptopurine (6MP) Pill-Taking Habits on Adherence, Thioguanine Nucleotide (TGN) Levels and Relapse Risk in Children with Acute Lymphoblastic Leukemia (ALL): Results from a Children’s Oncology Group (COG) Study (AALL03N1)

▪Background: Exposure to oral 6MP for ~2 years is critical for durable remissions in children with ALL. We have previously shown that poor 6MP adherence (rates <95%) is prevalent and increases relapse risk (JCO 30[17]:2094-101). 6MP is supplied in 50 mg tablets that may be swallowed, crushed, or chewed; patients are generally instructed to take 6MP with non-dairy liquid, in the evening on an empty stomach (~1-2h after a meal). These restrictive instructions could impose challenges in 6MP ingestion; the need for such restrictions with respect to clinical outcomes has not been examined systematically. This study examines the pill-taking habits of children with ALL and their association with adherence, risk of relapse and red cell TGN levels.Methods: Participants included 441 patients with ALL in first remission receiving oral 6MP (75 mg/m2/d) during maintenance. Adherence was measured using Medication Event Management System (MEMS) that recorded dates/times of each 6MP bottle opening for 6 mo/pt (58,086 patient-days of MEMS data). Average adherence rate (days MEMS bottle opened/days 6MP prescribed) over the 6m study period was computed to define non-adherence (adherence rate <95%). A 37-item questionnaire elicited pill-taking habits: i) 6MP never with food vs. with food; ii) 6MP never with dairy vs. with dairy; iii) 6MP pill swallowing (whole, crushed, chewed); iv) time of day 6MP always taken (evening, night, morning, midday, varying times); v) use of a routine: established routine vs. no routine. Monthly TGN levels (~6/pt) yielded 1,395 measurements. 6MP dose intensity [DI] was defined as dose prescribed/ protocol dose, and inherited capacity to metabolize 6MP was characterized by thiopurine methyltransferase (TPMT) genotype. Only TPMT wild-type patients were included in this analysis. 38 relapses occurred after a median follow-up of 6.1 years from start of maintenance (cumulative incidence of relapse: 8.9±1.4% at 6 years).Results: Median age at diagnosis was 6y (2-20); 67% were males; 40.3% had high-risk disease. 43.8% of the patients were non-adherent. Pill-taking habits and non-adherence: Time-varying generalized estimating equations (adjusted for age at study, ethnicity/race, income, and parental education) identified the following predictors of non-adherence: i) Taking 6MP with dairy (Odds Ratio [OR]=1.9, 95% Confidence Interval [CI]=1.3-2.9, p=0.003); ii) Taking 6MP at varying times vs. non-varying times (OR=2.4, 95%CI=1.2-4.7, p=0.008); iii) No routine (OR=1.7, 95%CI=1.1-2.7, p=0.02). Crushing the pill vs. swallowing whole was less likely to be associated with non-adherence (OR=0.7, 95%CI=0.4-0.99, p=0.05) [Fig 1A].Pill-taking habits and relapse: A proportional sub-distribution hazards model (adjusted for time from start of maintenance, NCI risk group, cytogenetics, race/ethnicity, age at study, and adherence) demonstrated no association between pill-taking habits and relapse risk: i) 6MP with food (Hazard Ratio [HR]=0.7, 95%CI=0.3-1.9, p=0.5); ii) 6MP with dairy (HR=0.3, 95%CI=0.07-1.5, p=0.2); iii) Swallowing tablet whole vs. crushing/chewing (HR=0.8, 95%CI=0.3-2.1, p=0.7); iv) 6MP in the evening/night vs. morning/mid-day (HR=1.1, 95%CI=0.2-7.5, p=0.9); v) 6MP at varying times vs. non-varying times (HR=1.0, 95%CI=0.2-4.5, p=0.9) [Fig 1B].Pill-taking habits and DI-adjusted TGN levels: Among adherent patients, there was no association between DI- and age-adjusted TGN levels and: i) 6MP with food (p=0.5); ii) 6MP with dairy (p=0.7); iii) Swallowing 6MP whole vs. chewing/crushing (p=0.4); iv) 6MP in the morning/midday vs. evening/night (p=0.5); v) 6MP at varying times vs. non-varying times (p=0.5) [Fig 1C].Conclusions: The association between non-adherence and certain pill-taking habits identifies a subgroup of patients that either did not receive appropriate instructions or did not adhere to them; our study was not designed to discern between the two scenarios. However, pill-taking habits were not associated with either relapse risk or TGN levels. These findings suggest that the commonly-practiced restrictions surrounding 6MP ingestion (i.e., taking only in the evening without food/dairy) might not influence outcome. [Display omitted] [Display omitted] [Display omitted] DisclosuresEvans:St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties. Relling:St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties.

