Thiopurine Methyltransferase Enzyme Research Articles

Assessing the Occurrence and Influence of Cancer Chemotherapy-Related Pharmacogenetic Alleles in the Chilean Population.

Pharmacogenomic knowledge as a biomarker for cancer care has transformed clinical practice, however, as current guidelines are primarily derived from Eurocentric populations, this limits their application in Latin America, particularly among Hispanic or Latino groups. Despite advancements, systemic chemotherapy still poses challenges in drug toxicity and suboptimal response. This study explores pharmacogenetic markers related to anticancer drugs in a Chilean cohort, filling a gap in Latin American research. Notably, the influence of native South American Mapuche-Huilliche ancestry. To explore pharmacogenetic markers related to anticancer drugs, we utilized an ethnically Admixed Chilean genome-wide association studies (GWAS) dataset of 1095 unrelated individuals. Pharmacogenomic markers were selected from PharmGKB, totaling 36 level 1 and 2 evidence single nucleotide polymorphisms (SNPs) and 571 level 3 SNPs. Comparative analyses involved assessing SNP frequencies across diverse populations from the 1000 Genomes Project. Haplotypes were estimated, and linkage disequilibrium was examined. Ancestry-based association analyses explored relationships between SNPs and Mapuche-Huilliche and European ancestries. Chi-square distribution with p ≤ 0.05 and Bonferroni's multiple adjustment tests determined statistical differences between allele frequencies. Our study reveals significant disparities in SNP frequency within the Chilean population. Notably, dihydropyrimidine dehydrogenase (DPYD) variants (rs75017182 and rs67376798), linked to an increased risk of severe fluoropyrimidine toxicity, exhibit an exceptionally low frequency (minor allele frequency (MAF) < 0.005). Nudix hydrolase 15 (NUDT15) rs116855232, associated with hematological mercaptopurine toxicity, is relatively common (MAF = 0.062), and is further linked to Mapuche-Huilliche ancestry. Thiopurine methyltransferase enzyme (TPMT), implicated in severe toxicity to mercaptopurines, SNPs rs1142345 and rs1800460 of TMPT gene demonstrate higher MAFs in Admixed Americans and the Chilean population (MAF range 0.031-0.057). Finally, the variant in the UDP-glucuronosyltransferase 1 gene (UGT1A1) rs4148323, correlated with irinotecan neutropenia, exhibits the highest MAF in East Asian (MAF = 0.136) and Chilean (MAF = 0.025) populations, distinguishing them from other investigated populations. This study provides the first comprehensive pharmacogenetic characterization of cancer therapy-related SNPs and highlights significant disparities in SNP frequencies within the Chilean population. Our findings underscore the necessity for inclusive research and personalized therapeutic strategies to ensure the equitable and effective application of precision medicine across diverse global communities.

SERS spectroscopy as a tool for the study of thiopurine drug pharmacokinetics in a model of human B leukemia cells

Thiopurine drugs are immunomodulatory antimetabolites relevant for pediatric patients characterized by dose-dependent adverse effects such as myelosuppression and hepatotoxicity, often related to inter-individual differences, involving the activity of important enzymes at the basis of their biotransformation, such as thiopurine S-methyltransferase (TPMT).Surface Enhanced Raman Scattering (SERS) spectroscopy is emerging as a bioanalytical tool and represents a valid alternative in terms of affordable costs, shorter analysis time and easier sample preparation in comparison to the most employed methods for pharmacokinetic analysis of drugs.The aim of this study is to investigate mercaptopurine and thioguanine pharmacokinetics by SERS in cell lysates of a B-lymphoblastoid cell line (NALM-6), that did (TPMT*1) or did not (MOCK) overexpress the wild-type form of TPMT as an in vitro cellular lymphocyte model to discriminate between cells with different levels of TPMT activity on the base of the amount of thioguanosine nucleotides (TGN) metabolites formed.SERS analysis of the cell lysates was carried out using SERS substrates constituted by Ag nanoparticles deposited on paper and parallel samples were used for quantification of thiopurine nucleotides with liquid chromatography-tandem mass spectrometry (LC-MS/MS).A direct SERS detection method has been set up that could be a tool to study thiopurine drug pharmacokinetics in in vitro cellular models to qualitatively discriminate between cells that do and do not overexpress the TPMT enzyme, as an alternative to other more laborious techniques.Results underlined decreased levels of TGN and increased levels of methylated metabolites when TPMT was overexpressed, both after mercaptopurine and thioguanine treatments.A strong positive correlation (Spearman's rank correlation coefficient rho = 0.96) exists between absolute quantification of TGMP (pmol/1 x 106 cells), obtained by LC-MS/MS, and SERS signal (intensity of TGN at 915 cm−1).In future studies, we aim to apply this method to investigate TPMT activity in pediatric patients’ leukocytes.

