Active Thiopurine Research Articles

P464 Rac1/pSTAT3 expression in T lymphocytes: A potential pharmacodynamic marker for thiopurines in the treatment of Inflammatory Bowel Disease patients?

Abstract Background The thiopurine derivatives, azathioprine (AZA), mercaptopurine (MP) and tioguanine (TG), remain standard treatment of Inflammatory Bowel Diseases (IBD). To date, therapeutic drug monitoring (TDM) is used to optimize thiopurine therapy. However, since this provides mainly pharmacokinetic information, TDM is of limited use to predict clinical effectiveness or to explain therapeutic failure. A specific pharmacodynamic marker would therefore be of more beneficial use. The immune suppressive mechanism of thiopurines, by the active thiopurine metabolite 6-thioguanine triphosphate, is primarily based on blocking the Ras-related C3 botulinum toxin substrate 1 (Rac1) causing T-cell apoptosis by inhibition of the phosphorylated downstream transcription factor signal transducer and activator of transcription 3 (pSTAT3) (Figure 1). The aim of this study was to explore whether expression of Rac1 and pSTAT3 in T-cells may be used as a pharmacodynamic marker for the therapeutic effect of thiopurine therapy in IBD patients. Methods To assess feasibility, T-cell Rac1 and pSTAT3 expression was evaluated in six parallel IBD groups: patients with active disease (1) and patients in remission on AZA/MP (2), TG (3), infliximab (IFX) (4), thiopurine and IFX (5) or without medication (6). Data of healthy subjects were used as reference values. Rac1 and pSTAT3 expression of patients in remission on AZA/MP or TG were compared to all other IBD groups and healthy subjects. Results Absolute Rac1 expression in T-cells did not differ between any of the IBD patient groups (Figure 2). A decrease in absolute pSTAT3 expression was found in IBD patients in remission on AZA/MP or TG, when compared to active disease patients, although not statistically significant. Notably, Rac1-corrected pSTAT3 expression was decreased in IBD patients in remission on AZA/MP or TG compared to active disease patients (p<0.01). This effect was not observed in the IBD groups in remission on other immunosuppressive therapy. Conclusion Rac1-corrected pSTAT3 expression in T-cells was significantly decreased in IBD patients in remission on thiopurine monotherapy compared to IBD patients with active disease, resulting in comparable values to healthy subjects. Rac1/pSTAT3 expression may serve as a potential, class specific pharmacodynamic marker to assess therapeutic efficacy of thiopurine monotherapy or as a marker to explain thiopurine resistance.

Current status and future perspectives on the use of therapeutic drug monitoring of thiopurine metabolites in patients with inflammatory bowel disease

Introduction Despite new treatment options for inflammatory bowel disease (IBD), conventional thiopurines remain a common treatment option for maintaining remission, particularly in non-Westernized countries. Therapeutic drug monitoring (TDM) is advised in standard care for optimizing therapy strategies to improve effectiveness, reveal nonadherence and reduce toxicity. Still, the rationale of TDM is debated. Areas covered Key insights on TDM of thiopurine metabolites are discussed. The pharmacology of thiopurines is described, emphasizing the interindividual differences in pharmacogenetics, pharmacokinetics and pharmacodynamics. Pharmacological differences between conventional thiopurines and tioguanine are outlined. Finally, several optimization strategies for thiopurine therapy in IBD are discussed. Expert opinion TDM has been a useful, but limited, tool to individualize thiopurine therapy. Pharmacokinetic data on the active thiopurine metabolites, derived from measurements in erythrocytes, associated with clinical response only partially predict effectiveness and toxicity. An additional pharmacodynamic marker, such as Rac1/pSTAT3 expression in leukocytes, may improve applicability of TDM in the future.

Rac1/pSTAT3 expression: A pharmacodynamic marker panel as a first step toward optimization of thiopurine therapy in inflammatory bowel disease patients.