Myelotoxicity after high-dose methotrexate in childhood acute leukemia is influenced by 6-mercaptopurine dosing but not by intermediate thiopurine methyltransferase activity.

Through enhancement of 6-mercaptopurine (6MP) bioavailability and inhibition of purine de novo synthesis, high-dose methotrexate (HD-MTX) may increase incorporation into DNA of 6-thioguanine nucleotides, the cytotoxic metabolites of 6MP. Patients with intermediate activity of thiopurine methyltransferase (TPMT(IA)) have higher cytosol 6-thioguanine nucleotide levels. We investigated toxicity following HD-MTX during MTX/6MP maintenance therapy in relation to 6MP and TPMT. Using linear mixed models, we explored myelo- and hepatotoxicity in relation to 6MP dosage and TPMT phenotype following 1,749 HD-MTX courses to 411 children with acute lymphoblastic leukemia on maintenance therapy. The degree of myelosuppression following HD-MTX was similar for patients with TPMT(IA) and patients with high TPMT activity (TPMT(HA)), when HD-MTX started with same blood counts and 6MP doses. However, since TPMT(IA) had lower blood counts at initiation of HD-MTX compared with TPMT(HA) patients (median WBC 2.8 vs. 3.3 × 10⁹/L, P = 0.01; median ANC 1.4 vs. 1.7 × 10⁹/L, P = 0.02), TPMT(IA) continued to have lower WBC and ANC levels compared with TPMT(HA) during all 28 days after HD-MTX [relative difference 9 % (95 % CI 2-17), P = 0.02 and 21 % (95 % CI 6-39), P = 0.005]. Still, the fractional decrease in WBC and ANC levels after HD-MTX did not differ between TPMT(IA) and TPMT(HA) patients (P = 0.47; P = 0.38). The degree of leukopenia, neutropenia, thrombocytopenia and rise in aminotransferases were all significantly related to 6MP dose (P < 0.001 for all analyses). For both TPMT(IA) and TPMT(HA) patients, dose of 6MP prior to HD-MTX should be guided by pre-HD-MTX blood counts, but not by TPMT activity.

Thiopurine metabolite ratios for monitoring therapy in pediatric Crohn disease.

Thiopurines (azathioprine, 6-mercaptopurine) are a mainstay of treatment in Crohn disease (CD). Monitoring intracellular metabolite (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine [6-MMP]) levels can help optimize therapeutic efficacy and minimize potential toxicity. Determination of 6-MMP/6-TGN ratios may provide additional useful information, such as the identification of individuals with excessive thiopurine methyltransferase activity and disadvantageous 6-MMP overproduction. These patients are at increased risk of therapeutic failure and hepatotoxicity. The aim of the study was to evaluate the correlation of 6-MMP/6-TGN ratios with therapeutic efficacy and risk of hepatotoxicity in CD. The present study was a single-center cross-sectional study including pediatric patients with CD studied prospectively with clinical and laboratory assessments along with serial measurements of 6-MMP and 6-TGN. Clinical response was determined using established clinical indices. The study included 238 pediatric patients with CD with a total of 1648 evaluation points. The patients were in steroid-free remission at 59.1% of the evaluation points. 6-MMP/6-TGN ratios of 4 to 24 were protective against relapse (odds ratio [OR] 0.52, 95% confidence interval [CI] -0.39 to 0.69, P = 0.001). Hepatotoxicity was associated with high 6-MMP levels (>3919 pmol/8 × 10 red blood cell count: OR 7.65, 95% CI 3.7-15.9, P = 0.001) and high 6-MMP/6-TGN ratios (>24: OR 5.35, 95% CI -3.43 to 8.43, P = 0.001). We observed significant associations between 6-MMP/6-TGN ratios and clinical response, and risk of hepatotoxicity. Our results suggest that determination of thiopurine metabolite ratios is a valuable tool for identification of patients at increased risk of therapeutic failure and hepatotoxicity.

Hematologic Indices as Surrogate Markers for Monitoring Thiopurine Therapy in IBD

Clinical efficacy and risk of complications are associated with intracellular levels of thiopurine metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurines (6-MMP) in patients with Crohn's disease. Therapeutic monitoring of thiopurine metabolites is not widely available. Surrogate markers such as hematologic indices (MCV, leukopenia) have been proposed. To evaluate accuracy of hematologic indices for prediction of therapeutic levels of thiopurine metabolites. A retrospective cross-sectional study. We included patients treated with thiopurines for IBD between February 2008 and November 2013. Hematologic indices were correlated with thiopurine metabolites and compared to pre-treatment levels. A total of 168 patients with 608 measurements were included. Macrocytosis was observed in 4.5 % of the patients. On multivariate analysis, macrocytosis was associated with 6-TGN levels >235 pmol/8 × 10(8) erythrocytes and 6-mmp levels >5,700 pmol/8 × 10(8) erythrocytes. Therapeutic 6-TGN levels were associated with MCV, ΔMCV, macrocytosis and lymphocyte count. Sensitivity and Spearman's r correlation for prediction of therapeutic metabolite levels were poor for all hematologic indices. Although macrocytosis and an elevated MCV are associated with therapeutic 6-TGN levels, the correlation is weak. None of the evaluated hematologic indices is a reliable surrogate marker for thiopurine metabolite levels.

