Prospective evaluation of the pharmacogenetics of azathioprine in the treatment of inflammatory bowel disease

Background One‐third of patients with inflammatory bowel disease (IBD) receiving azathioprine (AZA) withdraw treatment due to side effects or lack of clinical response. Aim To investigate whether pharmacogenetic loci or metabolite concentrations explain clinical response or side effects to AZA. Methods Patients with IBD were given 2 mg/kg of AZA without dose escalation or adjustment. Serial clinical response, thiopurine methyl transferase (TPMT) activity and thioguanine nucleotide (TGN) concentrations were measured over 6 months. All patients were genotyped for inosine triphosphatase (ITPase) and TPMT. Clinical response and side effects were compared to these variables. Results Two hundred and seven patients were analysed. Thirty‐nine per cent withdrew due to adverse effects. Heterozygous TPMT genotype strongly predicted adverse effects (79% heterozygous vs. 35% wild‐type TPMT, P < 0.001). The ITPA 94C>A mutation was associated with withdrawal due to flu‐like symptoms (P = 0.014). A baseline TPMT activity below 35 pmol/h/mg/Hb was associated with a greater chance of clinical response compared with a TPMT above 35 pmol/h/mg/Hb (81% vs. 43% respectively, P < 0.001). Patients achieving a mean TGN level above 100 were significantly more likely to respond (P = 0.0017). Conclusions TPMT testing predicts adverse effects and reduced chance of clinical response (TPMT >35 pmol/h/mg/Hb). ITPase deficiency is a predictor of adverse effects and TGN concentrations above 100 correlate with clinical response.     

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.     

Red Blood cell IMPDH activity in adults and children with or without azathioprine: Relationship between thiopurine metabolites,ITPA and TPMT activities

Inosine monophosphate dehydrogenase (IMPDH) is considered as the limiting enzyme of thiopurine metabolism for the formation of 6‐thioguanine nucleotides (6‐TGN). No data are available on the influence of RBC IMPDH activity on the metabolism of thiopurine drugs in individuals with IBD. The aims of this study were as follows: (a) to carry out a phenotypic study of RBC IMPDH activity in adults and children treated or not with azathioprine (AZA) for autoimmune diseases, and (b) to investigate the relationship between the activities of IMPDH, thiopurine metabolites, inosine triphosphatase (ITPA) and thiopurine methyltransferase (TPMT). IMPDH activity was determined in 97 adults and 67 children treated or not with AZA. 6‐Thioguanine nucleotides (6‐TGN), 6‐methylmercaptopurine nucleotide (6‐MeMPN) levels, and ITPA as well as TPMT activities were measured in RBCs by HPLC. Using the Gaussian mixture model, distribution of IMPDH activity was evaluated. Influence of age, sex and AZA treatment on IMPDH activity was also assessed. A bimodal distribution in IMPDH activity was found with 87% of patients exhibiting normal activity and 13% of patients with high activity. No influence of age, sex and AZA therapy was found. There is no relationship between TPMT, ITPA and IMPDH activities. A negative correlation between IMPDH activity and 6‐MeMPN was shown in adults and children (rs = ?0.335 P = 0.014 and rs = ?0.383 P = 0.012, respectively). Our results suggest that AZA‐treated patients exhibiting lower IMPDH activity could have higher Me‐6MPN levels with higher risk of hepatotoxicity. We demonstrated that RBC matrix could be an interesting alternative to lymphocyte matrix to monitor thiopurine metabolites and enzyme activity.     

Thiopurine methyltransferase genotype–phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia

Aims

In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype–genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation.

Methods

We measured TPMT (activity, as units ml⁻¹ packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10⁸ RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97.

Results

Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype–phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients.

Conclusions

In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy.     