Thiopurine derivatives, such as azathioprine and mercaptopurine, are standard conventional treatment options in inflammatory bowel disease (IBD). Unfortunately, approximately half of patients discontinue thiopurine therapy within 2 years. To improve the prediction of clinical effectiveness, thiopurine therapy is currently optimized using therapeutic drug monitoring. Ras-related C3 botulinum toxin substrate 1 (Rac1) has been suggested as a potential pharmacodynamic marker of the thiopurine effect in lymphocytes. The active thiopurine metabolite 6-thioguanine triphosphate (6-Thio-GTP) causes T cell apoptosis via Rac1 and the downstream transcription factor signal transducer and activator of transcription 3 (STAT3). The aim of this study was to develop and validate a functional pharmacodynamic multiparameter flow cytometric assay to determine Rac1/pSTAT3 expression in the various leukocyte subpopulations in peripheral blood in order to predict therapeutic response in IBD patients in the future. Peripheral blood samples of healthy subjects (no fever or clinical complaints of active disease, C-reactive protein < 10mg/L) were used for immunocytochemical labeling, applying an optimized fixation and permeabilization strategy. A gating procedure was performed to separate all leukocyte subpopulations. Quantitative data were obtained by measuring presence and median fluorescent intensity. In vitro, Rac1 presence and expression were detectable in all leukocyte subpopulations. After IL-6 stimulation, used as proxy for inflammation, a distinct pSTAT3 signal could be detected in T lymphocytes of healthy subjects. In vivo, an upregulated pSTAT3 signal was detected in nearly all IBD patients with active disease and differed substantially from the signal found in IBD patients in remission on thiopurines and healthy subjects. We developed and validated a functional flow cytometric assay to assess Rac1 and pSTAT3 presence and expression. This opens a venue for a pharmacodynamic assay to predict thiopurine effectiveness in IBD patients.

Development of a chemical probe against NUDT15.

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop the first small-molecule NUDT15 inhibitors to elucidate its biological functions, and potentially for improving NUDT15-dependent chemotherapeutics. Lead compound TH1760, demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We further employed thiopurine potentiation as a proxy functional read-out and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity is via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

Repeated Occurrences of Basal Cell Cancer in Patients With Inflammatory Bowel Disease Treated With Immunosuppressive Medications

There are limited data on repeated basal cell cancer (BCC) occurrences among patients with inflammatory bowel disease (IBD), especially the impact of continuing immunosuppressive medications. We conducted a retrospective cohort study of 54,919 patients with IBD followed in the Veterans Affairs Healthcare System. We identified patients who had an incident BCC after their IBD diagnosis. We defined patients' exposure based on their IBD medications use as follows: (i) only aminosalicylate (5-ASA) use, (ii) only active thiopurine (TP) use, (iii) past TP use (discontinued >6 months ago) and no antitumor necrosis factor (TNF) use, (iv) anti-TNF use after previous TP use, (v) only anti-TNF use, and (vi) active anti-TNF and TP use. The outcome of interest was the repeated occurrence of BCC. Adjusted and unadjusted hazard ratios with 95% confidence intervals were used to estimate the risk of repeated BCC occurrence. A total of 518 patients developed BCC after their IBD diagnosis. The numbers of repeated BCC occurrences per 100 person-years were 12.8 (5-ASA use only), 34.5 (active TP use), 19.3 (past TP use and no anti-TNF use), 25.4 (anti-TNF use after previous TP use), 17.8 (only anti-TNF use), and 22.4 (active anti-TNF and TP use). Compared with 5-ASA use alone, only active TP use was associated with an increased risk for repeated BCC occurrence (adjusted hazard ratio 1.65, 95% confidence interval 1.24-2.19; P = 0.0005). However, the increased risk was no longer present for other exposure categories. Among IBD patients who developed an incident BCC while taking a TP and continued it, there was an increased risk of repeated BCC occurrences.

Clinical Pharmacogenetics Implementation Consortium Guideline for Thiopurine Dosing Based on TPMT and NUDT15 Genotypes: 2018 Update.

Thiopurine methyltransferase (TPMT) activity exhibits a monogenic codominant inheritance and catabolizes thiopurines. TPMT variant alleles are associated with low enzyme activity and pronounced pharmacologic effects of thiopurines. Loss-of-function alleles in the NUDT15 gene are common in Asians and Hispanics and reduce the degradation of active thiopurine nucleotide metabolites, also predisposing to myelosuppression. We provide recommendations for adjusting starting doses of azathioprine, mercaptopurine, and thioguanine based on TPMT and NUDT15 genotypes (updates on www.cpicpgx.org).

Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells - Effects on gene expression levels and thiopurine metabolism.

Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP’s effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress.

Microbiota: relevant player in thiopurine metabolisation?

In this issue, Oancea et al 1 demonstrate for the first time that gut microbiota is able to convert the thiopurine drug thioguanine into therapeutically active thiopurine nucleotides (TGN). They thereby open new perspectives for an optimised use of thiopurines in the treatment of patients with IBD and inspire the vision of topical thiopurine delivery to the inflamed gut. During the last years, our understanding of the molecular mechanisms, which underlie the well-known efficacy of thiopurines in IBD therapy, has been improved significantly. In order to allow potent therapeutic effects, classic thiopurine drugs including azathiopurine, mercaptopurine and thioguanine have to undergo metabolisation towards TGN. In particular, 6-thioguanine triphosphate (6-thio-GTP) could be identified as a crucial mediator of azathioprine-induced immunosuppressive effects in activated T cells and also in innate immune cells.2–4 Through its capacity to compete with endogenous GTP for the binding to the small GTPase Rac1 and subsequent inhibition of Rac1/Vav1 interaction, 6-thioGTP is able to efficiently block Rac1 signalling and thereby impact on immune cell function.2 ,3 Thiopurine-driven Rac1 blockade was shown to trigger apoptosis of stimulated T cells and to impair the capacity of T cells to form complexes with antigen-presenting cells (T cell/APC synapse formation).2 ,3 Regarding innate immune cells, thiopurine exposure of macrophages and subsequent inhibition of Rac1 signalling result in a significantly decreased inducible nitric oxide synthase (iNOS) expression and thus affect macrophage functions.5 Collectively, these findings highlight the concept that azathioprine-derived metabolites directly affect functions of …

Use of Immunomodulators and Biologics Before, During, and After Pregnancy.

Immunomodulators and biologic medications, alone or in combination, form the core therapeutic strategy for managing moderate-to-severe inflammatory bowel disease (IBD). IBD incidence peaks during the prime reproductive years, raising concerns about the impact of disease and its treatment on fertility, maternal and fetal health during pregnancy, breastfeeding safety, and childhood development. Although IBD increases risk of pregnancy complications independent of disease activity, adverse pregnancy outcomes are more common when disease is active. To mitigate fetal risk, women should conceive while disease is quiescent. Aside from methotrexate, immunomodulators and biologics may be used during pregnancy to achieve and maintain disease control. Based on available safety data, there is no increased risk of congenital anomalies among infants exposed to these medications. Active thiopurine metabolites and most monoclonal antibodies cross the placenta and are detectable in neonates. They are detectable in breast milk in minute levels as well. The impact of this exposure on neonatal outcomes is discussed. Adjusted dosing schedules during gestation may reduce fetal drug exposure, though the maternal risks of such manipulation require careful consideration. Ongoing prospective studies will further inform risk assessment, including for newer medications such as the anti-integrin agents.

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.

New Genetic Associations in Thiopurine-Related Bone Marrow Toxicity Among Inflammatory Bowel Disease Patients

The toxicity related to thiopurine drug therapy for inflammatory bowel disease (IBD) varies widely among patients. Almost 15-30% of patients with IBD develop side effects during treatment, often bone marrow suppression. Several factors have been implicated in determining this toxicity, mainly individual genetic variation related to formation of active thiopurine metabolites. The aim was to identify genes involved in thiopurine-related myelosuppression. A two-stage investigation of 19,217 coding SNPs (cSNPs) was performed in a Spanish (Inflammatory Bowel Disease Group of Galicia [EIGA]) cohort of 173 IBD patients, 15 with bone marrow suppression. The top 20 cSNPs identified in the first stage with p < 10(-3) for allelic test association and SNPs that define the common TPMT alleles were replicated in a different Spanish (ENEIDA) cohort (87 patients, 29 with bone marrow suppression). Several cSNPs showed a significant p-value in the allelic joint analysis (p-Cochran-Mantel-Haenszel test ≤2.55 × 10(-3)) despite no cSNP passing correction for multiple testing in the first cohort. Of note is rs3729961 in the gene IL6ST, a transducer signal chain shared by many cytokines including IL6 (p-value combined = 2.36 × 10(-4), odds ratio [95% CI]: 3.41 [1.71-6.78]). In addition, we detected association with rs3749598 in the FSTL5 gene that appears to interact with metalloproteases at the extracellular matrix level (p-value combined = 4.89 × 10(-4)), odds ratio (95% CI): 3.67 (1.68-8.01). We have identified IL6ST and FSLT5 as new bone marrow suppression susceptibility candidate genes after thiopurine treatment in IBD patients. This is the first report of variants associated with thiopurine-related myelosuppression that was identified by a genome-wide association study. Its validation awaits functional analyses and replication in additional studies. Original submitted 14 September 2012; Revision submitted 13 February 2013.

