TPMT Activity Research Articles

Predictive role of ITPA genetic variants in thiopurine-related myelotoxicity in Crohn's disease patients.

Thiopurines, an effective therapy for Crohn's disease (CD), often lead to adverse events (AEs). Gene polymorphisms affecting thiopurine metabolism may predict AEs. This retrospective study in CD patients (n = 114) with TPMT activity > 5 Units/Red Blood Cells analyzed TPMT (c.238 G > C, c.460 G > A, c.719 A > G), ITPA (c.94 C > A, IVS2 + 21 A > C), and NUDT15 (c.415 C > T) polymorphisms. All patients received azathioprine (median dose 2.2 mg/kg) with 41.2% experiencing AEs, mainly myelotoxicity (28.1%). No NUDT15 polymorphisms were found, 7% had TPMT, and 31.6% had ITPA polymorphisms. AEs led to therapy modifications in 41.2% of patients. Multivariate analysis identified advanced age (OR 1.046, p = 0.007) and ITPA IVS2 + 21 A > C (OR 3.622, p = 0.015) as independent predictors of AEs. IVS2 + 21 A > C was also associated with myelotoxicity (OR 2.863, p = 0.021). These findings suggest that ITPA IVS2 + 21 A > C polymorphism and advanced age predict AEs during thiopurine therapy for CD with intermediate-normal TPMT activity.

TPMT gene polymorphisms (c.238G>C, c.460G>A and c.719A>G) in a healthy Venezuelan population.

Background: The presence of polymorphisms in the TPMT gene is associated with adverse effects in patients treated with standard doses of thiopurine drugs. Scientific evidence recognizes significant ethnic differences in their frequencies and how their early identification can prevent clinical complications. Methods: 150 healthy residents of Aragua, Venezuela were enrolled. The SNPs c.460G>A and c.719A>G were detected by PCR-restriction fragment length polymorphism assay and c.238G>C by allele-specific PCR. Results: All genotype polymorphisms were heterozygous. TPMT*1/*3A, TPMT*1/*3C and TPMT*1/*2 genotypes were found in 4.0, 2.0 and 0.7%, respectively. Conclusion: 6.7% of individuals have an intermediate TPMT activity. These findings support the importance of prior genotyping of TPMT in Venezuelan patients who require thiopurine drug therapy.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease

BackgroundThiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AimsThe aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. MethodsThe association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients’ erythrocytes by HPLC and associated with patients’ age group and TPMT DNA methylation. ResultsWhole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10−5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. ConclusionMethylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

IMPACT OF THIOPURINE DOSE IN COMBINATION THERAPY IN INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND Multiple studies have suggested that combination therapy with thiopurines and anti-TNFs is superior to monotherapy in Crohn’s disease (CD) and ulcerative colitis (UC). The optimal dose of thiopurines in combination therapy remains unclear. Our aim was to determine the impact of thiopurine dosing in combination therapy on the formation of anti-TNF antibodies and clinical outcomes in inflammatory bowel disease (IBD). METHODS This is a single-center, retrospective cohort study of all IBD patients treated with thiopurine and anti-TNF combination therapy between 1/1/2012 and 11/1/2020. Therapeutic dose of thiopurines was defined as ≥ 1 mg/kg for 6-mercaptopurine (6-MP) and ≥ 2 mg/kg for azathioprine (AZA). The primary outcome was anti-TNF antibody formation between patients on a therapeutic dose of thiopurines (TD) versus those on non-therapeutic doses (NTD). Secondary outcomes included steroid-free clinical remission (SFCR), endoscopic healing (absence of ulcers/erosions in CD and Mayo endoscopic score ≤ 1 for UC), and normal serum C-reactive protein (CRP) (defined as ≤ 5 mg/L) in patients who were on combination therapy. Continuous variables were analyzed using an unpaired student’s t-test. Categorical variables were analyzed using a chi-square test. RESULTS A total of 108 patients were included. The median age was 31.5 years (range 13-69), and 58.3% were male (Table 1). A total of 81 patients (75%) were on infliximab (IFX), while 27 patients (25%) were on adalimumab (ADA). Therapeutic dose of thiopurine was used in 19% (n=21). TPMT activity data was available for 56 patients. Normal TPMT activity was noted in 100% (n=7) in the TD group vs. 81.6% (n=40) in the NTD group (p=0.10). There were no significant differences between the TD vs. NTD groups in terms of baseline and disease characteristics (Table 2). Anti-TNF antibody formation was observed in 31 patients (32.1% of patients on IFX and 18.5% of patients on ADA). In the TD group 23.8% developed anti-TNF antibodies vs. 29.9% (p=0.58) in the NTD group. The mean anti-TNF drug level was 19.8 ± 13.8 vs. 16.9 ± 15.5 μg/mL (p=0.44) in the TD vs. NTD groups, respectively. SFCR was observed in 57.1% vs. 60.9% (p=0.75) and 38.1% vs. 37.9% discontinued anti-TNF treatment in the TD vs. NTD groups, respectively. Endoscopic healing was achieved in 55 % vs 60% (p=0.69), and normal CRP was observed in 52.4% vs. 46% (p=0.27) in the TD vs. NTD groups, respectively. CONCLUSION In our cohort, a therapeutic dose of thiopurine in combination therapy was not associated with lower rates of anti-TNF antibody formation compared to non-therapeutic doses. In addition, there were no significant differences in clinical outcomes such as normal CRP, SFCR, and endoscopic healing. Larger and prospective studies are needed to further elucidate the effect of thiopurine dosing in combination therapy for IBD.