The Role of Pharmacogenetics in the Therapeutic Response to Thiopurines in the Treatment of Inflammatory Bowel Disease: A Systematic Review.

This study focuses on the use of thiopurines for treating inflammatory bowel diseases (IBD). These drugs undergo enzymatic changes within the body, resulting in active and inactive metabolites that influence their therapeutic effects. The research examines the role of genetic polymorphisms in the enzyme thiopurine S-methyltransferase (TPMT) in predicting the therapeutic response and adverse effects of thiopurine treatment. The TPMT genotype variations impact the individual responses to thiopurines. Patients with reduced TPMT activity are more susceptible to adverse reactions (AEs), such leukopenia, hepatotoxicity, pancreatitis, and nausea, which are common adverse effects of thiopurine therapy. The therapeutic monitoring of the metabolites 6-thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine (6-MMP) is proposed to optimize treatment and minimize AEs. Patients with higher 6-TGN levels tend to have better clinical responses, while elevated 6-MMP levels are linked to hepatotoxicity. Genotyping for TPMT before or during treatment initiation is suggested to tailor dosing strategies and enhance treatment efficacy while reducing the risk of myelosuppression. In conclusion, this study highlights the importance of considering genetic variations and metabolite levels in optimizing thiopurine therapy for IBD patients, focusing on balance therapeutic efficacy with the prevention of adverse effects and contributing to personalized treatment and better patient outcomes.

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.

Improving outpatient care in adult inflammatory bowel disease: effect of implementation of a reminder checklist in the electronic health records (IBD-ERS)—a pilot study

Studies have shown that patients with inflammatory bowel disease (IBD) do not receive age appropriate preventive care services at the same rate as the general population. Providers extract information on...

Molybdenum Cofactor Catabolism Unravels the Physiological Role of the Drug Metabolizing Enzyme Thiopurine S-Methyltransferase.

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt-/- ) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt-/- mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the population-based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug- and endogenous substrates.

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] &lt; 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.

Importance of NUDT15 Polymorphisms in Thiopurine Treatments.

Thiopurines, mercaptopurine, and azathioprine are used as immunosuppressants in the treatments of inflammatory bowel disease, rheumatoid arthritis, and organ transplantation and as chemotherapeutic drugs for the treatment of acute leukemia and chronic myeloid leukemia. This drug class sometimes causes severe adverse reactions, including bone marrow suppression and hair loss. Genetic polymorphisms of the metabolizing enzyme thiopurine S-methyltransferase have been used for predicting these reactions in Caucasians, but these allele frequencies are less frequently observed in Asian populations. Recently, nudix hydrolase 15 (NUDT15) polymorphisms have been shown to play an important role in thiopurine-induced adverse reactions in Asians. In this review, we summarize the NUDT15 studies, mainly in Asian countries, and their implementation in several countries.

Thiopurines in Inflammatory Bowel Disease. How to Optimize Thiopurines in the Biologic Era?