Use of Pharmacogenetics, Enzymatic Phenotyping, and Metabolite Monitoring to Guide Treatment with Azathioprine in Patients with Systemic Lupus Erythematosus

Individualized therapy based on genetic background and monitoring of metabolites can optimize drug safety and efficacy. Such an approach is available for azathioprine (AZA), the thiopurine antimetabolite. AZA exerts therapeutic effects when metabolized to the active thiopurine nucleotide, 6-thioguanine (6-TGN). In inflammatory bowel disease (IBD), 6-TGN levels in the target range of 235-400 pmol/8x10(8) red blood cells (RBC) are associated with a high likelihood of response. Our objective was to evaluate whether drug escalation based on metabolite levels improves efficacy and maintains safety in patients with systemic lupus erythematosus (SLE). We conducted a 6-month open-label dose-escalation clinical study of patients with active SLE treated with azathioprine dosed by body weight and metabolite levels. The primary endpoint was >or=50% improvement in any one parameter of disease activity, or 50% decrease in glucocorticoid dose. Of 50 patients enrolled in the study, 21 achieved clinical responses, 13 of whom had 6-TGN<235 pmol/8 x10(8) RBC. Ten patients had no clinical response at 6 months, yet achieved either therapeutic IBD 6-TGN levels (>235, n=4) or received maximum AZA dose>or=3.5 mg/kg (n=6). In 19 patients the drug was discontinued prematurely due to side effects or SLE activity. For those patients in whom either liver function test or white blood cell count abnormalities were encountered, metabolites guided attribution to drug or disease activity. Clinical responses in SLE can occur at levels of 6-TGN lower than the target range established for IBD. During followup, measurements of AZA metabolites may provide a rational approach to safety.

The Role of Xanthine Oxidase in Thiopurine Metabolism: A Case Report

Azathioprine (AZA) is widely used in the treatment of autoimmune inflammatory diseases. AZA is normally rapidly and almost completely converted to 6-mercaptopurine (6-MP) in the liver, which is further metabolized into a variety of pharmacologic active thiopurine metabolites. 6-MP is catabolized by xanthine oxidase (XO) to the inactive metabolite 6-thiouric acid. The authors report the case of a woman with chronic autoimmune pancreatitis unable to form active thiopurine metabolites. The 55-year-old woman presented with weight loss, progressive elevation of liver transaminases, and serum amylase. She was treated with prednisolone 30 mg/day (1 mg/kg) and AZA was increased to 75 mg/day (2.5 mg/kg); this was later increased to 150 mg/day (5 mg/kg). Despite good patient compliance, the active metabolites of AZA, 6-thioguanine nucleotides (6-TGN), and 6-methylmercaptopurine ribonucleotides (6-MMPR) could not be detected in the erythrocytes (RBC). Subsequently, AZA was switched to high-dose 6-MP (2.5 mg/kg) and the XO inhibitor allopurinol was added. After 1 week, this combination led to a high 6-TGN level of 616 pmol/8 x 10(8) RBC and a 6-MMPR level of 1319 pmol/8 x 10(8) RBC. Three weeks after starting treatment, 6-TGN and 6-MMPR even reached toxic levels (1163 pmol/8 x 10(8) RBC and 10015 pmol/8 x 10(8) RBC, respectively) so that 6-MP treatment was discontinued. To elucidate this finding, 6-MP (1.7 mg/kg) was prescribed for 3 days without allopurinol. The woman was not able to form active thiopurine metabolites. According to the authors, this is the first report of a patient unable to form detectable active thiopurine metabolites on AZA and 6-MP therapy despite good patient compliance. High XO activity led to an inability to form detectable levels of active thiopurine metabolites 6-TGN and 6-MMPR. This finding emphasizes the important role of XO in the biotransformation of thiopurines.