P407 Thiopurines: use them or lose them? Results of an international survey

Abstract Background With the development of new drugs for inflammatory bowel disease (IBD), the role of thiopurines in the therapeutic algorithm of Crohn’s disease (CD) and Ulcerative Colitis (UC) is questioned. We aimed to investigate the current practice and future perspective of IBD physicians regarding the efficacy, safety, and role of precision medicine of thiopurines in IBD. Methods A 29-question web-based survey was developed and distributed to IBD physicians internationally, via national IBD associations and word of mouth. The final survey form was reviewed and approved by the clinical committee of ECCO. In the absence of a universal definition of an “IBD expert”, we defined them as senior physicians observing more than 20 patients per week. Results Complete answers from 408 physicians from 50 countries were obtained, the majority from European countries. Participants were mainly experienced physicians in IBD, with 26.0% meeting our definition of “IBD expert”. Only four physicians reported not using thiopurines in clinical practice. Azathioprine was the most used drug (97.1% of participants), while 61.3% of physicians reported using mercaptopurine; only 6.6% of participants used thioguanine. Most respondents used thiopurines in both monotherapy and in combination therapy, for both CD and UC, and consider them as effective treatments (Figures 1 and 2). Respondents tended to consider thiopurines as drugs with a good safety profile, with the agreement of 61.5% of the overall cohort; this was true especially in patients aged between 30 and 50 years old, with the agreement of 80.2% of the overall cohort (Figure 3). Access to TPMT activity assessment and metabolites measurement varied regionally. Physicians considered access to metabolites measurement as an important tool to improve thiopurines’ efficacy and safety, and to increase confidence when prescribing these drugs (Figure 4). Only a minority of the physicians (~6%) considered that thiopurines will not be used in the future in IBD, while 57.8% believed they will still be used both in mono and combination therapy in CD and UC. Conclusion Despite the many emerging treatments in IBD, thiopurines will still be part of the treatment algorithm of both CD and UC in the future. Access to tools for precision medicine varies around the globe, and wider implementation of these instruments, especially metabolite measurement, may improve confidence in thiopurines prescription, improving safety and efficacy.

The Examination of a TPMT Gene Before Administration of Azathioprine in Rheumatology Practice and Identification of a Novel Variant p.W29R.

Two-hundred patients were assessed for the presence of genetic allelic variants using PCR amplification and direct sequencing. In 19 patients, we detected genetic allelic variants affecting TPMT activity; in 1 case, it was an unpublished heterozygous variant c.85T>C (p.W29R); of those, 15 patients were switched from AZA to a different medication, and 1 patient was prescribed a reduced dose of AZA. Our findings show the importance of testing for variants of the TPMT gene before the administration of AZA in clinical rheumatology practice. Patients with documented episodes of leukopenia or elevated liver biochemical tests while on AZA should undergo TPMT genotype testing and/or TPMT enzyme activity testing.

Normal Ranges of Thiopurine Methyltransferase Activity Do Not Affect Thioguanine Nucleotide Concentrations With Azathioprine Therapy in Inflammatory Bowel Disease.

