Thiopurine Methyltransferase Enzyme Activity Research Articles

Implications of Tioguanine Dosing in IBD Patients with a TPMT Deficiency.

Tioguanine is metabolised by fewer enzymatic steps compared to azathioprine and mercaptopurine, without generating 6-methylmercaptopurine ribonucleotides. However, thiopurine S-methyl transferase (TPMT) plays a role in early toxicity in all thiopurines. We aimed to describe the hazards and opportunities of tioguanine use in inflammatory bowel disease (IBD) patients with aberrant TPMT metabolism and propose preventative measures to safely prescribe tioguanine in these patients. In this retrospective cohort study, all determined TPMT genotypes (2016-2021) were evaluated for aberrant metabolism (i.e., intermediate and poor TPMT metabolisers). Subsequently, all IBD patients on tioguanine with aberrant TPMT genotypes were evaluated for tioguanine dosages, adverse drug events, lab abnormalities, treatment duration and effectiveness. TPMT genotypes were determined in 485 patients, of whom, 50 (10.3%) and 4 patients (0.8%) were intermediate and poor metabolisers, respectively. Of these patients, 12 intermediate and 4 poor TPMT metabolisers had been prescribed tioguanine in varying doses. In one poor TPMT metaboliser, tioguanine 10 mg/day induced delayed pancytopenia. In general, reduced tioguanine dosages of 5 mg/day for intermediate TPMT metabolisers, and 10 mg two-weekly for poor TPMT metabolisers, resulted in a safe, long-term treatment strategy. Diminished or absent TPMT enzyme activity was related with a pharmacokinetic shift of tioguanine metabolism which is associated with relatively late-occurring myelotoxicity in patients on standard tioguanine dose. However, in strongly reduced dose regimens with strict therapeutic drug and safety monitoring, tioguanine treatment remained a safe and effective option in IBD patients with dysfunctional TPMT.

Exposure to Azathioprine Metabolites and Clinical Outcome in Indian Patients with Crohn’s Disease

Aim: To determine the concentration of 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP), the interpatient variability, and the relationship with disease activity in patients with Chron’s disease on long-term stable doses of azathioprine (AZA). Methods: This is a prospective, tertiary care single-center hospital study in adult Chron’s disease patients treated with AZA. The quantification of phenotypic thiopurine methyltransferase enzyme activity in red blood cells and the estimation of the concentration of 6-TGN and 6-MMP in whole blood was performed using the HPLC-UV detector method. A clinical response was categorized as remission (Harvey-Bradshaw Index [HBI] < 5) or improvement (drop from baseline of at least three points of HBI) based on HBI. Exposure to metabolite concentrations and the clinical response to AZA treatment was observed. Results: Study analysis included 30 patients who were initiated on AZA, and they were followed up with an estimation of metabolite concentrations to determine their clinical outcome. At six months, 93% of ( n = 28) patients continued to be on AZA and had clinical improvement. All the patients achieved remission of Chron’s disease. Only two patients developed adverse effects such as joint pain and thrombocytopenia. Conclusion: AZA is a safe and effective therapy in managing Chron’s disease when administered after determining thiopurine methyltransferase phenotype and with dose optimization performed using therapeutic drug monitoring of 6-TGN and 6-MMP.

LC-MS/MS Method for Measurement of Thiopurine Nucleotides (TN) in Erythrocytes and Association of TN Concentrations With TPMT Enzyme Activity.

