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Inflammatory bowel disease (IBD), once considered a disease of the Western hemisphere, has emerged as a global disease. As the disease prevalence is on a steady rise, management of IBD has come under the spotlight. 5-Aminosalicylates, corticosteroids, immunosuppressive agents and biologics are the backbone of treatment of IBD. With the advent of biologics and small molecules, the need for surgery and hospitalization has decreased. However, economic viability and acceptability is an important determinant of local prescription patterns. Nearly one-third of the patients in West receive biologics as the first/initial therapy. The scenario is different in developing countries where biologics are used only in a small proportion of patients with IBD. Increased risk of reactivation of tuberculosis and high cost of the therapy are limitations to their use. Thiopurines hence become critical for optimal management of patients with IBD in these regions. However, approximately one-third of patients are intolerant or develop adverse effects with their use. This has led to suboptimal use of thiopurines in clinical practice. This review article discusses the clinical aspects of thiopurine use in patients with IBD with the aim of optimizing their use to full therapeutic potential.

Herpes simplex virus 1 (HSV-1) infects eye corneal tissues leading to herpetic stromal keratitis (HSK), which is one of the leading causes of blindness. Here in our study, we found that 6-thioguanine (6-TG), a once clinically approved medication for child acute myelogenous leukemia, inhibited multiple strains of HSV-1 infection in vitro and in vivo. 6-TG is more potent than acyclovir (ACV) and ganciclovir (GCV), with the 50% inhibitory concentration (IC50) of 6-TG at 0.104 μM with high stimulation index (SI) (SI = 6,475.48) compared to the IC50 of ACV at 1.253 μM and the IC50 of GCV at 1.257 μM. In addition, 6-TG at 500 μM topically applied to the eyes with HSV-1 infection significantly inhibits HSV-1 replication, alleviates virus-induced HSK pathogenesis, and improves eye conditions. More importantly, 6-TG is effective against ACV-resistant HSV-1 strains, including HSV-1/153 and HSV-1/blue. Knockdown of Rac1 with small interfering RNA (siRNA) negatively affected HSV-1 replication, suggesting that Rac1 facilitated HSV-1 replication. Following HSV-1 infection of human corneal epithelial cells (HCECs), endogenous Rac1 activity was upregulated by glutathione S-transferase (GST) pulldown assay. We further found that Rac1 was highly expressed in the corneal tissue of HSK patients compared to normal individuals. Mechanistic study showed that 6-TG inhibited HSV-1 replication by targeting Rac1 activity in HSV-1 infected cells, and the Rac1 is critical in the pathogenesis of HSK. Our results indicated that 6-TG is a promising therapeutic molecule for the treatment of HSK. IMPORTANCE We reported the discovery of 6-TG inhibition of HSV-1 infection and its inhibitory roles in HSK both in vitro and in vivo. 6-TG was shown to possess at least 10× more potent inhibitory activity against HSV-1 than ACV and GCV and, more importantly, inhibit ACV/GCV-resistant mutant viruses. Animal model studies showed that gel-formulated 6-TG topically applied to eyes locally infected with HSV-1 could significantly inhibit HSV-1 replication, alleviate virus-induced HSK pathogenesis, and improve eye conditions. Further study showed that HSV-1 infection upregulated Rac1 expression, and knockdown of Rac1 using siRNA markedly restricted HSV-1 replication, suggesting that Rac1 is required for HSV-1 replication. In addition, we also documented that Rac1 is highly expressed in corneal tissues from HSK patients, indicating that Rac1 is associated with HSK pathogenesis. In view of the high potency of 6-TG, low cytotoxicity, targeting a distinct therapeutic target, we suggest that 6-TG is a potential candidate for development as a therapeutic agent for HSK therapy.

aintenance therapy containing methotrexate and 6-mercapto-purine is essential to cure acute lymphoblastic leukemia (ALL). Cytotoxicity is elicited by incorporation of thioguanine nucleotides into DNA (DNA-TG), and higher leukocyte DNA-TG is associated with increased relapse-free survival. As 6-thioguanine provides 6-fold higher cytosolic levels of thioguanine nucleotides than does 6-mercaptopurine, we added low-dose 6-thioguanine to methotrexate/6mercaptopurine maintenance therapy to explore if this combination results in significantly higher DNA-TG. The target population of the “Thiopurine Enhanced ALL Maintenance therapy” (TEAM) study was 30 patients with non-high-risk ALL, aged 1-45 years on methotrexate/6-mercaptopurine maintenance therapy receiving no other systemic chemotherapy. Incremental doses of 6-thioguanine were added to methotrexate/6-mercaptopurine maintenance therapy (starting 6-thioguanine dose: 2.5 mg/m2/day, maximum: 12.5 mg/m2/day). The primary endpoint was DNA-TG increments. Thirty-four patients were included, and 30 patients completed maintenance therapy according to the TEAM strategy. Of these 30 patients, 26 (87%) tolerated 10.0-12.5 mg/m2/day as the maximum 6-thioguanine dose. TEAM resulted in significantly higher DNA-TG levels compared to those in both TEAM patients before their inclusion in TEAM (on average 251 fmol/mg DNA higher [95% confidence interval: 160-341; P