Thiopurine methyltransferase (TPMT) activity influences azathioprine conversion into active metabolite 6-thioguanine nucleotide (6-TGN). Low TPMT activity correlates with high 6-TGN and risk for myelosuppression. Conversely, normal-to-high TPMT activity may be associated with low 6-TGN and drug resistance, the so-called hypermetabolizers. Our aim was to identify the effect of normal-to-high TPMT activity on 6-TGN concentrations in an inflammatory bowel disease population. A retrospective chart review of patients aged ≥18 with inflammatory bowel disease, on azathioprine, with documented TPMT activity and 6-TGN concentration was performed. Correlations were evaluated via the Spearman rho correlation coefficient. Linear regression was used to determine the effect of TPMT activity on 6-TGN accounting for confounders. Relationships between TPMT activity, drug dose, and 6-TGN levels were defined via average causal mediation effects. One hundred patients were included. No correlation was observed between TPMT activity, azathioprine dosing, and metabolite concentrations. Overall, 39% of the cohort had a therapeutic 6-TGN level of >230 pmol/8 × 108 red blood cells (RBCs). No patient under 1 mg/kg achieved a therapeutic 6-TGN level, whereas 42% of patients taking 2.5 mg/kg did. The median 6-TGN concentration was higher for those in remission (254 pmol/8 × 108 RBCs, interquartile range: 174, 309) versus those not in remission (177 pmol/8 × 108 RBCs, interquartile range: 94.3, 287.8), though not significantly (P = 0.08). Smoking was the only clinical factor associated with 6-TGN level. On multivariate linear regression, only age, azathioprine dose, and obese body mass index were predictive of metabolite concentration. Variations within the normal range of TPMT activity do not affect 6-TGN concentration.

2504 Successful Budesonide Monotherapy for Autoimmune Hepatitis/Hepatic Sarcoidosis Overlap Syndrome in a Patient With Drug Reaction With Eosinophilia and Systemic Symptoms (DRESS) From Azathioprine

INTRODUCTION: Drug reaction with eosinophilia and systemic symptoms (DRESS) is considered a severe idiosyncratic drug hypersensitivity which can be fatal if not promptly diagnosed and treated. Here, we describe a case of hepatic sarcoidosis and autoimmune hepatitis (AIH) overlap syndrome who was found to have DRESS from taking azathioprine (AZA). CASE DESCRIPTION/METHODS: A 63-year-old African-American female with a diagnosis of AIH and hepatic sarcoidosis overlap based on liver biopsy (chronic hepatitis with moderate activity (grade 2/4), mild fibrosis (stage 1/4), non-caseating granulomata, and interface hepatitis with dense lymphoplasmacytic infiltration) (Figure 1), positive ANA (1:160), and elevated IgG (2140) was started on treatment with AZA and budesonide (intolerance to prednisone). She had normal TPMT activity. Patient stopped taking AZA few days later because of feeling “jittery.” She presented to the ED five weeks later with lip swelling and desquamating rash and was discharged with antihistamines. She returned to the ED two days later with worsening rash, fever, and dyspnea. Labs showed leukocytosis, eosinophilia (56.1%, 13,400/mm3), acute hepatitis, acute kidney injury, and respiratory distress with concern for DRESS. Her initial LFTs were ALT 96, AST 65, and Alk Phos 633, which were increased from her baseline. Imaging ruled out biliary obstruction and bone marrow biopsy was negative for myeloproliferative disorders such as hypereosinophilic syndrome. She was diagnosed with DRESS secondary to AZA and was treated with a short course of prednisone with transition to budesonide monotherapy. Eosinophilia and other organ dysfunction resolved. Her most recent LFTs are ALT 37, AST 42, and Alk Phos 277. DISCUSSION: While AZA hypersensitivity syndrome is a well-known disorder presenting with drug eruption and flu-like symptoms, to our knowledge this is the first case of DRESS secondary to AZA. Additionally, this occurred in the setting of hepatic sarcoidosis and AIH overlap syndrome which itself is a rare entity. DRESS is characterized by a long latency time between exposure and disease (2-8 weeks) and presents with end organ dysfunction requiring steroid treatment. Based on the European Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) scoring system, our patient's score was 7, where a score of 6 or higher is considered definite for DRESS. This case also stresses the value of budesonide monotherapy for treating AIH and hepatic sarcoidosis overlap when other first line medications cannot be used.

Novel motif of variable number of tandem repeats in TPMTpromoter region and evolutionary association of variable number of tandem repeats with TPMT*3alleles.