Monitoring concentrations of thiopurine metabolites is used clinically to prevent adverse effects in patients on thiopurine drug therapy. We developed a LC–MS/MS method for the quantification of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in red blood cells (RBCs). This method utilizes an automated cell washer for RBC separation from whole blood samples and washing of the separated RBCs. The lower limit of quantification of the method was 0.2 μmol/L for 6-TG (∼50 pmol/8 × 108 RBC) and 4 μmol/L for 6-MMP (∼1,000 pmol/8 × 108 RBC). The total imprecision of the assay was <3.0%. The upper limit of linearity for 6-TG and 6-MMP was 7.5 μmol/L and 150 μmol/L, respectively. The stability of the thiopurine metabolites under pre- and post-analytically relevant conditions was also evaluated. A good agreement was observed between this method and validated LC–MS/MS methods from three laboratories, except for ∼40% low bias for 6-MMP observed in one of the methods. The assessment of the association between 6-TG and 6-MMP concentrations with thiopurine S-methyltransferase (TPMT) phenotype and genotype demonstrated a statistically significant difference in the thiopurine metabolite concentrations between the TPMT groups with normal and intermediate activity of 6-MMP (p < 0.0001), while the difference in 6-TG concentrations was statistically not significant (p = 0.096). Among the samples with normal TPMT activity, higher concentrations of 6-MMP (p = 0.015) were observed in pediatric samples than in the samples of adults. No statistically significant differences were observed in the distributions of 6-TG and 6-MMP concentrations among the evaluated genotypes.

Dosage of 6-Mercaptopurine in Relation to Genetic TPMT and ITPA Variants: Toward Individualized Pediatric Acute Lymphoblastic Leukemia Maintenance Treatment.

6-mercaptopurine (6-MP) is the mainstay in pediatric acute lymphoblastic leukemia (ALL) maintenance treatment. Variants in genes coding for thiopurine S-methyl transferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) are known to influence 6-MP metabolism. We determined TPMT and ITPA genotype and enzyme activity and the mean 6-MP doses during maintenance treatment in 40 children treated for ALL according to the Dutch Childhood Oncology Group (DCOG)-ALL11 protocol in the Radboudumc Amalia Children's Hospital, Nijmegen, The Netherlands. Patients with genetic variants in TPMT (N=3) had significantly lower TPMT enzyme activity (mean 0.46 vs. 0.72 µmol/mmol hemoglobin/h, P=0.005). Although the difference was not statistically significant, they were treated with lower mean 6-MP doses (28.1 mg/m [SD 25.5 mg/m] vs. 41.3 mg/m [SD 17.2 mg/m], P=0.375). In patients with genetic ITPA variants (N=21), ITPA enzyme activity was significantly lowered (mean 3.67 vs. 6.84 mmol/mmol hemoglobin/h, P<0.0005). The mean 6-MP doses did not differ between patients with and without variants in ITPA (40.0 mg/m [SD 20.3 mg/m] vs. 40.6 mg/m [SD 14.9 mg/m], P=0.663). The TPMT genotype, but not the ITPA genotype, should be considered as part of standard evaluation before starting ALL maintenance treatment.

Determination of Thiopurine S-methyltransferase Activity using Reverse-Phase High-Performance Liquid Chromatography Assay with Ultraviolet Detection: Reference Values for the Indian Population

Objective: To determine thiopurine S-methyltransferase (TPMT) activity and the range of TPMT activity values for the Indian population. Methods: An isocratic reversed-phase high-performance liquid chromatography method with ultraviolet detection for the determination of TPMT activity in red blood cells was developed and validated. TPMT activity (nmol of 6-methylmercaptopurine [6-MMP]/h/ml erythrocytes) was measured based on the conversion of 6-mercaptopurine to 6-MMP with S-adenosyl-l-methionine. The stability of erythrocyte lysate at −20°C and the whole-blood specimens for TPMT activity were determined at 4°C. TPMT enzyme activity was measured in 150 patients who were not on azathioprine (AZA), and population difference in TPMT enzyme activity was evaluated. Results: The assay was linear from 1.0 to 60.14 nmol 6-MMP concentrations. The chromatogram peaks obtained were baseline separated for 6-MMP and the internal standard. The bias and precision of low-, medium-, and high-quality controls (QC) were within acceptable limits. Stock and lysate specimens were found to be stable in −20°C. The Indian populations have a considerable number of patients with low- and intermediate-TPMT activity. The wide range of TPMT activity in the study population (3.85–35.68 nmol/h/ml erythrocytes) is suggestive of high inter-individual variability in the formation of 6-thioguanine nucleotide formation, and therefore clinical efficacy and safety of AZA. Conclusion: A simple isocratic method was developed and validated for measuring TPMT activity in erythrocytes. Measuring TPMT activity before initiating AZA may help to decide the initial dose of AZA in patient management.