Long-term outcome of using allopurinol co-therapy as a strategy for overcoming thiopurine hepatotoxicity in treating inflammatory bowel disease

Background  Hepatotoxicity results in the withdrawal of thiopurines drugs, azathioprine (AZA) and mercaptopurine (MP), in up to 10% of patients with inflammatory bowel disease. Our group previously demonstrated that allopurinol with AZA/ciclosporin/steroid 'triple therapy' improved renal graft survival.
Aim  To confirm the hypothesis that allopurinol may alleviate thiopurine hepatotoxicity by similar mechanisms as proposed in our renal study.
Methods  Unselected patients with acute thiopurine hepatotoxicity were offered allopurinol co-therapy with low-dose AZA or MP. The starting AZA/MP dose was determined by thiopurine methyltransferase (TPMT) activity (two patients were intermediate TPMT); then this dose was reduced to 25% for allopurinol co-therapy. Response to treatment was assessed by clinical severity indices, endoscopy and blood tests.
Results  Of 11 patients (three Crohn's disease, eight ulcerative colitis) treated, nine (82%) remain in long-term remission (median 42 months) with normal liver tests. One patient also successfully bypassed flu-like symptoms. Two stopped: one nausea, one abnormal liver function (steatosis on biopsy). Leucopenia occurred in two cases and resolved with minor dose reductions.
Conclusions  Allopurinol co-therapy with low-dose AZA/MP can alleviate thiopurine hepatotoxicity. It appears safe and effective for long-term use, but requires monitoring for myelotoxicity. Assessing the TPMT activity helps tailor the AZA/MP doses.     

Influence of xanthine oxidase on thiopurine metabolism in Crohn's disease

Background  The thiopurines, azathioprine (AZA) and mercaptopurine are extensively used in Crohn's disease (CD). Thiopurine bioactivation can be diverted by either thiopurine methyltransferase (TPMT), or by xanthine oxidase/dehydrogenase (XOD) which forms 6-thiouric acid (6TU).
Aim  To investigate whether chronic inflammation could influence small intestinal XOD activity using urinary excretion of 6TU as a surrogate marker of XOD activity.
Methods  6-Thiouric acid excretion was compared between 32 CD patients and nine dermatology patients (control group), on AZA. Six CD patients were interesting: five with low TPMT activity (one deficient, four intermediate), and one receiving AZA/allopurinol co-therapy.
Results  There was no statistical difference in 6TU excretion between the CD and control group. CD location, severity or surgery did not affect excretion. The TPMT-deficient patient excreted 89% of daily AZA dose as 6TU, but excretion by TPMT carriers was essentially normal. Concurrent 5-aminosalicylic acid therapy increased 6TU excretion significantly (median 32.9%), consistent with inhibiting TPMT. 6TU was undetectable in the patient on AZA/allopurinol co-therapy.
Conclusions  The results refuted our hypothesis, but fitted a model where most of an oral thiopurine dose effectively escapes first-pass metabolism by gut XOD, but is heavily catabolized by TPMT. Bioavailability of thiopurines may be competitively inhibited by dietary purines.     

Intraindividual variability in red blood cell thiopurine methyltransferase activity

Objective: Long-term (13 weeks) and circadian (24 hours) intraindividual variability in red blood cell (RBC) thiopurine methyltransferase (TPMT) activity in healthy subjects was studied. Methods: RBC TPMT activity was measured radiochemically. Results: The variability in RBC TPMT activity was low and was only slightly higher than the imprecision of the TPMT assay. Mean long-term intraindividual variability in RBC TPMT activity was 6.5% (CV) (n = 46). Mean intraindividual circadian variability in RBC TPMT activity was 6.4% (CV) (n = 18). Conclusions: In contrast to what has been observed in children with acute lymphoblastic leukaemia, the intra- individual variability in RBC TPMT activity in healthy subjects was low. The reported changes in baseline RBC TPMT activity in patients are probably therefore due to drugs, disease, assay variation or other, unidentified factors. Received: 9 October 1995/Accepted in revised form: 8 January 1996     