SNPs in the gene for TPMT exemplify one of the most successful translations of pharmacogenomics into clinical practice. This study explains the correlation between common SNPs and variable number of tandem repeats (VNTR) in promoter of the gene. We determined VNTR polymorphisms, as well as TPMT*2 and TPMT*3 SNPs and TPMT activity in Slovenian and Italian individuals and lymphoblastoid cell lines. We observed a previously unreported VNTR allele, AB7C, in a TPMT*3A heterozygous individual. VNTRs with two (AB2C) and three or more (ABnC, n≥3) B motifs were statistically significant in complete linkage disequilibrium (D'=1, r2=1, p<0.0001) with the TPMT*3C and TPMT*3A alleles, respectively. The study provides insights into the stepwise evolution of TPMT*3 alleles from *3C to *3A, with increasing number of B motifs in the VNTR region.

Association between TPMT*3C and decreased thiopurine S-methyltransferase activity in patients with neuromyelitis optica spectrum disorders in China

ABSTRACTAim of the study: Thiopurines are effective drugs in treating neuromyelitis optica spectrum disorders and other diseases. Thiopurines’ toxicity is mainly imputed to thiopurine S-methyltransferase activity. In Chinese population, the most common and important variation of thiopurine S-methyltransferase is TPMT*3C (rs1142345). This study aims to reveal the association between thiopurine S-methyltransferase activity and genetic polymorphisms of thiopurine S-methyltransferase in patients with neuromyelitis optica spectrum disorders in China.Material and methods: A liquid chromatography tandem mass/mass method was used to evaluate the thiopurine S-methyltransferase activity by using 6-mercapthioprine as the substrate in human erythrocyte haemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine. The amount of 6-methylmercaptopurine was adjusted by haematocrit and normalized to 8 × 108 erythrocytes. The selected polymorphisms of thiopurine S-methyltransferase were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique.Results: In 69 patients with neuromyelitis optica spectrum disorders, thiopurine S-methyltransferase activity was 80.29–154.53 (127.51 ± 16.83) pmol/h/8 × 108 erythrocytes. TPMT*3C (rs1142345) was associated with lower thiopurine S-methyltransferase activity (BETA = −25.37, P = 0.011). Other selected variants were not associated with thiopurine S-methyltransferase activity.Conclusions: TPMT*3C affects TPMT activity in Chinese patients with neuromyelitis optica spectrum disorders. Further studies are warranted to confirm the results.Abbreviations: TPRs = thiopurines; NMOSD = neuromyelitis optica spectrum disorders; TPMT = thiopurine S-methyltransferase; LC-MS/MS = liquid chromatography tandem mass/mass; 6-MMP = 6-methylmercaptopurine; IS = internal standard; SNP = single nucleotide polymorphism; MAF = minor allele frequency; HWE = Hardy–Weinberg equilibrium; BETA = regression coefficients; UTR-3 = untranslated region 3

Mercaptopurine and inflammatory bowel disease: the other thiopurine.

Data about use and effectiveness of mercaptopurine in inflammatory bowel disease are relatively limited. To assess the possible therapeutic indications, efficacy and safety of mercaptopurine as an alternative to azathioprine in inflammatory bowel disease. Retrospective observational study in patients treated with mercaptopurine in a total cohort of 1,574 patients with inflammatory bowel disease. One hundred and fifty-two patients received mercaptopurine, 15.7% of these patients as an initial thiopurine, 5.3% after azathioprine failure, and 79% after azathioprine intolerance. In 52.6% of patients (n = 80), adverse effects of mercaptopurine occurred, resulting in withdrawal in 49 of them. Mercaptopurine was effective in 39% of cases (95% CI 31-48%). In the remaining patients, failure was due mainly to withdrawal due to side effects (55.1%) and therapeutic step-up (33.7%). The average total time of mercaptopurine exposure was 36 months (IQR: 2-60). Myelotoxicity with mercaptopurine was more common in patients with intermediate TPMT activity than in those with normal activity (p = 0.046). In our setting, mercaptopurine is primarily used as a rescue therapy in patients with azathioprine adverse effects. This could explain its modest efficacy and the high rate of adverse effects. However, this drug is still an alternative in this group of patients, before a therapeutic step-up to biologics is considered.