Thiopurines have been a cornerstone in the treatment of inflammatory bowel disease (IBD). Although they have been used for more than 50 years, there are still some unsolved issues about their efficacy and, also, some safety concerns, mainly the risk of myelosuppression and life-threatening lymphoproliferative disorders. Furthermore, the development of biological therapy raises the question whether there is still a role for thiopurines in the IBD treatment algorithm. On the other hand, limited cost and wide availability make thiopurines a reasonable option in settings of limited resources and increasing prevalence of IBD. In fact, there is a growing interest in optimizing thiopurine therapy, since pharmacogenomic findings suggest that a personalized approach based on the genotyping of some molecules involved in its metabolism could be useful to prevent side effects. Polymorphisms of thiopurine methyltransferase enzyme (TPMT) that result in low enzymatic activity have been associated with an increased risk of myelotoxicity, especially in Caucasians; however, in Asians it is assumed that the variants of nudix hydrolase 15 (NUDT15) are more relevant in the development of toxicity. Age is also important, since in elderly patients the risk of complications seems to be increased. Moreover, the primo-infection of Epstein Barr virus and cytomegalovirus under thiopurine treatment has been associated with severe lymphoproliferative disorders. In addition to assessing individual characteristics that may influence thiopurines treatment outcomes, this review also discusses other strategies to optimize the therapy. Low-dose thiopurines combined with allopurinol can be used in hypermethylators and in thiopurine-related hepatotoxicity. The measurement of metabolites could be useful to assess compliance, identify patients at risk of adverse events and also facilitating the management of refractory patients. Thioguanine is also a rescue therapy in patients with toxicity related to conventional thiopurine therapy. Finally, the current indications for thiopurines in monotherapy or in combination with biologics, as well as the optimal duration of treatment, are also reviewed.

TPMT and HLA-DQA1-HLA-DRB genetic profiling to guide the use of azathioprine in the treatment of interstitial lung disease: First experience

BackgroundThe choice of immunosuppressive therapy in interstitial lung disease (ILD) other than idiopathic pulmonary fibrosis (IPF) is based on safety profile and expected efficacy. Azathioprine is one of the most commonly used agents to treat ILD. The immunosuppressive effect and pancreatitis risk of azathioprine are influenced by the activity of the enzyme thiopurine methyltransferase (TPMT) and by the genetic mutations in HLA-DQA1-HLA-DRB locus, respectively. We hypothesized that systematic genotyping prior to starting azathioprine improves the rate of discontinuation of immunosuppressive therapy and the total incidence of adverse drug reactions (ADRs). MethodsEighty-two patients with ILD other than IPF were included in the study. The rate of immunosuppressive therapy discontinuation due to major ADRs and the total incidence of ADRs were compared between a cohort of genotyped patients (n = 49) and an untested cohort of patients (n = 33). ResultsThirty-seven out of 49 patients in the genotyped cohort and 27 out of 33 patients in the untested cohort were started on azathioprine. The rate of immunosuppressive therapy discontinuation due to major ADRs was significantly lower (6/49) in the genotyped cohort compared to the untested cohort (11/33; p = 0.0276). All but one discontinuation due to severe ADRs occurred within a month of therapy. However, the total incidence rate of ADRs was very similar in the 2 cohorts (0.025 in the genotyped cohort vs. 0.023 in the untested cohort). ConclusionIn patients with ILD other than IPF, genotyping for azathioprine metabolism prior to starting therapy is associated with a significantly reduced rate of immunosuppressive therapy discontinuation due to major ADRs, with prevention of bone marrow suppression and pancreatitis, but without a reduction of the total incidence of ADRs. While these data support the use of genetic profiling prior to starting azathioprine to treat ILD, its cost effectiveness remains to be established.

Role of therapeutic drug monitoring of azathioprine and thiopurine methyltransferase enzyme status in patients with inflammatory bowel disease: Indian scenario

Inflammatory bowel disease is mainly caused by dysragulated immune system. Inflammatory bowel disease incidences are rising in Asian countries with difficulty in their diagnosis and managements. There is rising the incidences and prevalence rate in India. Inflammatory bowel disease has two major subtypes Ulcerative colitis and chron’s disease. In ulcerative colitis inflammation occurs in lower part of large intestine that extend from anal verge to proximal colon while in case of chron’s disease there is transmural inflammation of gastrointestinal tract. This review is to provide comprehensive review focused on the current status of therapeutic drug monitoring of azathioprine metabolites in patients of inflammatory bowel disease.