中国肾移植患者红细胞内嘌呤类药物活性代谢物6-硫鸟嘌呤核苷酸浓度监测及影响因素分析

目的：研究服用硫唑嘌呤（AZA）中国肾移植患者红细胞（RBC）内活性代谢物6-硫鸟嘌呤核苷酸（6-TGNs）分布特征及影响因素，为临床合理应用嘌呤类药物提供依据。方法：以89例中国肾移植患者为研究对象，关联分析年龄、性别、体质量、AZA剂量和TPMT活性对RBC内6-TGNs浓度的影响，并应用SPSS v20.0软件进行多元线性回归分析。结果：89例中国肾移植患者RBC内6-TGNs浓度呈非正态分布（P<0.000 1），6-TGNs浓度中位数为167.60（四分位间距，108.10~300.80） pmol/8×10⁸ RBC，个体间差异约24.3倍。关联分析显示患者年龄、性别、体质量、TPMT活性对6-TGNs浓度均无显著影响（P>0.05）；而AZA剂量与6-TGNs浓度间呈显著正相关性（r_s=0.307 1，P<0.01）。多元线性回归分析显示，RBC内6-TGNs浓度与AZA剂量间呈显著正相关（P<0.001），与TPMT活性呈显著负相关（P<0.05）。结论：AZA剂量和RBC内TPMT活性协同影响嘌呤类药物活性代谢物6-TGNs浓度，进而影响该类药物临床疗效和毒性反应。     

Evaluating the use of metabolite measurement in children receiving treatment with a thiopurine

Aliment Pharmacol Ther 2011; 34: 1106–1114

Summary

Background Clinical response to thiopurine medication is related to the concentration of its metabolites. Proxy measures are traditionally used to assess dose adequacy. We present our experience of using tioguanine (previously known/formerly referred to as thioguanine) metabolite measurements in paediatric patients and evaluate their effect on clinical practice. Aims To report our experience of using tioguanine metabolite measurements in paediatric patients and to evaluate their effects on clinical practice. Methods The 6‐tioguanine nucleotide (6‐TGN) and 6‐methylmercaptopurine (6‐MMP) were measured in children prescribed thiopurine medication for at least 3 months. Data were collected on thiopurine methyl transferase (TPMT) genotype, drug dose, laboratory indices and management changes. Therapeutic 6‐TGN levels were defined as 235–400 pmol/8 × 10⁸ RBCs. Seventy individuals (30 males) with a median age of 15 years. Underlying diagnoses were ‘IBD’ (68/70) and two cases of eosinophilic colitis. Sixty‐three were treated with azathioprine and seven with mercaptopurine. A total of 103 separate measurements were made. Results On initial measurement, 68% of patients had 6‐TGN levels outside therapeutic levels despite standard thiopurine dosing. Initial 6‐TGN levels were significantly higher in patients with TPMT mutations. Toxicity occurred in seven cases. The 6‐TGN levels were significantly higher in those with signs of marrow toxicity. The 6‐TGN level correlated with WBC, leukocyte count, mean corpuscular volume (MCV) and ΔMCV; however, the ability of each of these to predict therapeutic 6‐TGN levels was poor. After initial measurement, management was changed in 25/70 cases (36%). Conclusions 6‐TGN levels were therapeutic in a minority of those patients who were tested. Proxy measures perform poorly in predicting therapeutic 6‐TGN levels. Measuring thiopurine metabolites is useful for dosage adjustment in children, and for the detection of potential toxicity.     

The thiopurine S-methyltransferase gene locus -- implications for clinical pharmacogenomics

Thiopurine methyltransferase catalyzes the S-methylation of azathioprine (AZA), 6-mercapto-purine (6-MP) and thioguanine, medications widely used to treat malignancies, rheumatic diseases, dermatologic conditions, inflammatory bowel disease and solid organ transplant rejection. TPMT activity exhibits a genetic polymorphism in 10% of Caucasians, with 1/300 individuals having complete deficiency. Patients with intermediate or deficient TPMT activity are at risk for excessive toxicity, including fatal myelosuppression, after receiving standard doses of thiopurine medications. The molecular basis for low TPMT activity has been elucidated, leading to the development of assays for the three signature mutations, which account for the majority of mutant alleles. TPMT genotype is correlated with erythrocyte and leukemia blast cell TPMT activity and associated with a risk of toxicity after thiopurine therapy. Recent studies defined target starting doses for mercaptopurine based on TPMT genotypes. This polymorphism is one of the best models for the translation of genomic information to guide patient therapeutics.     