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational., Highlights • 6-TG inhibits rotavirus infection. • Rac1, the cellular target of 6-TG, supports rotavirus infection. • 6-TG inhibits the active form of Rac1 (GTP-Rac1) to exert the anti-rotavirus effect.

Item does not contain fulltext Background: Patients with active ulcerative colitis (UC) failing conventional therapies are in need of rescue strategies. Due to the fact that accepted step-up therapy with biologicals is expensive and sometimes unavailable, alternative therapies are warranted. Methotrexate (MTX) and thioguanine (TG) have both been suggested as alternative maintenance strategies in conventional thiopurine failing UC patients. In this multicenter database study, we compared safety and effectiveness (drug-survival) of MTX and TG in UC patients. Methods: We collected data from the Parelsnoer database, a prospective Dutch national database consisting of inflammatory bowel disease patients from all university hospitals in The Netherlands. Additional data were collected from detailed chart review. Results: In total, 99 UC patients were included, of which 48 used TG, 43 used MTX, and 8 patients had a history of both TG and MTX use. In 12% of the patients, biological therapy had failed. Roughly 70% of the patients in both groups were able to continue therapy for over 1 year. Adverse events were noted in 33% of all the patients and were mainly elevated liver enzymes or gastrointestinal complaints. Twenty-eight patients (28%) continued therapy (15 TG, 13 MTX) without the need of escalation therapy (eg, corticosteroids, biologicals, or surgery). Drug survival curves of both drugs were comparable, just as the number of patients with sustained clinical benefit of therapy (P > 0.05). Conclusion: Both MTX and TG may be used and maintained as rescue therapy with sustained clinical benefit in one-third of the UC patients failing conventional therapies.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in southern China in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. Ten years later, a second highly pathogenic human CoV, MERS-CoV, emerged in the Middle East and has spread to other countries in Europe, North Africa, North America and Asia. As of November 2017, MERS-CoV had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against MERS-CoV. Here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (PLpros) of MERS-CoV and SARS-CoV. Our findings suggest that disulfiram acts as an allosteric inhibitor of MERS-CoV PLpro but as a competitive (or mixed) inhibitor of SARS-CoV PLpro. The phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of SARS-CoV PLpro by disulfiram, while synergistic inhibition of MERS-CoV PLpro by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs., Highlights • Disulfiram, a drug for use in alcohol aversion therapy, can inhibit the papain-like proteases of MERS-CoV and SARS-CoV. • Disulfiram is a noncompetitive inhibitor of MERS-CoV papain-like protease. • Disulfiram, 6-thioguanine and mycophenolic acid can synergistically inhibit MERS-CoV papain-like protease. • Disulfiram is a competitive inhibitor of SARS-CoV papain-like protease. • Disulfiram is a slow-binding inhibitor that forms a covalent adduct at the active site of SARS-CoV papain-like protease.

Inflammatory bowel diseases (IBD) are chronic relapsing disorders of the gastrointestinal tract. Several mouse models for IBD are available, but the acute dextran sulfate sodium (DSS)-induced colitis model is mostly used for preclinical studies. However, this model lacks chronicity and often leads to significant loss of mice. The aim of this study was to establish a refined and translationally relevant model of DSS chronic colitis in BALB/c mice. In the first part, we compared several standard therapeutic (ST) treatments for IBD in the acute DSS colitis model to identify the optimal treatment control for a DSS colitis model as compared to literature data. In the second part, we tested the two most effective ST treatments in a refined model of chronic DSS colitis. Cyclosporine A (CsA) and 6-thioguanine (6-TG) caused considerable reduction of clinical scores in acute DSS colitis. The clinical outcome was confirmed by the results for colon length and by histopathological evaluation. Moreover, CsA and 6-TG considerably reduced mRNA expression of several pro-inflammatory cytokines in spleen and colon. Both compounds also showed a substantial therapeutic effect in the refined model of chronic DSS colitis with regard to clinical scores and histopathology as well as the expression of inflammatory markers. The refined model of chronic DSS colitis reflects important features of IBD and is well suited to test potential IBD therapeutics.