Structural and functional impact of missense mutations in TPMT: An integrated computational approach

BackgroundThiopurine S-methyltransferase (TPMT) detoxifies thiopurine drugs which are used for treatment of various diseases including inflammatory bowel disease (IBD), and hematological malignancies. Individual variation in TPMT activity results from mutations in TPMT gene. In this study, the effects of all the known missense mutations in TPMT enzyme were studied at the sequence and structural level MethodsA broad set of bioinformatic tools was used to assess all the known missense mutations affecting enzyme activity. The effects of these mutations on protein stability, aggregation propensity, and residue interaction network were analyzed. ResultsOur results indicate that the missense mutations have diverse effects on TPMT structure and function. Stability and aggregation propensities are affected by various mutations. Several mutations also affect residues in ligand binding site. ConclusionsIn vitro study of missense mutation is laborious and time-consuming. However, computational methods can be used to obtain information about effects of missense mutations on protein structure. In this study, the effects of most of the mutations on enzyme activity could be explained by computational methods. Thus, the present approach can be used for understanding the protein structure-function relationships.

Physiologically based pharmacokinetic model for 6-mercpatopurine: exploring the role of genetic polymorphism in TPMT enzyme activity.

To extend the physiologically based pharmacokinetic (PBPK) model developed for 6-mercaptopurine to account for intracellular metabolism and to explore the role of genetic polymorphism in the TPMT enzyme on the pharmacokinetics of 6-mercaptopurine. The developed PBPK model was extended for 6-mercaptopurine to account for intracellular metabolism and genetic polymorphism in TPMT activity. System and drug specific parameters were obtained from the literature or estimated using plasma or intracellular red blood cell concentrations of 6-mercaptopurine and its metabolites. Age-dependent changes in parameters were implemented for scaling, and variability was also introduced for simulation. The model was validated using published data. The model was extended successfully. Parameter estimation and model predictions were satisfactory. Prediction of intracellular red blood cell concentrations of 6-thioguanine nucleotide for different TPMT phenotypes (in a clinical study that compared conventional and individualized dosing) showed results that were consistent with observed values and reported incidence of haematopoietic toxicity. Following conventional dosing, the predicted mean concentrations for homozygous and heterozygous variants, respectively, were about 10 times and two times the levels for wild-type. However, following individualized dosing, the mean concentration was around the same level for the three phenotypes despite different doses. The developed PBPK model has been extended for 6-mercaptopurine and can be used to predict plasma 6-mercaptopurine and tissue concentration of 6-mercaptopurine, 6-thioguanine nucleotide and 6-methylmercaptopurine ribonucleotide in adults and children. Predictions of reported data from clinical studies showed satisfactory results. The model may help to improve 6-mercaptopurine dosing, achieve better clinical outcome and reduce toxicity.

PWE-044 Exploring the efficacy of weight-based dosing of thiopurine therapy in relation to metabolite levels at week 12 in patients with inflammatory bowel disease

<h3>Introduction</h3> Traditionally patients commencing on thiopurine therapy in IBD have been dosed according to their body weight with data showing less than 70% achieve long term steroid free remission.¹The ability to measure thiopurine metabolites has shown that 6-thioguanine nucleotide (6-TGN) levels inversely correlate with disease activity and patients with sub-therapeutic concentrations are less likely to achieve remission. Also very high 6-TGN concentrations risk myelotoxicity and hepatic nodular regernative hyperplasia whilst 6-methyl mercaptopurine (6-MMP) concentrations above 5700 pmol/8 × 10⁸correlate with hepatotoxicity.² <h3>Method</h3> The primary aim of this study was to assess how effective weight based dosing is in achieving therapeutic 6-TGN levels after 12 weeks of therapy, in patients with normal TPMT activity, when commencing thiopurines for the first time at our institution for both Crohn’s and Ulcerative Colitis between Oct 2013 and July 2014. The incidence of potentially toxic thiopurine levels was also assessed along with symptoms, MCV, LFTs and disease activity indicies. All patients with normal TMPT levels were dosed at 2.5 mg/kg (azathioprine) or 1.5 mg/kg (mercaptopurine) with any dose adjustments made clinically based on blood results and symptoms. <h3>Results</h3> From 37 patients with normal TPMT activity (19 male) at week 12 on stable thiopurine weight based therapy 15 (41%) did not achieve therapeutic 6-TGN concentrations. 7 (19%) were under-dosed, 6 (16%) were overdosed and 2 (6%) preferentially metabolised to excessive 6-MMP levels. Disease activity indices, faecal calprotectin, MCV and routine blood monitoring for leucopenia and abnormal LFTs did not consistently correlate with metabolite levels at week 12. <h3>Conclusion</h3> Weight-based dosing does not appear to be an accurate method for establishing patients on thiopurine maintenance therapy and this may explain the previous perceived lack of efficacy of thiopurines in a significant number of patients. MCV cannot be used as a surrogate for sufficient 6-TGN levels at week 12 and of the 2 “shunters” neither had significantly abnormal LFTs highlighting the need to identify these group using metabolite monitoring to enable allopurinol co-prescribing. The lack of correlation with disease activity indicies at week 12 likely reflects the patients having recently completed an induction course of steroids and that thiopurines themselves are not induction agents. Starting patients on weight-based dosing plus metabolite monitoring at week 12 to achieve therapeutic ranges may increase long term maintenance rates and increase the safety of treatment by preventing toxicity and a larger long term outcome study is warranted. <h3>Disclosure of interest</h3> None Declared. <h3>References</h3> D9Haens G, <i>et al</i>. 1999;50:667–71 Moreau AC, <i>et al</i>. 2014;20:464–71