Thiopurine Methyltransferase Genetic Polymorphisms and Activity and Metabolic Products of Azathioprine in Patients with Inflammatory Bowel Disease.

Thiopurine S-methyltransferase (TPMT) is a cytoplasmic enzyme that catalyzes thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. There is a correlation between thiopurine drug metabolism, response, and toxicity and genetic polymorphism of TPMT. The aim of this study is to assess TPMT genetic polymorphisms activity and metabolic products of AZA in patients with IBD. Blood samples were obtained from 50 IBD unrelated patients from a private laboratory. We used polymerase chain reaction-restriction length polymorphism (PCR-RFLP) and allele-specific PCRbased assays to determine the TPMT gene for the different variants. A high-performance liquid chromatography system (HPLC) was carried out to determine the whole blood 6-TGN concentration. Determination of serum TMPT activity was done by ELISA kit. In IBD patients, 46/50 (92%) subjects were homozygous for the wild-type allele (TPMT*1/*1). Mutant TPMT*1/*2 and TPMT*1/*3C alleles were found in 4/46 (8%) and 3/47 (6%) of IBD patients, respectively. TPMT*1/*3B variant was not detected in any of the IBD patients. TPMT enzyme activity was higher in wild-type than that mutant variants TPMT*1/*2 and TPMT*1/*3C, suggesting that there are statistically significant differences between 6-TG levels and polymorphisms of TMPT enzyme. 6-TG levels significantly increased in IBD patients mutant variants TPMT*1/*2 and TPMT*1/*3C. Our results showed that TPMT polymorphisms are associated with 6-TGN levels in patients using AZA. This study suggests that AZA dosage may be determined according to the high or low prevalence of a TPMT genotype. Moreover, the results present the determination of metabolite for assessing possible safe effective dosage of the drug.

Development and validation of a UPLC-UV method for the quantification of thiopurine methyltransferase enzyme activity in human erythrocytes

Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime.After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 μm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision.The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.

Combined evaluation of genotype and phenotype of thiopurine S-methyltransferase (TPMT) in the clinical management of patients in chronic therapy with azathioprine.

Background The thiopurine S-methyltransferase (TPMT)/azathioprine (AZA) gene-drug pair is one of the most well-known pharmacogenetic markers. Despite this, few studies investigated the implementation of TPMT testing and the combined evaluation of genotype and phenotype in multidisciplinary clinical settings where patients are undergoing chronic therapy with AZA. Methods A total of 356 AZA-treated patients for chronic autoimmune diseases were enrolled. DNA was isolated from whole blood and the samples were analyzed for the c.460G>A and c.719A>G variants by the restriction fragment length polymorphism (RFLP) technique and sequenced for the c.238G>C variant. The TPMT enzyme activity was determined in erythrocytes by a high-performance liquid chromatography (HPLC) assay. Results All the patients enrolled were genotyped while the TPMT enzyme activity was assessed in 41 patients. Clinical information was available on 181 patients. We found no significant difference in the odds of having adverse drug reactions (ADRs) in wild-type patients and variant allele carriers, but the latter had an extra risk of experiencing hematologically adverse events. The enzyme activity was significantly associated to genotype. Conclusions TPMT variant allele carriers have an extra risk of experiencing hematologically adverse events compared to wild-type patients. Interestingly, only two out of 30 (6.6%) patients had discordant results between genotype, phenotype and onset of ADRs.

Retrospective Data Analysis of the Influence of Age and Sex on TPMT Activity and Its Phenotype-Genotype Correlation.