Non‐detectable levels of 6‐thioguanine nucleotides and 6‐methylmercaptopurine in a patient treated with azathioprine: a case report

Introduction: Azathioprine (AZA) is a thiopurine prodrug clinically used for immunosuppression in the treatment of auto‐immune inflammatory diseases and in regimens of organ transplantations. The pharmacological action of AZA is based on the formation of 6‐mercaptopurine (6‐MP), which is metabolised into a variety of active thiopurine‐nucleotide metabolites. We report the case of a 55 year old woman (bodyweight 30 kg) with chronic pancreatitis, weight loss, and progressive elevation of liver transaminases and serum amylase.Case description: The woman was treated with prednisolone (30 mg 1 dd; tapered 5 mg each week) and AZA (75 mg 1 dd; 5 weeks later 150 mg 1 dd). Despite good patient‐compliance verified during hospital‐stay, none of the active metabolites of AZA, 6‐thioguanine‐nucleotides (6TGN) and 6‐methylmercaptopurine ribonucleotides (6MMPR), were detected in erythrocytes. After two months of treatment clinical improvement was achieved, but no normalisation of laboratory parameters. Subsequently, AZA was switched to 6‐MP 75 mg 1 dd, and allopurinol 100 mg 1 dd was added. After one week the 6TGN level was 616 pmol/ 8 × 108 red blood cells (RBC), the 6MMPR level was 1319 pmol/ 8 × 108 RBC.Two weeks later the 6TGN level was 1163 pmol/ 8 × 108 RBC and the 6MMPR level 10015 pmol/ 8 × 108 RBC.These 6TGN and 6MMPR levels were higher than the upper limits of the therapeutic ranges (500 pmol/ 8 × 108 RBC and 5700 pmol/ 8 × 108 RBC, respectively). Therefore, treatment with 6‐MP was discontinued. A week later the 6TGN and 6MMPR levels decreased to 686 pmol/ 8 × 108 RBC and 4027 pmol/ 8 × 108 RBC, respectively.Genotyping of the enzym thiopurine S‐methyl transferase (TPMT) revealed a wild‐type TPMT (*1/*1) genotype.Discussion: To our knowledge this is the first report of a patient who was not able to form detectable active thiopurine metabolites on the treatment with AZA.AZA is normally rapidly and almost completely converted to 6‐MP and methylnitroimidazole in the liver. 6‐MP is then further catabolised by xanthine‐oxidase (XO) and TPMT or anabolised by hypoxanthine phosphoribosyl transferase (HPRT). Remarkably, treatment with 6‐MP in combination with the XO‐inhibitor allopurinol resulted in a myelotoxic 6TGN level in the first week after start of treatment.There are two hypotheses on the mechanism by which this finding could be explained. First, an ‘ultra‐high’ XO activity results in a direct and complete conversion of 6‐MP into 6‐thiouric acid. Secondly, AZA is not converted into 6‐MP. Consequently, there is no formation of active thiopurine nucleotides out of 6‐MP.Also, a combination of these two mechanisms could lead to a decreased formation of active thiopurine metabolites.Conclusion: Our finding indicates non‐conventional insights in the biotransformation of AZA contributing to an interindividual variation in thiopurine metabolism.

Pharmacogenomics in Inflammatory Bowel Disease

Inherited variations in the nucleotide sequence of genes influence how individual patients respond to drugs. Most commonly, clinically significant genetic variations consist of single nucleotide polymorphisms (SNPs) within genes that affect drug disposition or drug targets. Up to now, relatively few clinically important examples of inherited traits that affect drug responses have been studied in detail. However, one of the well-characterized examples is highly relevant to inflammatory bowel disease therapeutics, that of thiopurine methyltransferase pharmacogenetics. Individuals with 2 normal alleles of the gene encoding thiopurine methyltransferase metabolize and clear thiopurines such as azathioprine and 6-mercaptopurine rapidly. Individuals with 1 normal and 1 variant allele are intermediate, whereas those with 2 variant alleles clear thiopurines very slowly. Intermediate and slow metabolizers are predisposed to have high active thiopurine drug levels and develop bone marrow suppression. Genomic era technology permits determination of large numbers of SNPs in large numbers of individuals. This capability is allowing the field of pharmacogenomics to become one of the most productive interfaces in translational biomedical research at present. By using high-throughput SNP genotyping, combined with careful phenotypic characterization of disease, pharmacogenomic research carries the potential of identifying individual biomarkers that predict the relative likelihood of benefit or risk from a therapeutic intervention. If this promise can be realized, pharmacogenomics will deliver the opportunity for personalized medicine.