THU0410 COMPANION IMMUNOSUPPRESSION WITH AZATHIOPRINE INCREASES THE FREQUENCY OF PERSISTENT RESPONSIVENESS TO PEGLOTICASE IN PATIENTS WITH CHRONIC REFRACTORY GOUT

Background:Pegloticase is a mammalian recombinant uricase coupled to monomethoxy polyethylene glycol that is approved in the US for treatment of patients with chronic refractory gout and causes profound reductions in serum urate. However, treatment with pegloticase is limited by the induction of anti-drug antibodies and loss of responsiveness in nearly half of treated patients.Objectives:The goal of this study was to determine whether co-therapy with azathioprine (AZA) would increase the frequency of chronic refractory gout patients who had persistent urate lowering from pegloticase therapy.Methods:This open label multicenter study enrolled subjects with chronic gout who failed to lower serum urate to &lt;6 mg/dL despite medically indicated doses of urate lowering therapy (NCT02598596). Patients were screened for adequate levels of the AZA metabolizing enzyme thiopurine methyl transferase and then started on daily oral AZA 1.25 mg/kg for 1 week and then 2.5 mg/kg for the remainder of the trial. Blood levels of AZA metabolites 6-thioguanine and 6-methylmercaptopurine were measured biweekly. After receiving 2 weeks of AZA, patients were started on pegloticase (8 mg IV) and were treated biweekly for 24 weeks. The primary endpoint was the persistent lowering of serum urate to &lt;6 mg/dL at the last three consecutive study visits. Patients who had an increase in serum urate to &gt;6 mg/dL while on therapy did not receive additional pegloticase. All patients received infusion prophylaxis with hydrocortisone as well as gout flare prophylaxis.Results:To date, 12 patients have been enrolled. All patients were male, 75% white and 25% African American. Mean age was 62.4 ± 14.7 years, the mean BMI was 31.1 ± 4.5 and the mean duration of gout was 13.8 ± 9.2 years. At baseline, all patients had visible tophi; 58.3% suffered from gout flares; 81.8% had hypertension; 45.5% had dyslipidemia and 9.0% had coronary artery disease. Of the 12 patients, 6 have completed the full course of treatment with persistent urate lowering and 2 remain on treatment also with persistent urate lowering (figure). 2 patients lost the urate lowering effect, both after 2 doses of pegloticase, and did not receive additional therapy. 1 patient experienced an infusion reaction during the first dose (1 infusion reaction in 90 infusions [1.1%] in the entire trial to date) and 1 subject had subjective symptoms of AZA intolerance with no laboratory abnormalities; these subjects discontinued the study and were not evaluable for the endpoint. No adverse events related to AZA were reported and gout flares were noted in 6 subjects (mean 1.5 flares/patient with flares).Conclusion:AZA can be used safely in subjects with chronic refractory gout and appears to increase the frequency of subjects experiencing long term lowering of serum urate.

P709 Is azathioprine safe as long-term treatment in paediatric patients with inflammatory bowel disease?

Abstract Background The toxicity of azathioprine (AZA) includes myelosuppression, infections, pancreatitis, photosensitivity, and hepatotoxicity. The aim of this study was to describe the adverse effects profile of azathioprine as long-term treatment in paediatric inflammatory bowel disease (IBD). Methods An observational, descriptive and retrospective study was performed in the paediatric IBD Unit of a tertiary care hospital from September 2008 to December 2018. It was included patients under 18 diagnosed with IBD who were treated with AZA during their follow-up. We recorded epidemiological data, thiopurine methyltransferase (TPMT) enzyme activity, AZA side effects, and the dosage the patients were receiving when these effects took place. Bone marrow suppression (BMS) was defined as leukopenia, thrombocytopenia and/or anaemia. Acute pancreatitis (AP) induced by azathioprine was considered when two of these criteria (Atlanta 2012) were met: lipase increase (&amp;gt; 3 times normal value), congruent signs and symptoms and/or echographic findings, without other possible aetiology and with complete recovery after AZA withdrawal. Results We included 52 patients, being 31 men (59.6%). They were diagnosed with Crohn′s disease (CD) (73%), ulcerative colitis (UC) (21%) and IBD-unclassified (6%). The median TPMT activity was 17 U/ml (14.2–19.2). Up to 63.5% developed adverse effects by AZA with a median time from the beginning of treatment of 11.4 months (2.6–26.4) and a median dosage of 2 mg/kg/day (1.7–2.3). The most frequent side effect was BMS (52%). These patients had a median TPMT activity of 16.9 U/ml (14.2–18.9), the median duration of treatment was 14 months (3.9–27.7), and the median dosage was 2 mg/kg/d (1.8–2.5). BMS was more frequent in patients with UC (p 0.04) and longer treatment (p 0.08). No differences were found according to age, sex or TPMT activity. Up to 11.5% developed AP, the median duration of treatment until its appearance was 1.5 months (0.7–43.3) and the median dosage was 2 mg/kg/d (1.5–2.5). No differences were found related to age, sex, diagnosis or dosage. Other side effects were: 3 flu-like symptoms, 3 opportunistic infections, 2 hypertransaminasemia, and 1 patient with elevated pancreatic enzymes and hyperbilirubinemia. AZA was discontinued in 14 patients (43.8%): in 6 due to AP, in 4 due to severe lymphopenia, in 2 because of Epstein-Barr virus infection, in 1 due to flu-like symptoms and in 1 with several adverse effects. Conclusion More than half of the patients treated with AZA presented side effects, mainly BMS, although most of them were mild and temporary, and the withdrawal of the drug was not necessary. It seems that TPMT activity is not useful to predict BMS, but this adverse effect could be related to a longer treatment.