Clinical pharmacogenomics of thiopurine S-methyltransferase

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine (6-MP), thioguanine and azathioprine (AZA). These drugs are used to treat conditions such as acute lymphoblastic leukemia, inflammatory bowel disease, rheumatoid arthritis, and organ transplant rejection. This review highlights the polymorphisms of TPMT gene and their clinical impact on the use of thiopurine drugs. To date, there are 18 known mutational TPMT alleles. The three main TPMT alleles, namely TPMT *2, *3A and *3C, account for 80 - 95% of the intermediate and low enzyme activity. The TPMT gene exhibits significant genetic polymorphisms among all ethnic groups studied. Patients who inherited very low levels of TPMT activity are at greatly increased risk for thiopurine-induced toxicity such as myelosuppression, when treated with standard doses of these drugs, while subjects with very high activity may be undertreated. Moreover, clinical drug interactions may occur due to TMPT induction or inhibition. Identification of the TPMT mutant alleles allows physicians to tailor the dosage of the thiopurine drugs to the genotype of the patient or to use alternatives, improving therapeutic outcome.     

Determination of 6-thioguanine and 6-methylmercaptopurine metabolites in renal transplantation recipients and patients with glomerulonephritis treated with azathioprine

The metabolism of azathioprine (AZA) was studied by monitoring the concentrations of red blood cell (RBC) 6-thioguanine nucleotides (6-TGN) and of 6-methylmercaptopurine metabolites (6-mMP) in 27 renal transplantation recipients and in 10 patient subjects with glomerulonephritis (GN). Concentrations of 6-TGNs and 6-mMP metabolites were measured using high-performance liquid chromatography (HPLC). Six patients from the group of renal transplantation recipients were also administered allopurinol. Median values of RBC 6-TGN and of 6-mMP metabolites concentrations in 21 renal transplantation recipients (without allopurinol) were 122 pmol/8x10(8) RBCs (range, <60-298) and 280 pmol/8x10(8) RBC (range, <150-1330), respectively; there was no correlation between concentrations of 6-TGN and of 6-mMP metabolites. The group of 21 renal transplantation recipients received different AZA doses (100 or 50 mg/d) related to clinical symptoms of AZA intolerance. The median values of 6-TGN concentrations in these subgroups were 131 and 122 pmol/8x10(8) RBCs and were not significantly different. Median values of 6-TGN concentrations in patients given allopurinol were significantly higher, despite AZA dose reduction, compared with the group without allopurinol and were equal to 363 and 122 pmol/8x10(8) RBC, p < 0.004, respectively. No significant differences were found between the concentrations of 6-mMP metabolites in either group. In the group of renal transplantation recipients, a significant correlation between white blood cell (WBC) count and 6-TGN concentration was established (r(s) = -0.59, p < 0.005). In the group of GN patients, the median values of 6-TGN and of 6-mMP metabolites concentrations were 108 pmol/8x10(8) RBCs (range, 0-297) and 420 pmol/8x10(8) RBC (range, 0-1440), respectively. There were no significant correlations between either the WBC count and 6-TGN concentrations or between 6-TGN concentrations and 6-mMP metabolites. We expect the results of our study to provide indications for better individualization of AZA therapy.     

IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - relation to TPMT activity and metabolite concentrations

AIMS

Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the role of inosine-5′-monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines.

METHODS

IMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5′-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blod cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics.

RESULTS

A wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7–24.2 nmol mg⁻¹ protein h⁻¹) and IBD patients (median 14.0, range 7.0–21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (r_s = −0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations.

