High Thiopurine Methyltransferase Activity Research Articles

797 High Thiopurine Methyltransferase Activity Does Not Affect Concentration of Azathioprine Metabolite 6-Thioguanine Nucleotide in Inflammatory Bowel Disease

INTRODUCTION: Azathioprine (AZA) is commonly used to treat inflammatory bowel disease (IBD). Thiopurine methyltransferase (TPMT) converts AZA to key active metabolite 6-thioguanine nucleotide (6-TGN). Low TPMT activity is associated with high 6-TGN and side effects. Conversely, low 6-TGN is associated with lack of clinical response and has been associated with high TPMT activity but has not been examined in an IBD population. The aim of this study was to identify the independent effect of normal to high TPMT activity on 6-TGN concentrations in an IBD population. METHODS: We performed a retrospective chart review of patients aged >18 with IBD treated with AZA and with documented TPMT activity and 6-TGN levels from 2010 to 2017. To account for different assays, TPMT activity was normalized against the established low-normal TPMT activity, with low activity defined as less than 1 and high activity defined as above the 75th quantile of TPMT activity in the cohort. Those with low TPMT were excluded. Linear regression was used to determine the effect of TPMT activity on 6-TGN accounting for potential confounders. Further assessment of the interaction between dose and TPMT activity on 6-TGN was measured through the Average Causal Mediation Effects (ACME). Statistics performed in R v3.6.0 using mediation package. This study was IRB approved. RESULTS: Dosing of AZA in mg/kg, TPMT activity, and 6-TGN concentration were documented in 63 patients, of which 10 had low TPMT activity and were subsequently excluded, leaving 53 patients for analysis. Overall 37% of the cohort had a therapeutic 6-TGN level of >240 pmol/8 × 108 RBC. No patient under 1mg/kg achieved a therapeutic 6-TGN level, whereas 50% of patients taking 2.5 mg/kg did. The median 6-TGN in the normal TPMT range was 191.5 (IQR: 78.8, 296.3) versus 201 (IQR: 45, 312) in the high group (P = 0.57). There was a weak positive correlation between dosing (mg/kg) and 6-TGN (R2 = 0.08), while no correlation was seen between TPMT and 6-TGN (R2 = 0.0). Linear regression to assess the effect of TPMT activity and dose on 6-TGN concentrations including potential confounders of sex, age, smoking status only identified dose (mg/kg) to have a significant effect on 6-TGN concentration (Table 1). CONCLUSION: There was no effect on 6-TGN as TPMT activity increased, although 6-TGN was positively affected by dose. Despite normal TPMT activity, therapeutic 6-TGN concentrations were only found in 50% of those dosed at 2.5 mg/kg.

Should Thiopurine Methyltransferase Genotypes and Phenotypes be Measured Before Thiopurine Therapy in Patients With Inflammatory Bowel Disease?

Not all of the adverse effects to thiopurine therapy can be explained by thiopurine methyltransferase (TPMT) polymorphisms. This study was intended to evaluate the value of TPMT genotype and phenotype measurement during the first year of thiopurine therapy. Consecutive patients with inflammatory bowel disease (IBD) who were receiving azathioprine or 6-mercaptopurine were followed up for 12 months. TPMT genotypes and phenotypes were examined in patients with IBD before thiopurine therapy and in unrelated healthy volunteers by polymerase chain reaction and high-performance liquid chromatography. A total of 199 patients and 300 healthy volunteers were included at 2 centers. Forty-seven of the 199 patients (23.62%) exhibited adverse effects during the entire course of thiopurine therapy. Two (1%) patients carrying TPMT*3C developed leucopenia at week 4 of azathioprine treatment. The TPMT*3C had a specificity of 100% (163/163) but a sensitivity of 5.56% (2/36) for predicting leucopenia. The calculated optimal cutoff activity for high TPMT activity and decreased TPMT activity was 4.75 U/mL red blood cells. The risk of leucopenia increased in the decreased TPMT group (odds ratio: 20.25; 95% confidence interval: 2.19-187.17; P = 0.004) and increased more during the initial 3 months of thiopurine therapy (odds ratio: 34.80; 95% confidence interval: 3.71-326.77; P = 0.001). Leucopenia occurred more frequently in the patients cotreated with 5-aminosalicylates than in those not cotreated (32.81% versus 11.11%, respectively, P < 0.001). The results of this study suggest that the value of TPMT genotyping before thiopurine therapy is limited in Chinese patients with IBD, considering the low sensitivity of predicting leucopenia, and that phenotyping is a more cost-effective tool that can be successfully used in patients. The coadministration of 5-aminosalicylates results in a high frequency of leucopenia in patients receiving azathioprine or 6-mercaptopurine.