Novel assay to improve therapeutic drug monitoring of thiopurines in inflammatory bowel disease

Background and aimsThe 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. MethodsSamples 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. ResultsTPMT 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 1500pmol/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 (R2>0.88). ConclusionsThe 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.

Transperineal ultrasound (TPUS) in pediatric perianal disease

titratedon therapeutic efficacy (response andadverse effects). Clinical efficacy was defined as disease activity score below 10. Results: Frequency of clinical efficacy was not different among early-onset and control group (respectively 40% and 57%, pvalue =0.50). However, early-onset patients required higher doses of azathioprine (median 2.8 vs 2.0mg/kg/day, p-value =0.00062), which resulted in comparable azathioprine active thioguaninenucleotide (TGN) metabolites concentrations (median 292 vs 345 U, p-value =0.96) and higher methyl-mercaptopurine-nucleotides (MMPN)metabolites in early-onset patients (median 1978 vs 1427 U, p-value =0.020). Interestingly, early-onset patients presented also higher MMPN/TGN ratio (median 7.6 vs 4.2, p-value =0.026). Conclusions: Early-onset IBD patients require higher azathioprine doses to achieve therapeutic response and present augmented inactivating azathioprine metabolism, putatively through increased activity of the enzyme thiopurine-S-methyl transferase.

P-099: Single center experience with infliximab in Japanese children with ulcerative colitis

P-099: Single center experience with infliximab in Japanese children with ulcerative colitis

Correlation of Pharmacokinetics of 6Mp and Its Metabolites with Haematological Toxicity: Step Towards Development of Pharmacokinetics Based Model for Optimal 6 Mp Dosing in Adult All

ABSTRACT Aim: Dosing of 6MP has remained empirical in acute lymphoblastic leukemia (ALL) with leucocyte count and thiopurine methyl transferase (TPMT) being the main determinants. However, TPMT alone cannot predict for marked inter-individual differences. In our experience, adult ALL patients tolerate standard 6- MP doses poorly, resulting in frequent dose interruptions or subtherapeutic doses. To address this issue we are conducting a study to develop pharmacokinetic model (PK) for optimal 6 MP dosing. Herein we report the preliminary data for correlationof PK parameters of 6 MP and its metabolites with toxicity (dose interruptions and dose reduction). Methods: Adult ALL patients (18 years or more) who were receiving 6 MP during interim maintenance (IM)or maintenance (M) phase of MCP841 protocol were enrolled after informed consent. Plasma samples were collected on day 1 for measuring 6-mercaptopurine levels using HPLC. TPMT activity was calculated by measuring 6-mMP (6-methylmercaptopurine) and 6-methyl thioguanine nucleotides (6-mTGN) levels in erythrocytes on day 1.Levels for 6-mMP and 6-TGN were also measured in erythrocytes using HPLC on day 8 and day 22 of the IM and on day 1 of M phase upto 6 months post enrollment. Statistical analysis was performed with receiver operating characteristic curve to assess the discrimination potential of level of 6-mp, 6-mMP (day 8 and day 22), 6-TGN (day 8 and day 22), 6-mMP and 6-mTGN levels (day 1-IM) for toxicity. Mean AUC has been compared for the above parameters using Mann-Whitney test. Binomial logistic regression was performed to assess correlation of various covariates with the toxicity. Results: 29 patients (males-24, females-5) with median age of 28 years (range,16-50) were enrolled.14 patients experienced dose interruptions (median-1, range1-2)) and 10 required dose reduction due to grade 4 cytopenias. OnDay 22 levels, a concentration of 0.315 ug/ml for 6-mMP (AUC = 0.73, p = 0.2) and 0.17 ug/ml for 6-TGN (AUC = 0.92,p value =0.01)in erythrocytes could successfully discriminate between patients who suffered from toxicity. Day 8 levels were not discriminatory. Hemoglobin (p= 0.05) and TPMT activity (p = 0.08) were found to be significant covariates for toxicity. Conclusions: Preliminary data does suggest the value of PK variables on 6 MP toxicity. Disclosure: All authors have declared no conflicts of interest.