Molecular insight into thiopurine resistance: transcriptomic signature in lymphoblastoid cell lines.

BackgroundThere has been considerable progress in the management of acute lymphoblastic leukemia (ALL) but further improvement is needed to increase long-term survival. The thiopurine agent 6-mercaptopurine (6-MP) used for ALL maintenance therapy has a key influence on clinical outcomes and relapse prevention. Genetic inheritance in thiopurine metabolism plays a major role in interindividual clinical response variability to thiopurines; however, most cases of thiopurine resistance remain unexplained.MethodsWe used lymphoblastoid cell lines (LCLs) from healthy donors, selected for their extreme thiopurine susceptibility. Thiopurine metabolism was characterized by the determination of TPMT and HPRT activity. We performed genome-wide expression profiling in resistant and sensitive cell lines with the goal of elucidating the mechanisms of thiopurine resistance.ResultsWe determined a higher TPMT activity (+44%; P = 0.024) in resistant compared to sensitive cell lines, although there was no difference in HPRT activity. We identified a 32-gene transcriptomic signature that predicts thiopurine resistance. This signature includes the GTPBP4 gene coding for a GTP-binding protein that interacts with p53. A comprehensive pathway analysis of the genes differentially expressed between resistant and sensitive cell lines indicated a role for cell cycle and DNA mismatch repair system in thiopurine resistance. It also revealed overexpression of the ATM/p53/p21 pathway, which is activated in response to DNA damage and induces cell cycle arrest in thiopurine resistant LCLs. Furthermore, overexpression of the p53 target gene TNFRSF10D or the negative cell cycle regulator CCNG2 induces cell cycle arrest and may also contribute to thiopurine resistance. ARHGDIA under-expression in resistant cell lines may constitute a novel molecular mechanism contributing to thiopurine resistance based on Rac1 inhibition induced apoptosis and in relation with thiopurine pharmacodynamics.ConclusionOur study provides new insights into the molecular mechanisms underlying thiopurine resistance and suggests a potential research focus for developing tailored medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0150-6) contains supplementary material, which is available to authorized users.

Tu1332 Exploring the Efficacy of Weight-Based Dosing of Thiopurine Therapy in Relation to Metabolite Levels at Week 12 in Patients With Inflammatory Bowel Disease