Therapeutic efficacy and toxicity of thiopurine drugs (used as anticancer and immunosuppressant agents) are affected by thiopurine S-methyltransferase (TPMT) enzyme activity. TPMT genotype and/or phenotype is used to predict the risk for adverse effects before drug administration. Inosine triphosphate pyrophosphatase (ITPA) is another enzyme involved in thiopurine metabolism. In this study, we aimed to evaluate (a) frequency of various TPMT phenotypes and genotypes, (b) correlations between them, (c) influence of age and sex on TPMT activity, and (d) distribution of ITPA variants among various TPMT subgroups. TPMT enzyme activity was determined by LC-MS/MS. TPMT (*2,*3A-C) and ITPA (rs1127354, rs7270101) genotypes were determined using a customized TaqMan® OpenArray®. TPMT enzyme activity varied largely (6.3-90 U/mL). The frequency of low, intermediate, normal, and high activity was 0.5% (n = 230), 13.1% (n = 5998), 86.1% (n = 39448), and 0.28% (n = 126), respectively. No significant difference in TPMT activity in relation to age and sex was found. Genotype analysis revealed the frequency of variant TPMT alleles was 6.73% (*3A, n = 344), 0.05% (*3B, n = 2), 2.22% (*3C, n = 95), and 0.42% (*2, n = 19). Analysis of paired phenotype and genotype showed that TPMT activity in samples with variant allele(s) was significantly lower than those without variant alleles. Lastly, an equal distribution of ITPA variants was found among normal and abnormal TPMT activity. This retrospective data analysis demonstrated a clustering of variant TPMT genotypes with phenotypes, no significant influence of age and sex on TPMT activity, and an equal distribution of ITPA variants among various TPMT subgroups.

Tu1700 - Ethnic Variation of Thiopurine Methyltransferase Enzyme Activity and Risk of Adverse Event in Patients with Inflammatory Bowel Disease

Tu1700 - Ethnic Variation of Thiopurine Methyltransferase Enzyme Activity and Risk of Adverse Event in Patients with Inflammatory Bowel Disease

Thiopurine Therapy Risk Evaluation for Inflammatory Bowel Disease Patients Using TPMT Enzyme Expression Method

Introduction: Thiopurine drugs, what are widely used medications for the treatment of inflammatory bowel diseases (IBD), have remarcable adverse effects like myelosuppression, leading to life threatening complications (severe infection or bleeding). Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurine drugs. Low TPMT activity in body is associated with pathological thiopurine drug metabolisms, overproduction of cytotoxic metabolites and myelosuppression. Methods: A purpose of our study was to compare TPMT enzyme expression analysis in patients with ulcerative colitis (UC) and Crohn's disease (CD) who are using azathioprine therapy with patients who have not yet begun this therapy. A prospective pilot study included 20 patients (55% female n=11 and 45% male n=9) having an age ranging from 22 to 79 years. All participants where admitted to Pauls Stradins Clinical university hospital (Riga, Latvia) from January 2017 to May 2017. All included patients received standard treatment of 5-ASA, steroid or azathioprine; leucocyte level was no lower than 4 x 109/L. The expression of TPMT in each blood sample was determined using ELISA (MyBioSource, USA) method. Results: All patient was previously diagnosed with IBD and now was admitted to hospital because of IBD exacerbation. 70 % of patients (n=14) was diagnosed with UC, 30 (n=6) with CD. 75% (n=15) of patients had not previously received azathioprine. 15% (n=3) had received azathioprine therapy, but stopped using it because of negative side effects like dyspepsia, acute pancreatitis, rash or exacerbation symptoms. 10% (n=2) was still receiving azathioprine therapy. Patients TPMT expression was in the range of 1.4 to 50 U/mL. All respondents were divided into the three TPMT enzyme activity groups. Activity of TPMT was low (< 5.5 U/mL) in 10% (n=2), intermediate (5.6-15.5 U/mL) in 5% (n=1) and normal/high (>15.6 U/mL) ir 85% (n=17) of patients. Conclusion: The results of this study confirmed the important role of the TPMT enzyme activity in the therapeutic drug monitoring. 15% of examined patients had decreased TPMT activity. Further studies in combination with TPMT genotype analysis are necessary for additional evaluation of treatment risks.