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.

Correlation between Thiopurine S-Methyltransferase Genotype and Adverse Events in Inflammatory Bowel Disease Patients.

Background and Objectives: In patients with inflammatory bowel diseases (IBD), the use of azathioprine results in adverse events at a rate of 5% to 20%. The aim of the study was to assess a possible correlation between genetic variability of the enzyme thiopurine S-methyltransferase (TPMT) and the development of toxicity to azathioprine. Materials and Methods: A retrospective, single center, blind, case-control study was conducted on 200 IBD patients, of whom 60 cases suspended azathioprine due to toxicity (leukopenia, pancreatitis, hepatitis, and nausea or vomiting), and 140 controls continued treatment with the drug without adverse events. Results: In the entire cohort, only 8 cases of heterozygous mutations of TPMT were observed, corresponding to 4% mutated haplotype rate, much lower than that reported in literature (close to 10%). No homozygous mutation was found. Regarding the TPMT allelic variants, we did not find any statistically significant difference between patients who tolerated azathioprine and those who suffered from adverse events. (OR = 0.77, 95% CI = 0.08–7.72; p = 0.82). Conclusions: According to our study, in IBD patients, the search for TPMT gene mutations before starting treatment with azathioprine is not helpful in predicting the occurrence of adverse events. Importantly, patients with allelic variants should not be denied the therapeutic option of azathioprine, as they may tolerate this drug.

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.

Comprehensive study of thiopurine methyltransferase genotype, phenotype, and genotype-phenotype discrepancies in Sweden

Thiopurines are widely used in the treatment of leukemia and inflammatory bowel diseases. Thiopurine metabolism varies among individuals because of differences in the polymorphic enzyme thiopurine methyltransferase (TPMT, EC 2.1.1.67), and to avoid severe adverse reactions caused by incorrect dosing it is recommended that the patient’s TPMT status be determined before the start of thiopurine treatment. This study describes the concordance between genotyping for common TPMT alleles and phenotyping in a Swedish cohort of 12,663 patients sampled before or during thiopurine treatment. The concordance between TPMT genotype and enzyme activity was 94.5%. Compared to the genotype, the first measurement of TPMT enzyme activity was lower than expected for 4.6% of the patients. Sequencing of all coding regions of the TPMT gene in genotype/phenotype discrepant individuals led to the identification of rare and novel TPMT alleles. Fifteen individuals (0.1%) with rare or novel genotypes were identified, and three TPMT alleles (TPMT*42, *43, and *44) are characterized here for the first time. These 15 patients would not have been detected as carrying a deviating TPMT genotype if only genotyping of the most common TPMT variants had been performed. This study highlights the benefit of combining TPMT genotype and phenotype determination in routine testing. More accurate dose recommendations can be made, which might decrease the number of adverse reactions and treatment failures during thiopurine treatment.

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.