The recently published American Gastroenterological Association (AGA) guideline on therapeutic drug monitoring (TDM) in inflammatory bowel disease (IBD) provides important and useful perspectives for individuals caring for and managing individuals with IBD and also for researchers focusing on this area (1). The guideline reviews key aspects of the roles that TDM increasingly has in the management of individuals with IBD and highlights key concepts. It also illustrates that many of these areas are supported by scanty evidence. Consequently, this also serves to prompt further study that might expand the evidence base accordingly.

BackgroundThe recently established association between higher levels of DNA-incorporated thioguanine nucleotides and lower relapse risk in childhood acute lymphoblastic leukaemia (ALL) calls for reassessment of prolonged 6-thioguanine (6TG) treatment, while avoiding the risk of hepatotoxicity.ObjectivesTo assess the incidence of hepatotoxicity in patients treated with 6TG, and to explore if a safe dose of continuous 6TG can be established.Data sourcesDatabases, conference proceedings, and reference lists of included studies were systematically searched for 6TG and synonyms from 1998–2018.MethodsWe included studies of patients with ALL or inflammatory bowel disorder (IBD) treated with 6TG, excluding studies with 6TG as part of an intensive chemotherapy regimen. We uploaded a protocol to PROSPERO (registration number CRD42018089424). Database and manual searches yielded 1823 unique records. Of these, 395 full-texts were screened for eligibility. Finally, 134 reports representing 42 studies were included.Results and conclusionsWe included data from 42 studies of ALL and IBD patients; four randomised controlled trials (RCTs) including 3,993 patients, 20 observational studies including 796 patients, and 18 case reports including 60 patients. Hepatotoxicity in the form of sinusoidal obstruction syndrome (SOS) occurred in 9–25% of the ALL patients in two of the four included RCTs using 6TG doses of 40–60 mg/m2/day, and long-term hepatotoxicity in the form of nodular regenerative hyperplasia (NRH) was reported in 2.5%. In IBD patients treated with 6TG doses of approximately 23 mg/m2/day, NRH occurred in 14% of patients; SOS has not been reported. At a 6TG dose of approximately 12 mg/m2/day, NRH was reported in 6% of IBD patients, which is similar to the background incidence. According to this review, doses at or below 12 mg/m2/day are rarely associated with notable hepatotoxicity and can probably be considered safe.

Methotrexate polyglutamates (MTXpg) facilitate incorporation of thioguanine nucleotides into DNA (DNA-TG, the primary cytotoxic thiopurine metabolite and outcome determinant in MTX/6-mercaptopurine treatment of childhood ALL). We hypothesized that mapping erythrocyte levels of MTXpg with 1–6 glutamates and their associations with DNA-TG formation would facilitate future guidelines for maintenance therapy dosing. Summed MTX with 1–6 glutamates resolved by LCMS [median (interquartile): 5.47 (3.58–7.69) nmol/mmol hemoglobin] was in agreement with total MTX by radio ligand assay. In 16,389 blood samples from 1426 ALL maintenance therapy patients, MTXpg3 21.0 (15.2–27.4)% was the predominant metabolite, and MTXpg1 (the maternal drug) constituted 38.6 (27.2–50.2)% of MTXpg1–6. All subsets correlated; the strongest associations were between metabolites with similar polyglutamate lengths. Correlations of MTXpg1 with MTXpg2 and MTXpg3,4,5,6 were rs = 0.68 and rs = 0.25–0.42, respectively. Intercorrelations of MTXpg3,4,5,6 were all rs ≥ 0.51. MTXpg4 accounted for 29.8 (24.7–33.3)% of MTXpg3–6, yet explained 96% of the summed MTXpg3–6 variation. MTXpg1–4, MTXpg1–6, MTXpg2–6 and MTXpg3 were all associated with DNA-TG levels (p

Background Azathioprine is widely used for the maintenance of remission in children with inflammatory bowel disease (IBD). Measuring thiopurine metabolites 6-thioguanine (6-TGN) and 6-methyl-mercaptopurine (6-MMP) can aid in optimizing treatment and preventing toxicity. We report a proactive approach combining early metabolite measurements with IBD activity index to achieve optimal azathioprine dosing. Methods The reporting of azathioprine dosing, IBD activity indexes and thiopurine metabolites was evaluated retrospectively in 40 children with IBD. Additional treatments and the effect of azathioprine on blood counts were also examined. Results Forty children (40% female) with IBD (26 Crohn's disease, 12 ulcerative colitis, and 2 unclassified IBD), mean age 12.2±3.4 years, were included in the study. The mean azathioprine dose was 1.3±0.4 mg/kg; mean 6-TGN level was 280±151 pmol/8 × 108 red blood cells (RBC) and mean 6-MMP level 1022±1007 pmol/8 × 108 RBC. Disease activity index (Crohn's and ulcerative colitis, pediatric specific) at the time of metabolite measurement was 6.5±8. Twenty-eight children did not require azathioprine dose adjustment, while it was increased in 12. Data from children with azathioprine monotherapy were analyzed separately and the results were similar. Conclusion Timely measurement of thiopurine metabolites and clinical assessment can provide a powerful tool to optimize azathioprine dosing and reduce serious adverse effects in children with IBD.