TPMT genotype and the use of thiopurines in paediatric inflammatory bowel disease

Background Thiopurines are used in the treatment of inflammatory bowel disease. They are metabolised via methylation by thiopurine-S-methyltransferase (TPMT), which displays a genetically determined polymorphic activity. Subjects with reduced TPMT activity have a higher concentration of active thiopurine metabolites and may be at increased risk of bone-marrow suppression. Aims To evaluate the relevance of TPMT genotyping in the management of thiopurines therapy in inflammatory bowel disease patients. Patients and methods Adverse effects and clinical response were determined retrospectively and correlated with TPMT genotype in 70 paediatric inflammatory bowel disease patients. Results Nineteen patients (27.1%) developed adverse effects; of the 51 who did not, 34 (66.7%) responded to treatment. Five patients (7.1%) were heterozygous for a variant TPMT allele; two of these (40%) were intolerant to thiopurines, compared to 17 of the 65 patients (26.2%) with a wild type gene (O.R. 1.88, 95% CI 0.29–12.2, p = 0.61); among the 34 responders, the median dosage of the drug required to obtain remission was lower for mutated than for wild type patients (1.6 mg kg −1 day −1 versus 2.0 mg kg −1 day −1, p = 0.043). Conclusions There was no significant association between adverse effects of thiopurines and TPMT heterozygous genotype, but TPMT genotyping could be useful in establishing the most appropriate dose of thiopurines to start treatment.

Rapid Detection of ITPA 94C&amp;gt;A and IVS2 + 21A&amp;gt;C Gene Mutations by Real-Time Fluorescence PCR and in Vitro Demonstration of Effect of ITPA IVS2 + 21A&amp;gt;C Polymorphism on Splicing Efficiency

Inosine triphosphatase (ITPA; EC 3.6.1.19) catalyzes the hydrolysis of ITP to inosine monophosphate, thereby recycling purines that might otherwise be trapped in the form of ITP (1)(2). Two single-nucleotide polymorphisms associated with ITPA deficiency have been identified in the ITPA gene. Individuals who are homozygous for a 94C>A (P32T) mutation have a total deficiency of enzyme activity and accumulate ITP intracellularly, whereas 94C>A heterozygotes have decreased ITPA activity that is 22.5% of the control mean value (2). A second mutation, IVS2 + 21A>C, was detected in ITPA-deficient families. This intronic mutation has a more subtle effect on ITPA activity, and heterozygotes have activities that are, on average, ∼60% of the control mean. It was presumed that the IVS2 + 21A>C mutation alters the relatively conserved adenine of a putative splicing branch site, leading to abnormal mRNA splicing (2). Although ITPA deficiency is not related to any defined pathology in humans, it was recently demonstrated that polymorphisms in the ITPA gene associated with ITPA deficiency have pharmacogenomic implications for patients treated with thiopurines (3). In a retrospective study involving patients with inflammatory bowel disease receiving azathioprine, Marinaki et al. (3) observed that the 94C>A deficient allele was significantly related to the adverse drug reactions (ADRs) flu-like symptoms, rash, and pancreatitis. The purine analog 6-mercaptopurine and its prodrug azathioprine (AZA) are widely used in the treatment of leukemia and autoimmune disease, and in transplantation. ADRs to these drugs have been related to a genetic deficiency of thiopurine S -methyltransferase (TPMT; EC 2.1.1.67), which is a key enzyme of thiopurine drug catabolism (4). TPMT deficiency leads to life-threatening myelosuppression by accumulation of active thiopurine metabolites (5). Most ADRs to thiopurines, however, cannot be explained by TPMT deficiency. Thiopurines are more frequently discontinued because of non-dose-dependent ADRs (fever, pancreatitis, nausea) than …