Thiopurine monitoring in children with inflammatory bowel disease: a systematic review

The aim was to systematically review the evidence on the clinical usefulness of thiopurine metabolite and white blood count (WBC) monitoring in the assessment of clinical outcomes in children with inflammatory bowel disease (IBD). Medline, Embase, Cochrane Central Register of controlled trials and http://www.clinicaltrials.gov were screened in adherence to the PRISMA statement by two independent reviewers for identification of eligible studies. Eligible studies were randomized controlled trials (RCTs), cohort studies and large case series of children with inflammatory bowel disease (IBD) (<18 years) who underwent monitoring of thiopurine metabolites and/or WBC. Fifteen papers were identified (n = 1026). None of the eligible studies were RCTs. High 6-thioguanine nucleotide (6TGN) concentrations were not consistently associated with leucopenia. Leucopenia was not associated with achievement of clinical remission. A positive but not consistent correlation between 6TGN and clinical remission was reported. Haematological toxicity could not be reliably assessed with 6TGN measurements only. A number of studies supported the use of high 6-methylmercaptopurine ribonucleotides (6MMPR) as an indicator of hepatotoxicity. Low thiopurine metabolite concentration may be indicative of non-compliance. Thiopurine metabolite testing does not safely predict clinical outcome, but may facilitate toxicity surveillance and treatment optimization in poor responders. Current evidence favours the combination of thiopurine metabolite/WBC monitoring and clinic follow-up for prompt identification of haematologic/hepatic toxicity safe dose adjustment, and treatment modification in cases of suboptimal clinical outcome or non-compliance. Well designed RCTs for the identification of robust surrogate markers of thiopurine efficacy and toxicity are required.

Development of a rapid clinical TPMT genotyping assay

ObjectivesThiopurine compounds are commonly used in the treatment of childhood acute lymphoblastic leukemia, and as immunosuppressants following organ transplantation or for treatment of various autoimmune disorders. Thiopurine S-methyltransferase (TPMT) is required for detoxification, through S-methylation, of 6-thioguanine nucleotides (TGNs), a byproduct of thiopurine metabolism. Single nucleotide polymorphisms (SNPs) in the TPMT gene have been shown to affect its function, with some variants associated with serious clinical manifestations including severe to fatal myelosuppression and organ transplant rejection following treatment with standard thiopurine doses. In this study, we describe a TaqMan real time PCR allelic discrimination assay requiring minimal DNA input for TPMT genotyping. Design and methodsWe designed controls for the homozygous wild type and allelic variants of TPMT*2, *3B, and *3C. Genomic DNA was extracted from an additional 412 human blood samples. The samples were tested for the TPMT*2, *3B, *3C, and *3A polymorphisms by TaqMan genotyping assays using the AB 7500 FAST Real-Time PCR instrument. Allelic discrimination plots were used to identify each mutation. ResultsThe TaqMan assay correctly genotyped all custom control DNA samples. Of the 412 tested samples, our assay identified 375 samples as wild-type *1/*1 (91.02%), 3 as *1/*2 (0.73%), 1 as *1/*3B (0.24%), 3 as *1/*3C (0.73%), 27 presumed to be *1/*3A (6.55%), and 3 as *3B/*3A (0.73%). ConclusionsThe clinical implications of TPMT genotyping, along with the simplicity and specificity of the TaqMan genotyping assays make this test highly suitable for use in a clinical laboratory.

Harmful effects of the azathioprine metabolite 6-mercaptopurine in vascular cells: induction of mineralization.

Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP) on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans-differentiation of rat vascular smooth muscle cells in vitro. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in in vitro and ex vivo models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis.

PWE-088 The Impact Of Thiopurine Hypermethylation On Clinical Outcomes In Patients With Ibd

Introduction15% of patients with inflammatory bowel disease (IBD) prescribed azathioprine (AZA)/mercaptopurine (MP) demonstrate a skewed drug metabolism with low thioguanine nucleotides (TGN) and unexpectedly high methylmercaptopurine (MeMP) levels. This predicts...

PWE-080 Beware The Interaction Between Thiopurines And Warfarin

IntroductionIt is not uncommon for patients to require both immunosuppression and anticoagulation and for warfarin and thiopurines to be prescribed concurrently. There is limited evidence that thiopurines can inhibit the...

PWE-100 Increasing Weight And Body Mass Index Adversely Affect Thioguanine Nucleotide Levels In Inflammatory Bowel Disease

IntroductionInflammatory bowel disease (IBD) often requires long term immunosuppressive therapy with thiopurines such as azathioprine (AZA) or mercaptopurine (MP) and anti-tumour necrosis factor (TNF) agents. Despite the variable response to...