Pancreatic cancer is one of the most difficult cancers to cure due to the lack of early diagnostic tools and effective therapeutic agents. In this study, we aimed to isolate new bioactive compounds that effectively kill pancreatic ductal adenocarcinoma (PDAC) cells, but not untransformed, human pancreatic ductal epithelial (HPDE) cells. To this end, we established four primary PDAC cell lines and screened 4141 compounds from four bioactive‐compound libraries. Initial screening yielded 113 primary hit compounds that caused over a 50% viability reduction in all tested PDAC cells. Subsequent triplicate, dose‐dependent analysis revealed three compounds with a tumor cell‐specific cytotoxic effect. We found that these three compounds fall into a single category of thiopurine biogenesis. Among them, 6‐thioguanine (6‐TG) showed an IC50 of 0.39–1.13 μm toward PDAC cells but had no effect on HPDE cells. We propose that this cancer selectivity is due to differences in thiopurine methyltransferase (TPMT) expression between normal and cancer cells. This enzyme is responsible for methylation of thiopurine, which reduces its cytotoxicity. We found that TPMT levels were lower in all four PDAC cell lines than in HPDE or Panc1 cells, and that knockdown of TPMT in HPDE or Panc1 cells sensitized them to 6‐TG. Lastly, we used a patient‐derived xenograft model to confirm that 6‐TG has a significant antitumor effect in combination with gemcitabine. Overall, our study presents 6‐TG as a strong candidate for use as a therapeutic agent against PDAC with low levels of TPMT.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6chi Ly6g− monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6clo Ly6g+ granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1int Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6chi macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8+ T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies. Electronic supplementary material The online version of this article (doi:10.1007/s00262-015-1702-8) contains supplementary material, which is available to authorized users.

Methotrexate/6-mercaptopurine maintenance therapy of childhood acute lymphoblastic leukemia is challenged by treatment-related hepatotoxicity, failure to achieve the myelosuppressive target, and lack of direct parameters for monitoring treatment efficacy or even intensity. Patients with low thiopurine methyltransferase (TPMT) activity have lower levels of hepatotoxic methylated thiopurine metabolites (MeMPs), higher levels of thioguanine nucleotides (TGNs), and reduced relapse rates. Addition of 6-thioguanine to maintenance therapy of a child with ALL and high TPMT activity increased the TGN/MeMP index in erythrocytes 5.5-fold, mimicking the more favorable thiopurine metabolism seen in patients with low TPMT activity.

Background: Thiopurine S-methyltransferase (TPMT) enzyme metabolizes thiopurine drugs which are widely used in various disciplines as well as in leukemias. Individual enzyme activity varies depending on the genetic polymorphisms of TPMT gene located at chromosome 6. Up to 14% of popu-lation is known to have a decreased enzyme activity, and if treated with standard doses of thiopurines, these individuals are at a high risk of severe Adverse Drug Reactions (ADR) as myelosuppression, gas-trointestinal intolerance, pancreatitis and hypersensitivity. However, TPMT-deficient patients can suc-cessfully be treated with decreased thiopurine doses if enzyme status is identified by a prior testing. TPMT status identification is a pioneering experience in application of pharmacogenetic testing in clini-cal settings. 4 TPMT (*2, *3A, *3B, *3C) alleles are known to account for 80-95% of a decreased en-zyme activity, and therefore, identifying the presence of these alleles supported by phenotypic measure-ment of the enzyme activity can reveal patient’s TPMT status. Evaluation of the levels of thiopurine me-tabolites further supports the practice of appropriate dose adjustment by providing the efficient monitor-ing of drug cytotoxicity. Conclusion: We hereby review the thiopurine pharmacogenetics and the methods applied in common practice to evaluate patient’s TPMT status.