Letter: chronic Salmonella typhi carrier status and gall‐bladder cancer – authors’ reply

We thank Dr Arya and colleagues for their interest in our article1, 2 and highlighting the relationship between HLA and susceptibility to typhoid fever. The momentum for the implementation of personalised medicine in clinical practice is gaining. Recently, HLA genetic screening has been used to prevent carbamazepine-induced toxic effects among persons of Han Chinese descent3 and hypersensitivity to abacavir.4 Similarly, Ivacaftor, an orally available CFTR modulator that is specific for the G551D mutation has come up as a potential treatment for cystic fibrosis,5 thiopurine methyltransferase enzyme activity is predictive of azathioprine related life-threatening myelotoxicity in inflammatory bowel disease6 and reduced-function of CYP2C19 genotype has been related to adverse clinical outcomes among patients treated with clopidogrel.7 Imperial College of Science investigated the association between class II and class III major histocompatibility complex and typhoid fever in Vietnam. It is interesting to see that HLADRB1* 0301/6/8, HLA-DQB1*0201-3 and TNFA*2 (-308)) were associated with susceptibility to typhoid fever and HLA-DRB1*04, HLA-DQB1*0401/2 and TNFA*1 (-308) were linked with disease resistance.8 It would be a good strategy to utilize this characteristic to help prevent typhoid in this cohort of patients, especially in South East Asia. The endemic regions for typhoid can implement more stringent vaccination policies to reduce the health care burden of chronic Salmonella typhi carrier status-related gall-bladder cancer with the help of HLA screening. Another strategy would be to identify the HLA association between chronic Salmonella typhi carriage and gall-bladder cancer. However, whether this will be a cost-effective strategy is a question for the future. We suggest further research in this area to determine this association with the help of genome-wide association studies and animal studies. The authors’ declarations of personal and financial interests are unchanged from those in the original article.2

Implementation of TPMT testing

The activity of the enzyme thiopurine methyltransferase (TPMT) is regulated by a common genetic polymorphism. One in 300 individuals lack enzyme activity and 11% are heterozygous for a variant low activity allele and have an intermediate activity. The thiopurine drugs azathioprine, mercaptopurine and thioguanine are substrates for TPMT; these drugs exhibit well documented myelosuppressive effects on haematopoietic cells and have a track record of idiosyncratic drug reactions. The development of severe bone marrow toxicity, in patients taking standard doses of thiopurine drugs, is associated with TPMT deficiency whilst the TPMT heterozygote is at an increased risk of developing myelosuppression. Factors influencing TPMT enzyme activity, as measured in the surrogate red blood cell, are discussed in this review to enable an appreciation of why concordance between TPMT genotype and phenotype is not 100%. This is particularly important for lower/intermediate TPMT activities to avoid misclassification of TPMT status. TPMT testing is now widely available in routine service laboratories. The British National Formulary suggests TPMT testing before starting thiopurine drugs. Dermatologists were quick to adopt routine TPMT testing whilst gastroenterologists do not specifically recommend TPMT screening. TPMT testing is mandatory prior to the use of mercaptopurine in childhood leukaemia. Thiopurine drug dose and other treatment related influences on cell counts explain some of the differing recommendations between clinical specialities. TPMT testing is cost-effective and the major role is in the identification of the TPMT deficient individual prior to the start of thiopurine drugs.