PWE-251 District general hospital experience of optimising treatment outcome on thiopurines by co-prescription of allopurinol in patients with inflammatory bowel disease

IntroductionNumerous patients, especially those with elevated thiopurine methyltransferase (TPMT) activity, selectively methylate thiopurine drugs, generating high levels of methylated metabolites and low thioguanine nucleotides. This pattern of metabolism is related...

MRI Guided Biologic Therapy for Crohn's Perianal Fistulae: 3 Year Follow up Data

Background: Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of azathioprine and 6-mercaptopurine. Higher TPMT activity is associated with lower levels of the therapeutic metabolite 6-thioguanine (6-TG). Some therapeutic studies in patients with inflammatory bowel disease have excluded those with intermediate TPMT activity. A small difference in TPMT enzyme activity has been reported between AfricanAmericans and Caucasians. Our own preliminary data suggested that this difference may be more pronounced. We sought to further assess racial differences in TPMT activity to help determine optimal treatment strategies for patients with inflammatory bowel disease. Methods: A retrospective chart review from the electronic medical records of Henry Ford Health System was conducted for patients who had undergone testing for TPMT enzyme activity from June 2004 through September 2010. Testing was performed at Prometheus Laboratories (San Diego, CA). TPMT activity was categorized into low ( 21 EU). Results: 394 patients underwent TPMT testing during the study period. Race was available for 388 patients (246 women and 142 men). The population was composed of 223 Caucasians, 134 African-Americans, and 31 patients of other race. Mean TPMT level was 28.3 EU (SD 9.6) for Caucasians, 23.3 EU(SD 8.2) for African-Americans, and 27.5 EU (SD 9.0) for others (p <0.001). Mean TPMT activity for the 13 Middle Eastern patients in the population was 22.1 (SD 7.8) which was significantly lower than that of Caucasians (p = 0.024). Analysis by TPMT activity category showed that low activity was seen in only 2 patients, and both were Caucasians. Intermediate activity was seen in 37 of the 223 Caucasians (16.6%), 52 of 134 African-Americans (38.8%), and 7 of 31 other race patients (22.6%). Normal TPMT activity was seen for 184 of the 223 Caucasians (82.5%), 82 of the 134 African-Americans (61.2%), and 24 of the 31 patients of other race (77.4%). This distribution was statistically significant (p <0.001). Conclusions: Mean TPMT activity is lower in African-American and Middle Eastern patients compared to Caucasians. African-Americans are more likely to have intermediate TPMT activity compared to Caucasians. Therapeutic studies using 6-mercaptopurine and azathioprine that exclude patients with intermediate TPMT activity may exclude a high proportion of AfricanAmerican patients.

Significant Racial Differences in Thiopurine Methyltransferase Enzyme Activity in a Metropolitan Population

Background: Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of azathioprine and 6-mercaptopurine. Higher TPMT activity is associated with lower levels of the therapeutic metabolite 6-thioguanine (6-TG). Some therapeutic studies in patients with inflammatory bowel disease have excluded those with intermediate TPMT activity. A small difference in TPMT enzyme activity has been reported between AfricanAmericans and Caucasians. Our own preliminary data suggested that this difference may be more pronounced. We sought to further assess racial differences in TPMT activity to help determine optimal treatment strategies for patients with inflammatory bowel disease. Methods: A retrospective chart review from the electronic medical records of Henry Ford Health System was conducted for patients who had undergone testing for TPMT enzyme activity from June 2004 through September 2010. Testing was performed at Prometheus Laboratories (San Diego, CA). TPMT activity was categorized into low ( 21 EU). Results: 394 patients underwent TPMT testing during the study period. Race was available for 388 patients (246 women and 142 men). The population was composed of 223 Caucasians, 134 African-Americans, and 31 patients of other race. Mean TPMT level was 28.3 EU (SD 9.6) for Caucasians, 23.3 EU(SD 8.2) for African-Americans, and 27.5 EU (SD 9.0) for others (p <0.001). Mean TPMT activity for the 13 Middle Eastern patients in the population was 22.1 (SD 7.8) which was significantly lower than that of Caucasians (p = 0.024). Analysis by TPMT activity category showed that low activity was seen in only 2 patients, and both were Caucasians. Intermediate activity was seen in 37 of the 223 Caucasians (16.6%), 52 of 134 African-Americans (38.8%), and 7 of 31 other race patients (22.6%). Normal TPMT activity was seen for 184 of the 223 Caucasians (82.5%), 82 of the 134 African-Americans (61.2%), and 24 of the 31 patients of other race (77.4%). This distribution was statistically significant (p <0.001). Conclusions: Mean TPMT activity is lower in African-American and Middle Eastern patients compared to Caucasians. African-Americans are more likely to have intermediate TPMT activity compared to Caucasians. Therapeutic studies using 6-mercaptopurine and azathioprine that exclude patients with intermediate TPMT activity may exclude a high proportion of AfricanAmerican patients.