Background: Nodular regenerative hyperplasia is an uncommon liver condition associated with several autoimmune disorders and drugs. The clinical symptoms of nodular regenerative hyperplasia vary from asymptomatic to severe complications of portal hypertension (nodular regenerative hyperplasia-syndrome).Objective: The purpose of this study was to identify the prognosis and optimal management, as well as the role of liver transplantation, in nodular regenerative hyperplasia.Methods: The pathology databases of all three Dutch liver transplant units were retrospectively scrutinised for explanted livers diagnosed with nodular regenerative hyperplasia or without clear diagnosis. Pre- and post-transplantation clinical, biochemical, radiological and histological information was obtained from electronic and paper records.Results: In total, 1886 patients received a liver transplant. In 255 patients, nodular regenerative hyperplasia could not be excluded. After detailed chart review, the native livers of 11 patients (0.6%) (82% male, median age: 44 years) displayed nodular regenerative hyperplasia. Seven patients (64%) had underlying disorders or drug exposure which possibly caused nodular regenerative hyperplasia. Laboratory and imaging abnormalities were present in all patients but did not contribute to the diagnosis of nodular regenerative hyperplasia. Five-year survival was 73% (median follow-up: four years, range: 2-248 months).Conclusion: Nodular regenerative hyperplasia is a rare finding in patients, predominantly young males, transplanted for end-stage liver disease with unknown aetiology. Nonetheless, liver transplantation may have an important role in end-stage nodular regenerative hyperplasia-syndrome.

Contains fulltext : 169966.pdf (Publisher’s version ) (Closed access) BACKGROUND: Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related to high steady-state 6-methylmercaptopurine ribonucleotide (6-MMPR) metabolite concentrations. AIM: To determine the predictive value of 6-MMPR concentrations 1 week after treatment initiation (T1) for the development of these adverse reactions, especially hepatotoxicity, during the first 20 weeks of treatment. METHODS: The cohort study consisted of the first 270 IBD patients starting thiopurine treatment as part of the Dutch randomised-controlled trial evaluating pre-treatment thiopurine S-methyltransferase genotype testing (ClinicalTrials.gov NCT00521950). Blood samples for metabolite assessment were collected at T1. Hepatotoxicity was defined by alanine aminotransaminase elevations >2 times the upper normal limit or a ratio of alanine aminotransaminase/alkaline phosphatase >/=5. RESULTS: Forty-seven patients (17%) presented hepatotoxicity during the first 20 weeks of thiopurine treatment. A T1 6-MMPR threshold of 3615 pmol/8 x 108 erythrocytes was defined. Analysis of patients on stable thiopurine dose (n = 174) showed that those exceeding the 6-MMPR threshold were at increased risk of hepatotoxicity: OR = 3.8 (95% CI: 1.8-8.0). Age, male gender and BMI were significant determinants. A predictive algorithm was developed based on these determinants and the 6-MMPR threshold to assess hepatotoxicity risk [AUC = 0.83 (95% CI: 0.75-0.91)]. 6-MMPR concentrations above the threshold also correlated with gastrointestinal complaints: OR = 2.4 (95% CI: 1.4-4.3), and general malaise: OR = 2.0 (95% CI: 1.1-3.7). CONCLUSIONS: In more than 80% of patients, thiopurine-induced hepatotoxicity could be explained by elevated T1 6-MMPR concentrations and the independent risk factors age, gender and BMI, allowing personalised thiopurine treatment in IBD to prevent early failure.

Background: Mutations in the oncosuppressor gene BReast CAncer susceptibility gene 2 (BRCA2) predispose to aggressive forms of prostate cancer which show poor response to taxane-based therapy, the standard treatment for castration-resistant, aggressive prostate cancer. Herein, we addressed the question whether changes in BRCA2 expression, a potential surrogate marker for BRCA2 activity, may affect the response of castration-resistant prostate cancer cells to 6-thioguanine (6-TG), a thiopurine used in the treatment of haematological malignancies. Methods: Yeast, normal prostate cells and castration-resistant prostate cancer cells were treated with 6-TG or its analogues, in presence or absence of paclitaxel, or with olaparib, a poly-(ADP-ribose) polymerase (PARP) inhibitor currently in clinical trials for treatment of metastatic castration-resistant prostate cancer, and cell proliferation, apoptosis and androgen receptor (AR) levels were measured. Results: 6-TG inhibited cell proliferation in yeast, normal and castration-resistant prostate cancer cells but promoted apoptosis only in cancer cells. Suppression of BRCA2 expression by siRNA or shRNA increased the sensitivity to 6-TG- and olaparib-induced apoptosis but did not affect cancer cell response to taxane. Intriguingly, 6-TG reduced AR expression levels independently on BRCA2 expression. Instead, olaparib decreased AR levels only in BRCA2-knockdown prostate cancer cells. Notably, overexpression of BRCA2 resulted in resistance of castration-resistant prostate cancer cells to 6-TG-, taxane- and olaparib-based treatment but promoted sensitivity to apoptosis induced by 2-amino-6-bromopurine and 2,6&ndash, dithiopurine, two 6-TG analogues. Conclusions: Our results provide a pre-clinical rationale for the use of 6-TG in the treatment of BRCA2-deficient castration-resistant prostate cancers, and of certain 6-TG analogues for treatment of BRCA2-proficient prostate cancers.