Association of thiopurine methyltransferase status with azathioprine side effects in Chinese patients with systemic lupus erythematosus

Azathioprine (AZA) is indicated for the treatment of systemic lupus erythematosus (SLE). Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in the steps of AZA metabolization. Heritable deficiency of TPMT enzyme activity and polymorphisms may lead to leukopenia. This study aims to detect TPMT polymorphisms and TPMT enzyme activity in Chinese SLE patients and to describe the association between TPMT genotypes and adverse effects of AZA. One hundred and twenty-six SLE patients with present or previous thiopurine therapy were identified from a local database. Adverse effects were documented. No TPMT*2, TPMT*3A, or TPMT*3B mutant alleles were detected. TPMT*3C was detected in four patients (3.17%). The heterozygotes had significantly lower mean TPMT activity as compared to the homozygotes (2.38 ± 1.24 vs. 12.56 ± 7.02U/mL, P < 0.001). Twenty-seven cases (21.42%) exhibited adverse effects. All of the heterozygotes (4/4, 100%) developed severe leukopenia, and three cases (3/4, 75%) of whom exhibited alopecia simultaneously. The specificity of TPMT*3C for predicting leukopenia and alopecia was 100 and 99.17%, respectively, and the sensitivity was 28.57 and 60.00%, respectively. The mean value of TPMT activity with leukopenia (4.67 ± 3.01 vs. 13.2 ± 6.94U/mL RBC, P < 0.001) or alopecia (2.31 ± 1.16 vs. 12.65 ± 6.98U/mL RBC, P < 0.001) was significantly lower than those without. TPMT*3C was the most common mutant polymorphism found in the study group. TPMT activity is reduced in TPMT*3C mutant. AZA-induced leukopenia and alopecia were partly correlated to TPMT*3C heterozygotes and low TPMT activity. The results of this study suggest that the value of TPMT genotyping before AZA therapy was limited in Chinese SLE patients, considering the low sensitivity. Routine monitoring of TPMT activity before prescribing and continuous hematological monitoring dose were recommended.

TPMT genetic variants are associated with increased rejection with azathioprine use in heart transplantation

Azathioprine (AZA) is an important immunosuppressant drug used in heart transplantation (HTX). Consensus guidelines recommend that patients with thiopurine S-methyltransferase (TPMT) genetic variants be started on lower AZA dose because of higher active metabolite levels and risk of adverse events. However, in-vitro lymphocyte proliferation assays performed in participants with inactive TPMT alleles have suggested that AZA use may result in decreased immunosuppressant efficacy as compared with wild-type (WT) individuals. The objective of this study was therefore to determine the effect of TPMT genetic variation on AZA efficacy or prevention of rejection in HTX recipients treated with AZA. We genotyped 93 HTX recipients treated with AZA and measured erythrocyte TPMT enzyme activity. Acute rejection was monitored by routine endomyocardial biopsies. There were 83 WT and 10 heterozygote (HZ) HTX recipients. TPMT activity level was lower in HZ compared with WT (13.1±2.8 vs. 21±4.5 U/ml red blood cell, P<0.001). Despite similar AZA dose, HZ developed severe rejection earlier (P<0.001), and the total rejection score was higher (P=0.02) than WT. AZA was discontinued more frequently in HZ (P=0.01) because of rejection. The incidence of leukopenia was similar between the groups (40 vs. 43%, P=1.0). HTX recipients with TPMT genetic variant alleles who are treated with AZA develop acute rejection earlier, more frequently, and of greater severity. These patients, despite having lower TPMT enzymatic activity, should be monitored carefully for possible increased risk of acute rejection.

The pharmacokinetic effect of adalimumab on thiopurine metabolism in Crohn's disease patients

Background and aimsA 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 1–3weeks 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. MethodsCrohn'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 12weeks 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. ResultsTwelve Crohn's disease patients were analyzed. During the follow-up period of 12weeks 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 4weeks. In two patients (17%) myelotoxicity was observed within 2–4weeks, in whom both low therapeutic 6-TGN and 6-MMPR concentrations were found. ConclusionsIn this study in Crohn's disease patients no pharmacokinetic interaction was shown between adalimumab and the conventional thiopurines, azathioprine and mercaptopurine.