CONCLUSION

Even though the meTIMP concentrations correlated inversely to the IMPDH activity, the role of IMPDH in balancing the formation of methylated and phosphorylated metabolites was not evident. Taken together, the results give cause to question established opinions about thiopurine metabolism.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Up to 30% of inflammatory bowel disease patients treated with the thiopurine drugs azathioprine and 6-mercaptopurine do not respond properly to therapy.
• Genetic variation in the polymorphic enzyme thiopurine S-methyltransferase (TPMT) is associated with adverse events if patients are treated with standard doses.
• However, not all adverse events or metabolite patterns can be explained by genetic variations in TPMT, therefore we investigated the role of another thiopurine-metabolizing enzyme, inosine-5′-monophosphate dehydrogenase (IMPDH).

WHAT THIS STUDY ADDS

• There was a negative correlation of mononuclear cell (MNC) IMPDH activity with red blood cell (RBC) 6-methylthioinosine 5′-monophosphate, but not with RBC 6-thioguanine nucleotide (6-TGN).
• The results indicate either that measuring thiopurine metabolites in RBC, as is the current practice in clinical monitoring, is not an appropriate surrogate compartment for MNC metabolite concentrations, or that IMPDH in MNC is not as important a rate-limiting enzyme in the interconversion of thioinosine monophosphate to 6-TGN as has been hypothesized.
• All metabolite concentrations and enzymatic activities should preferably be measured in the same compartment.

     

Thiopurine drugs in the treatment of childhood leukaemia: the influence of inherited thiopurine methyltransferase activity on drug metabolism and cytotoxicity

Aims The response to 6-mercaptopurine (6MP) is highly variable. Its antileukaemic effect can be related to drug derived 6-thioguanine nucleotides (TGNs). The inherited level of thiopurine methyltransferase (TPMT) activity may be a major factor in the clinical response to 6MP because TPMT forms methylmercaptopurine metabolites (MeMPs) at the expense of TGNs. The aim of this study was to explore the clinical importance of TPMT phenotype.
Methods Thiopurine metabolism was studied in a consecutive cohort of children with acute lymphoblastic leukaemia (ALL) treated according to the Medical Research Council trial UK ALL XI. TPMT phenotype was measured in 38 children at diagnosis, and thiopurine metabolites were measured at defined times during 2 years treatment in 29 of these children.
Results TPMT activities at diagnosis ranged from 5.5 to 18.5 units  ml⁻¹ packed RBCs, no different from the range of activities reported in healthy children. TGNs and MeMPs measured during the first 6MP cycle at 75  mg  m⁻² ranged from 187 to 594  pmol 6TGNs, median 327, and 0.5 to 22.0  nmol MeMPs, median 4.5, per 8×10⁸ RBCs. TPMT activity was not significantly related to the generation of MeMPs ( r _s=0.06), but was negatively correlated to 6TGNs ( r _s=−0.44, P <0.025, n =29). TGNs were related to neutropenia at the point of dose reduction ( r _s=−0.5, P <0.01). TPMT activity was also inversely related to the duration of cytopenia driven 6MP withdrawal ( r _s=−0.41, P <0.05).
Conclusions These findings support the suggestion that the inherited activity of TPMT in a given individual can modulate the cytotoxic effect of 6MP, and this information may help in clinical management.     

Human thiopurine methyltransferase activity varies with red blood cell age

AIMS: Inherited differences in thiopurine methyltransferase (TPMT) activity are an important factor in the wide interindividual variations observed in the clinical response to thiopurine chemotherapy. The aim of this study was to establish a population range for red blood cell (RBC) TPMT activity in children with acute lymphoblastic leukaemia (ALL) at disease diagnosis. An additional aim was to investigate factors that can influence TPMT activity within the RBC. METHODS: Blood samples were collected from children with ALL at disease diagnosis, prior to any blood transfusions, as part of the nationwide UK MRC ALL97 therapeutic trial. RBC TPMT activity was measured by h.p.l.c. RBCs were age-fractionated on Percoll density gradients. RESULTS: Pretreatment blood samples were received from 570 children within 3 days of venepuncture. TPMT activities at disease diagnosis ranged from 1.6 to 23.6 units/ml RBCs (median 7.9) compared with 0.654-18.8 units (median 12.9), in 111 healthy control children (median difference 4.5 units, 95% CI 3.9, 5.1 units, P < 0.001). A TPMT quality control sample, aliquots of which were assayed in 60 analytical runs over a 12 month period, contained a median of 11.98 units with a CV of 11.6%. Seven children had their RBCs age-fractionated on density gradients. TPMT activities in the top gradient (young cells) ranged from 4.2 to 14.1 units (median 7.5) and in the bottom gradient (old cells) 1.5-12.6 units (median 4.7 units), median difference 2.3 units, 95% CI 0.7, 4.1, P = 0.035. CONCLUSIONS: Circulating RBCs do not constitute a homogeneous population. They have a life span of around 120 days and during that time undergo a progressive ageing process. The anaemia of ALL is due to deficient RBC production. The results of this study indicate that RBC TPMT activities are significantly lower in children with ALL at disease diagnosis. This may be due, at least in part, to a relative excess of older RBCs.     