Background: Previous studies have reported no or only a very poor correlation between 6-methylmercaptopurine/6-thioguanine nucleotide (6-MeMPN/6-TGN) and azathioprine (AZA) dose in the treatment of inflammatory bowel disease (IBD). However, metabolite levels are often repeatedly measured yielding a hierarchical data structure that requires more appropriate data analysis.Methods: This study explored the relationship between the weight-based dosage of AZA and metabolites levels in 86 pediatric IBD patients using multilevel analysis. Other covariates related to patient characteristics and treatment were evaluated.Results: This is the first study to demonstrate positive correlations between AZA dose and 6-TGN and 6-MeMPN levels and 6-MeMPN/6-TGN ratio (P Conclusions: The reliable AZA dose-metabolites relationship is useful for clinicians to guide the dosing regimen to maximize clinical response and minimize side effects or to consider alternative therapies when patients have preferential production of the toxic 6-MeMPN. These results may be of potential interest for optimizing thiopurine therapy to achieve safe and efficacious AZA use in pediatric IBD patients.

Long-term follow-up of 11 children with 6-thioguanine-induced hepatoportal toxicity is described. Features of persistent portal hypertension in eight patients after 9.7 +/- 3.4 years (mean +/- SD) of treatment were more common in late presenters. Splenomegaly, thrombocytopenia and altered hepatic echotexture were seen in six, eight and seven patients, respectively. One of the thrombocytopenic patients had heavy menstrual bleeding and pregnancy loss. Five of six patients who underwent upper gastrointestinal endoscopy had esophageal varices and four underwent banding. Late presentation in a subset of patients mandates long-term surveillance and follow-up for all patients treated with 6-thioguanine for early detection and management of hepatoportal complications.

Contains fulltext : 108348.pdf (Publisher’s version ) (Open Access) BACKGROUND: Infliximab-induced hepatotoxicity is reported in several case studies involving patients with inflammatory bowel disease (IBD) and a direct hepatotoxic effect has been proposed. OBJECTIVE: The aim of this study was to determine the direct in vitro toxicity of infliximab. As a proof of principle the in vitro toxicity of thiopurines and methotrexate was also determined. METHODS: Cell survival curves and the half maximal inhibitory concentrations (IC(50)) were obtained after 24, 48 and 72 h of incubation in HepG2 cells with the IBD drugs azathioprine, 6-mercaptopurine, 6-thioguanine, methotrexate or infliximab by using the WST-1 cytotoxicity assay. RESULTS: No in vitro hepatotoxicity in HepG2 cells was seen with infliximab, while concentration-dependent cytotoxicity was observed when HepG2 cells were incubated with increasing concentrations of azathioprine, 6-mercaptopurine and 6-thioguanine. CONCLUSION: Infliximab alone or given in combination with azathioprine showed no direct hepatotoxic effect in vitro, indicating that the postulated direct hepatotoxicity of infliximab is unlikely. 01 juni 2012

SCIRO Preventative Health National Research Flagship and Division of Livestock Industries; The University of Queensland; Mater Medical Research Institute

Resume L’azathioprine, la 6-mercaptopurine et la 6-thioguanine sont utilisees pour leur activite immunosuppressive dans la prevention des rejets d’organes transplantes, les traitements des maladies auto-immunes et inflammatoires digestives ainsi que pour leurs proprietes anti-nucleotidiques dans le traitement des leucemies aigues. Leur metabolisme est soumis au polymorphisme genetique de la thiopurine-methyl-transferase, enzyme qui convertit ces molecules en 6-thioguanine-nucleotides (6-TGN), metabolites actifs. Chez les patients traites par ces molecules dans le cadre d’une maladie inflammatoire cryptogenique de l’intestin, il n’y a pas de consensus sur les valeurs de 6-TGN a atteindre. Un suivi therapeutique pharmacologique est toutefois propose dans 4 situations : evaluation de l’observance au traitement, bilan d’echec therapeutique, suivi des sujets presentant une deficience en thiopurine-methyl-transferase, co-administration de traitements modifiant l’activite de la thiopurine-methyl-transferase. La revue des donnees de la litterature confirme que des concentrations elevees de 6-thioguanine-nucleotides et de leurs derives methyles augmentent considerablement le risque de myelotoxicite, auquel s’ajouterait un risque d’hepatotoxicite pour les derives methyles. Ces deux complications peuvent etre largement evitees par un genotypage/phenotypage prealable de la thiopurine-methyl-transferase et une adaptation de la posologie, d’emblee optimisee selon les resultats.