Preanalytical stringency: what factors may confound interpretation of thiopurine S-methyl transferase enzyme activity?

Measurement of red blood cell thiopurine S-methyl transferase (TPMT) enzyme activity before commencing thiopurine therapy is recommended to avoid severe bone marrow suppression in TPMT-deficient patients. Patient's samples go through preanalytical transportation and storage steps before measurement. We studied patient's TPMT activity sample data to assess the effect of preanalytical variables including transportation time. A total of 8524 TPMT enzyme activity analyses were conducted from 2002 to 2010 in a single laboratory, with samples sent from seven centres throughout New Zealand. TPMT activity was correlated with time of arrival at the reference laboratory, patient gender and age and centre of sample collection. The 6348 (74%) selected TPMT measurements that fulfilled selection criteria ranged from 0 to 25.8 IU/mL. Median delay to sample analysis was 42 h. Median TPMT activity was significantly lower for all centres compared with the reference centre (P < 0.001). Delay in sample arrival was significantly and independently correlated with TPMT enzyme activity (ANCOVA; P < 0.001), which showed a 0.011 (95% CI, 0.008-0.014) IU/mL decrease per extra hour of delay. After correcting for these data, one centre still had a significantly lower TPMT enzyme activity compared with the reference centre. There was a significant negative correlation between TPMT enzyme activity and delay from sample collection to analysis. Transportation time is therefore an important preanalytical variable influencing TPMT activity. Samples from one centre had a lower TPMT activity after correcting for transportation delay, suggesting that other factors related to sample processing may also be relevant.

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients. Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line. Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (-9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %). Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

Cisplatin-induced ototoxicity and the role of pharmacogenetic testing.

A 10-year-old white male presented with a chief complaint of swelling and pain in the left distal femur that worsened when he participated in sports at school. After several weeks of continued pain and swelling an x-ray was performed and revealed the presence of a large mass. Magnetic resonance imaging confirmed a large primary bone tumor located on the left femur, and open biopsy subsequently confirmed the diagnosis of osteosarcoma. The tumor was 9 cm long in the axis and 7 cm in diameter. Further tests revealed that the disease was localized to that area. The patient weighed 36 kg and was 145 cm tall at the time of diagnosis, placing him in the 75th percentile for age and sex. The patient was started on chemotherapy based on a standard Children’s Oncology Group (COG) treatment protocol of high-dose methotrexate, doxorubicin, and cisplatin. He received cisplatin at a dose of 120 mg/m2 approximately every 5 weeks for a total of 4 treatments. For each treatment, cisplatin 60 mg/m2/day was administered via intravenous infusion on 2 consecutive days. Based on the patient’s body surface area of 1.2 m2, he received a cumulative dose of 576 mg of cisplatin (480 mg/m2). He was also placed on an appropriate antiemetic regimen per COG protocol. He was not on any other medications known to cause ototoxicity or nephrotoxicity. His renal function was normal at baseline and all subsequent lab tests thereafter. Genotyping for thiopurine S-methyltransferase (TPMT) mutations was not performed prior to initiation of cisplatin therapy. Standard audiometric monitoring was performed prior to administration of the first cisplatin dose, and the results were all within the normal range (Brock Grade 0 [Table]).1 The patient completed the full cisplatin treatment course, and audiometric monitoring was performed prior to administration of the second and third cisplatin treatments with no significant hearing impairment noted. On follow-up 2 months after completing the third treatment course, the patient reported having trouble hearing. The loss manifested initially as bilateral tinnitus and ultimately progressed to bilateral hearing loss. Audiometric monitoring performed at this visit revealed moderate to severe hearing loss, defined as a Brock score ≥2 (Table). This translates to educationally significant hearing loss presumed to be caused by the ototoxic effects of cisplatin. Hearing tests performed 6 months and 1 year later revealed that the patient’s hearing had declined slightly but remained in Brock Grade 2, suggesting that the initially observed hearing deficit was likely progressive and irreversible.