Introduction Traditionally patients commencing on thiopurine therapy in IBD have been dosed according to their body weight with data showing less than 70% achieve long term steroid free remission. 1 The ability to measure thiopurine metabolites has shown that 6-thioguanine nucleotide (6-TGN) levels inversely correlate with disease activity and patients with sub-therapeutic concentrations are less likely to achieve remission. Also very high 6-TGN concentrations risk myelotoxicity and hepatic nodular regernative hyperplasia whilst 6-methyl mercaptopurine (6-MMP) concentrations above 5700 pmol/8 × 10 8 correlate with hepatotoxicity. 2 Method The primary aim of this study was to assess how effective weight based dosing is in achieving therapeutic 6-TGN levels after 12 weeks of therapy, in patients with normal TPMT activity, when commencing thiopurines for the first time at our institution for both Crohn’s and Ulcerative Colitis between Oct 2013 and July 2014. The incidence of potentially toxic thiopurine levels was also assessed along with symptoms, MCV, LFTs and disease activity indicies. All patients with normal TMPT levels were dosed at 2.5 mg/kg (azathioprine) or 1.5 mg/kg (mercaptopurine) with any dose adjustments made clinically based on blood results and symptoms. Results From 37 patients with normal TPMT activity (19 male) at week 12 on stable thiopurine weight based therapy 15 (41%) did not achieve therapeutic 6-TGN concentrations. 7 (19%) were under-dosed, 6 (16%) were overdosed and 2 (6%) preferentially metabolised to excessive 6-MMP levels. Disease activity indices, faecal calprotectin, MCV and routine blood monitoring for leucopenia and abnormal LFTs did not consistently correlate with metabolite levels at week 12. Conclusion Weight-based dosing does not appear to be an accurate method for establishing patients on thiopurine maintenance therapy and this may explain the previous perceived lack of efficacy of thiopurines in a significant number of patients. MCV cannot be used as a surrogate for sufficient 6-TGN levels at week 12 and of the 2 “shunters” neither had significantly abnormal LFTs highlighting the need to identify these group using metabolite monitoring to enable allopurinol co-prescribing. The lack of correlation with disease activity indicies at week 12 likely reflects the patients having recently completed an induction course of steroids and that thiopurines themselves are not induction agents. Starting patients on weight-based dosing plus metabolite monitoring at week 12 to achieve therapeutic ranges may increase long term maintenance rates and increase the safety of treatment by preventing toxicity and a larger long term outcome study is warranted. Disclosure of interest None Declared. References D9Haens G, et al . 1999;50:667–71 Moreau AC, et al . 2014;20:464–71

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.

From pharmacogenetics to pharmacometabolomics: SAM modulates TPMT activity.

In the present study, the influence of SAM on TPMT activity in vivo on human subjects was investigated. A total of 1017 donors from the Estonian Genome Center of the University of Tartu (Estonia) were genotyped for common TPMT variants, evaluated for TPMT activity, SAM levels, a set of 19 biochemical and ten hematological parameters and demographic data. After adjustment in multiple regression models and correction for multiple testing, from the 43 factors that were tested, only TPMT genotype (p = 1 × 10(-13)) and SAM levels (p = 1 × 10(-13)) were found to significantly influence TPMT activity. The influence of SAM on TPMT activity was more pronounced in TPMT-heterozygous than wild-type individuals. SAM represents a potential pharmacometabolomic marker and therapeutic agent in TPMT-heterozygous subjects.

PTU-106 Successful Pregnancies With Thiopurine-allopurinol Co-therapy For Inflammatory Bowel Disease

IntroductionCombination of low dose thiopurine with allopurinol can improve the clinical efficacy and bypass some of the adverse reactions of thiopurine monotherapy. Thiopurines can be used safely during pregnancy but...

Su1389 Successful Pregnancies With Thiopurine-Allopurinol Co-Therapy for Inflammatory Bowel Disease

Introduction Combination of low dose thiopurine with allopurinol can improve the clinical efficacy and bypass some of the adverse reactions of thiopurine monotherapy. Thiopurines can be used safely during pregnancy but there is scarce data regarding allopurinol. We report twelve cases of safe use of thiopurine and allopurinol co-therapy to manage IBD during pregnancy. Methods Patients were retrospectively identified at two hospitals in the UK and in Australia, using our local IBD databases. All pregnancies of co-therapy patients were included. TPMT activity and pre-pregnancy weight were used to calculate thiopurine dosing. Data regarding pregnancy and fetal outcomes were collected from patient notes. Results Eleven females on co-therapy became pregnant, totalling twelve pregnancies with eight live births (Table 1) and four ongoing pregnancies. There were no reported terminations, miscarriages or spontaneous pre-term deliveries ( Conclusion All twelve cases were treated successfully with co-therapy without any adverse pregnancy related events or adverse fetal outcomes. Intrauterine exposure of the fetus to thiopurine metabolites is not greater with combination therapy compared with thiopurine monotherapy. There are only two reports of congenital malformations with maternal allopurinol use. The case for an association based on two cases is weak, moreover a negative publication bias with respects to successful maternal allopurinal use is suspected. Our study provides support for clinicians and patients wishing to continue thiopurine-allopurinol co-therapy during pregnancy. Disclosure of Interest None Declared.