Background The thiopurine drugs azathioprine and 6-thioguanine have been shown to induce significant clinical improvement in patients with moderate to severe psoriasis and/or psoriatic arthritis. Objective The aim of this paper is to present a comprehensive review of the available literature concerning the off-label role of thiopurine drugs in the treatment of psoriasis and psoriatic arthritis. Methods Studies published with English abstracts between 1960 and 2008 identified in MEDLINE and PubMed with the keywords purine synthesis inhibitors, thiopurine, 6-thioguanine, azathioprine, psoriasis and psoriatic arthritis were reviewed. Results Although 6-thioguanine and azathioprine have shown significant efficacy, their side effect profiles have discouraged their use for moderate to severe psoriasis. Conclusion For treatment of psoriasis and psoriatic arthritis, purine synthesis inhibitors should not be forgotten. They represent an entirely different option and provide an alternative therapy, especially for difficult cases.

Resumen: Introducción: la determinación de la enzima tiopurina metiltransferasa (TPMT) nos permite pautar la dosis inicial individualizada de azatioprina (AZA). Las determinaciones de los metabolitos tiopurínicos de la AZA, la 6-tioguanina (6-TGN) y la 6-metilmercaptopurina (6-MMP) se han descrito como nuevos marcadores de la actividad del fármaco. Objetivos: describir el fenotipo de TPMT en nuestra población y relacionar los valores de los metabolitos tiopurínicos con la actividad terapéutica y los efectos adversos. Material y métodos: se recogieron retrospectivamente los valores de TPMT de 107 pacientes y de 6-TGN y 6-MMP de 18 pacientes en tratamiento con AZA (8 con enfermedad de Crohn, 5 con colitis ulcerosa y 5 con hepatitis autoinmune). Resultados: la media de determinación de TPMT fue 20,19U/ml. Ninguno presentó actividad de TPMT menor que 5U/ml. De los 18 pacientes, 13 mostraron concentraciones subterapéuticas de 6-TGN (

none 9 si non disponibile none Marasco, Giovanni; Scaioli, Eleonora; Renzulli, Matteo; Colecchia, Antonio; Golfieri, Rita; Festi, Davide; Bazzoli, Franco; Digby, Richard John; Belluzzi, Andrea Marasco, Giovanni; Scaioli, Eleonora; Renzulli, Matteo; Colecchia, Antonio; Golfieri, Rita; Festi, Davide; Bazzoli, Franco; Digby, Richard John; Belluzzi, Andrea

There is a growing interest in the use of thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) for the management of inflammatory bowel disease. The genetically controlled thiopurine (S)-methyltransferase enzyme is involved in the metabolism of these agents and is hypothesised to determine the clinical response to thiopurines. Diminished activity of this enzyme decreases the methylation of thiopurines, theoretically resulting in potential overdosing, while high thiopurine (S)-methyltransferase status leads to overproduction of toxic metabolites and ineffectiveness of azathioprine and 6-mercaptopurine. In practice, controversies exist regarding the utility of standard thiopurine (S)methyltransferase pheno- and genotyping. Current pharmacogenetic insights suggest that another enzyme system may participate in the efficacy and toxicity of thiopurines; inosine triphosphate pyrophosphatase. Other topics discussed in this review are the utilisation of therapeutic drug monitoring and the experimental use of 6-thioguanine in the treatment of inflammatory bowel disease. 6-Thioguanine has a less genetically controlled metabolism and skips genetically determined metabolic steps. On theoretical basis, 6-thioguanine might therefore have a more predictable profile than azathioprine and 6-mercaptopurine. However, the use of 6-thioguanine has been associated with an increased risk of nodular regenerative hyperplasia of the liver and veno-occlusive disease. Further research is warranted before 6-thioguanine can be considered as a treatment option for inflammatory bowel disease. (c) 2004 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

WOS: 000219056100012, The association between 6-Thioguanine (6-TG) treatment and drug-induced liver injury has been reported since 1976. Recent reports have suggested that treatment of patients with 6-TG can lead to chronic hepatotoxicity and portal hypertension. Most of the cases reported to have 6-TG related hepatotoxicity had acute lymphoblastic leukemia or inflammatory bowel disease. In this case report, we discussed the diagnosis and approach in an acute myeloid leukemia patient who developed 6-TG related chronic hepatotoxicity.