Host Thiopurine Methyltransferase Status Affects Mercaptopurine Antileukemic Effectiveness

AbstractAbstract 3560Thiopurines are routinely used to treat many hematopoietic malignancies, including acute lymphoblastic leukemia (ALL), as well as inflammatory and autoimmune diseases. Common polymorphic null alleles among humans in thiopurine methyltransferase (TPMT) render 1 in 400 patients equivalent to homozygous knock-outs (−/−), 10% of patients equivalent to hemizygous (+/−), and 90% of patients comparable to wild-type (+/+) status for TPMT enzyme activity. TPMT activity is inversely related to the accumulation of active thioguanine nucleotide metabolites among patients, resulting in a higher risk for myelosuppressive adverse effects if routine doses are given to −/− compared to +/− compared to +/+ patients. However, the impact of the host TPMT polymorphism on antileukemic efficacy in the clinic is less clear.We generated a murine model of ALL by transducing bone marrow cells of Arf-null mice on the 129X1SvJ background (Tpmt +/+) with a retroviral vector encoding p185 BCR-ABL and luciferase, and culturing them for 7–10 days. We injected 2000 BCR-ABL+ pro/pre-B cells into Tpmt-deficient (−/−), heterozygous (+/−), or wild type (+/+) unconditioned syngenic recipients and compared the antileukemic efficacy when treating with a relatively low dose of mercaptopurine (equivalent to 7–9 mg/m2—such as would be tolerated by patients homozygous for null TPMT alleles). Treatment was started one day after injection of leukemia cells and continued for 70 days or until mice were sacrificed due to moribund state (usually due to illness from leukemia). To mimic clinically relevant treatment regimens, methotrexate was injected intraperitoneally (IP) at 1 mg/kg, once a week. Mercaptopurine was administered in the drinking water at 10 mg/L, for an estimated delivered dose of 2 – 2.5 mg/kg/day of mercaptopurine.Erythrocyte TPMT activity segregated by Tpmt genotype as expected and was comparable to the differences observed in patients: 0.6 U/ml in Tpmt −/−, 6.7 U/ml in Tpmt +/−, and 11.6 U/ml in Tpmt +/+ mice (p =0.008, n=2 for each genotype). As expected, the median survival (± standard error) was similar among Tpmt genotypes in untreated mice: 16 (± 0.4) days for Tpmt +/+, 17 (± 0.7) days for Tpmt +/−, and 18 (± 0.4) days for Tpmt −/−. The difference in leukemia-free survival by Tpmt genotype was profound: at 30 days after the start of treatment, 5% (± 9%) of Tpmt +/+ mice, 47% (± 26%) of Tpmt +/− mice, and 85% (± 14%) of Tpmt −/− mice were surviving (p = 5×10−8, Figure), indicating a substantial impact of host Tpmt status on thiopurine antileukemic effectiveness. In vivo imaging of luciferase-marked ALL cells confirmed an intermediate response phenotype for the Tpmt heterozygous mice (p = 0.00001). With higher doses (closer to the doses tolerated by patients homozygous for wild-type alleles, equivalent to 50–70 mg/m2/day), leukemia-free survival improved in the Tpmt +/+ and Tpmt +/− mice, comparable to the survival of Tpmt −/− mice treated with the low dose of mercaptopurine. These findings support the notion that germline polymorphisms in Tpmt, even in the heterozygous state, affect not only host tissue toxicity, but antitumor effectiveness as well. [Display omitted] Disclosures:Evans:St. Jude Children’s Research Hospital: Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms., Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms. Patents & Royalties. Relling:St. Jude Children’s Research Hospital: Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms., Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms. Patents & Royalties; Sigma-Tau Pharmaceuticals: Research Funding.