Extended thiopurine metabolite assessment during 6-thioguanine therapy for immunomodulation in Crohn's disease

The proposed metabolic advantage of 6-thioguanine (6-TG) is the direct conversion into the pharmacologically active 6-thioguaninenucleotides (6-TGN). The authors assessed metabolic characteristics of 6-TG treatment in patients with Crohn's disease (N = 7) on therapy with 20 mg 6-TG. 6-thioguanine-monophosphate (6-TGMP), 6-thioguanine-diphosphate (6-TGDP), and 6-thioguanine-triphosphate (6-TGTP) were measured by high-performance liquid chromatography analysis in erythrocytes. Thiopurine S-methyltransferase activity and total 6-TGN levels were determined by standard methods. High interindividual variance in metabolite measurements was observed. Main metabolites were 6-TGTP (median = 531 pmol/8 x 10(8) red blood cells) and 6-TGDP (median = 199 pmol/8 x 10(8) red blood cells). Traces of 6-TGMP (median = 39 pmol/8 x 10(8) red blood cells) and 6-TG (2 patients) could be detected. 6-TGN levels correlated with 6-TGTP levels (r = 0.929, P = .003) and with the sum of separate nucleotides (r = 0.929, P = .003). No correlations were established between TPMT activity (median = 13 pmol/h/10(7)) and 6-TG metabolites. The 1-step metabolism of 6-TG still leads to high interindividual variance in metabolite concentrations. Total 6-TGN level monitoring may suffice for clinical practice.     

The impact of thiopurine s-methyltransferase polymorphism on azathioprine-induced myelotoxicity in renal transplant recipients

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability, mainly as a result of genetic polymorphism. Patients with intermediate or deficient TMPT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles: TPMT*2, *3A, and *3C are responsible for over 95% cases of low enzyme activity. The purpose of this study was to explore the association between these polymorphisms and the occurrence of azathioprine adverse effects in 112 renal transplant recipients undergoing triple immunosuppressive therapy including azathioprine, cyclosporine, and prednisone. TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. Azathioprine dose, leukocyte, erythrocyte, and platelet counts, graft rejection episodes, as well as cyclosporine levels were analyzed throughout the first year after organ transplantation. We found the frequency of leukopenia episodes (WBC < 4.0 x 10(9)/L) significantly higher in heterozygous patients (53.8%) compared with those with TPMT wild-type genotype (23.5%). One patient, who was a compound homozygote (3A/*3C), experienced severe azathioprine-related myelotoxicity each time after receiving the standard drug dose. Our results suggest that polymorphisms in TPMT gene may be responsible for approximately 12.5% of all leukopenia episodes in renal transplant recipients treated with azathioprine. Genotyping for the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.     