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

Azathioprine (AZA) is one of the oldest immunosuppressive agents available today. It is used as an immunomodulator in different disciplines such as hematology, dermatology, rheumatology, gastroenterology, and transplant. The present article presents a summary of AZA metabolism and the role of AZA metabolite measurements in the current clinical practice.

We studied whether isotretinoin potentiated the effects of vincristine (VCR), daunorubicin (DNR), and 6-thioguanine (6-TG) against cells obtained from 24 patients with acute lymphoblastic leukemia (ALL). Treatment with 5 micrograms/ml isotretinoin alone resulted in a leukemic cell survival of 82% +/- 28.1%. So isotretinoin is toxic to ALL cells. Dose-response curves were obtained for VCR, DNR and 6-TG in the presence and absence of isotretinoin Isotretinoin showed additive leukemic cell kills in combination with VCR and DNR. When corrected for cell kill by isotretinoin alone, it appeared that isotretinoin did not significantly enhance leukemic cell kills by VCR, DNR and 6-TG. No differences were found between samples from patients at initial diagnosis and at relapse with respect to cell kill by isotretinoin alone and with respect to a possible synergistic effect of isotretinoin and the cytostatic drugs. It is concluded that isotretinoin has additive antileukemic effects in combination with VCR or DNR. However, isotretinoin does not potentiate the antileukemic effects of VCR, DNR and 6-TG against leukemic cells obtained from patients with ALL.

BACKGROUND: At least one-third of patients with inflammatory bowel disease do not respond or are intolerant to therapy with 6-mercaptopurine (6-MP). A subgroup fails to attain optimal levels of 6-thioguanine nucleotide (6-TGN) and instead shunts to 6-methylmercaptopurine nucleotide (6-MMPN).PATIENTS AND METHODS: A retrospective chart review was conducted, and four patients are described who had been previously unable to achieve optimal 6-TGN metabolite levels until allopurinol was added to their treatment.RESULTS: All four patients achieved optimal 6-TGN levels and undetectable 6-MMPN with a mean 6-MP dose of 0.49 mg/kg. Three achieved steroid-free clinical remission. Two of those three patients had normalization of liver enzymes; one patient had baseline normal liver enzymes despite an initial 6-MMPN level of 27,369 pmol/8×108red blood cells. Two patients experienced reversible leukopenia.CONCLUSIONS: Combination allopurinol and low-dose 6-MP is an effective means to achieve optimal metabolite levels and steroid-free clinical remission in previously refractory patients. Caution is advised.

Granulocytic sarcoma (GS), also called myeloid sarcoma is an extramedullary tumor of the immature granulocytic cells. It is a rare entity and mostly accompanied by acute myeloid leukemia (AML). Very rarely, it is detected before clinical signs of leukemia or other diseases. When the bone marrow biopsy reveals no other hematologic malignancies, the GS is described as aleukemic, primary or isolated. Here, we report two rare cases, one of which presented as aleukemic GS of lymph nodes with aleukemic leukemia cutis, and the other with aleukemic GS of lung. Both cases posed diagnostic dilemma in view of their atypical presentations and site of involvement. Final diagnosis was made by immunohistochemistry (IHC). Both patients were treated with standard induction chemotherapy for AML. One patient had relapsed on treatment and was further treated with only 6-thioguanine leading to complete remission. Our cases emphasize the importance of early suspicion and use of IHC in diagnosis of aleukemic GS and also potential role of oral thioguanine alone in relapsed cases not eligible for hematopoietic stem cell transplant.

International audience; OBJECTIVE: Drug-induced liver injury was recently reported as a major complication leading to hepatic nodular regenerative hyperplasia (NRH) in patients with inflammatory bowel disease (IBD) and 6-thioguanine (6-TG) therapy. The aim of the study was to evaluate the prevalence of 6-TG-related hepatotoxicity in a large multi-centered IBD population by means of a systematic online survey. METHODS: Clinical and laboratory data, imaging techniques (sonography, CT, MRI) and histology of liver biopsies were surveyed in IBD patients treated with 6-TG. The decision on whether liver imaging and/or liver biopsy were performed was exclusively at the discretion of the investigator. RESULTS: 6-TG use was fully documented in 296 patients (median treatment duration 56 weeks, range < 1-207). Laboratory signs of drug-induced liver injury were found in 43 patients (14.5%). Liver imaging revealed pathologic results in 68/176 patients (38.6%). Liver biopsy was performed in a subset of 60 patients; using silver-reticulin staining (n = 59), NRH was considered in 16 patients (27.1%). Age was the only independent, albeit weak, risk factor for development of NRH. CONCLUSION: This large online survey confirms the strong association between 6-TG treatment and the significant risk of development of NRH in patients with IBD. The definitive diagnosis of NRH depends solely upon liver biopsy.