Identification of factors regulating thiopurine methyltransferase activity in a Norwegian population

Summary Red blood cell (RBC) thiopurine methyltransferase (TPMT), an inactivating pathway of 6-mercaptopurine, is controlled by genetic polymorphism and is subject to ethnic variation. RBC TPMT is a good predictor of clinical outcome in children with acute lymphoblastic leukemia. RBC TPMT activity was determined in 226 patients, 176 of them living in northern Norway (of which 123 were Saami (Lapps)). Demographic variables, use of drugs and presence of chronic diseases were evaluated as possible predictors of RBC TPMT activity by a multiple regression model.Men had higher RBC TPMT activity compared to women. Living in the northernmost county of Norway was associated with increased RBC TPMT activity irrespective of ethnicity. The use of diuretics was associated with increased RBC TPMT activity.The gender difference in RBC TPMT activity may indicate a need to treat male subjects more aggressively with thiopurine drugs compared to female subjects.This study was supported by the Erna and Olav Aakre Foundation for Cancer Research and the Norwegian Cancer Society     

Canine red blood cell thiopurine S-methyltransferase: companion animal pharmacogenetics

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. In humans, a common genetic polymorphism for TPMT is a major factor responsible for individual variation in the toxicity and therapeutic efficacy of these drugs. Dogs (Canis familiaris) are also treated with thiopurine drugs and, similar to humans, they display large individual variations in thiopurine toxicity and efficacy. We set out to determine whether dogs might also display genetically determined variation in TPMT activity. As a first step, we observed that canine red blood cell (RBC) TPMT activity in samples from 145 dogs varied over a nine-fold range. That variation was not associated with either the age or sex of the animal. Subsequently, we cloned the canine TPMT cDNA and gene. The canine cDNA encoded a protein that was 81.2% identical to the enzyme encoded by the most common TPMT allele in humans. A genotype-phenotype correlation analysis was performed by resequencing the canine gene using DNA samples from 39 animals selected for high, low or intermediate levels of RBC TPMT activity. We observed nine polymorphisms in these 39 DNA samples, including three insertion/deletion events and six single nucleotide polymorphisms (SNPs), one of which was a nonsynonymous cSNP (Arg97Gln). However, when the variant allozyme at codon 97 was expressed in COS-1 cells, it did not display significant differences in either basal levels of TPMT activity or in substrate kinetics compared with the wild-type allozyme. Six of the nine canine TPMT polymorphisms were associated with 67% of the variation in level of RBC TPMT activity in these 39 blood samples. When those six SNPs were assayed using DNA from all 145 animals studied, 40% of the phenotypic variance in the entire population sample could be explained by these polymorphisms. Therefore, inheritance is a major factor involved in the regulation of variation in RBC TPMT in the dog, just as it is in humans. These observations represent a step towards the application of pharmacogenetic and pharmacogenomic principles to companion animal drug therapy.     

Thiopurine methyl-transferase activity and azathioprine metabolite concentrations do not predict clinical outcome in thiopurine-treated inflammatory bowel disease patients

Aliment Pharmacol Ther 2011; 34: 544–554

Summary

Background Low thiopurine‐methyl‐transferase (TPMT) activity and high 6‐thioguanine‐nucleotide (6TGN) concentrations have been linked to therapeutic success in inflammatory bowel disease patients treated with thiopurines; however, this has not been implemented in clinical practice. Aim To identify a therapeutic threshold value for TPMT or 6TGN concentrations, and their capability to predict treatment safety and efficacy. Methods Prospective multicentre study including steroid‐resistant/dependent patients starting thiopurines. The TPMT activity was determined at inclusion (>5 U/mL required). Azathioprine metabolites [6TGN, 6‐methyl‐mercaptopurine ribonucleotides (6MMP), and 6TGN/6MMP and 6TGN/TPMT ratios] were periodically monitored during steroid tapering and after withdrawal for 6 months or until a new flare occurred. Results A total of 113 patients were analysed (62% clinical response). Areas under the receiver operating characteristic (ROC) curve (AUC) relating clinical response and metabolite levels at 2, 4 and 6 months after steroid withdrawal were less than 0.7. The AUCs relating final response and initial TPMT activity or metabolite concentrations at 2, 4, 8 and 16 weeks after starting thiopurines were less than 0.7. No cut‐off point with worthwhile sensitivity/specificity was found. Eight (7%) patients developed thiopurine‐related toxicity that could not be linked to TPMT activity or 6TGN levels. Conclusions Our results do not support determination of TPMT activity or 6TGN concentrations to predict treatment outcome, and no useful serum metabolites threshold value to adjust the drug’s dose was identified.