6-Mercaptopurine pharmacokinetics after use of azathioprine in renal transplant recipients with intermediate or high thiopurine methyl transferase activity phenotype.

Prevention of allograft transplant rejection by the immunosuppressive 6-thiopurine drug azathioprine is limited by haematological toxicity (leucopenia or agranulocytosis). This toxicity is particularly apparent in subjects with low thiopurine methyltransferase activity (TPMTase) phenotype (1% in the Caucasian population). The thiopurine derivative 6-mercaptopurine is the active metabolite of azathioprine, and it would be of interest to measure, after validation of plasma measurements, the mean values of the pharmacokinetic parameters in transplant patients with high or intermediate TPMTase phenotypes (85 and 14% of the Caucasian population, respectively). We measured erythrocyte TPMTase activity in 103 kidney transplant recipients of high or intermediate phenotype and calculated, after a test dose of azathioprine, the mean values of the pharmacokinetic parameters for 6-mercaptopurine. We also compared these values with the same parameters from one subject with low TPMTase activity phenotype. The mean observed area under the plasma concentration-time curve (AUC) was 190+/-140 ng mL(-1) h and the elimination rate constant (Kel) was 1.92+/-1. The pharmacokinetic parameters (AUC, Kel, t1/2el (the elimination half-life)) of 6-mercaptopurine in transplant patients are normally distributed and suitable for acceptance as a gold standard value for this population of Caucasian transplant patients. It seems useful to calculate these parameters, representative of the systemic exposure of individual patients to the drug, before prescribing these subjects azathioprine immunosuppressive treatment. In subjects with low TPMTase phenotype these pharmacokinetic measurements could also be an index of dose reduction.

Allopurinol Salvage Therapy in Pediatric Overlap Autoimmune Hepatitis‐Primary Sclerosing Cholangitis With 6‐MMP Toxicity

Allopurinol Salvage Therapy in Pediatric Overlap Autoimmune Hepatitis‐Primary Sclerosing Cholangitis With 6‐MMP Toxicity

W1915 Further Experience of Optimising Treatment Outcome on Azathioprine by Co-Prescription of Allopurinol in Patients With Inflammatory Bowel Disease

Introduction Many patients, particularly those with high thiopurine methyltransferase (TPMT) activity, preferentially methylate thiopurine drugs, resulting in high levels of methylated metabolites and low thioguanine nucleotides (TGNs). This metabolite pattern is associated with hepatotoxicity and non-response to treatment. Co-prescription of thiopurines (at 25–50% of standard dose) with allopurinol, (which blocks xanthine oxidase) appears to circumvent this problem, optimising both metabolite profile and clinical outcome. Data on the use of allopurinol and thiopurines remain limited. We previously reported our experience of using this combination in thiopurine-related hepatotoxicity. In this study we report further experience for a broader indication, including toxicity (mainly hepatic) non-response with adverse metabolite profile, and very high TPMT activity. Methods Patients prescribed combination treatment were identified from notes in the IBD clinic, TGN monitoring results and hospital pharmacy records. Data were collected retrospectively. We were particularly interested in whether combination treatment overcame the specific problem that precluded thiopurine monotherapy. We also analysed changes in metabolite profiles and subsequent clinical outcome. Results 35 patients have been co-prescribed allopurinol and thiopurines since our previous report. 20 had Crohn9s disease and 15 ulcerative colitis, age range 20–65 years. Their average TPMT activity was 40 pmol/h/mgHb, (vs 32.5 in our population overall). In those receiving co-prescription for adverse effects (14 hepatotoxicity and three other: rash, nausea, fatigue) 94% were able to tolerate combination treatment with complete resolution of liver function tests abnormalities where relevant, of these 88% achieved clinical remission. In 10 patients failing thiopurine monotherapy 50% achieved clinical response on combination treatment. In eight started on combination treatment as primary therapy six (75%) achieved remission, comparing favourably with historical controls. The only adverse event was a transient lymphopaenia in a patient that had mistakenly continued full dose thiopurine. TGN levels increased from an average of 192 to 449 pmol/8×108 RBC on co-treatment, while methylmercaptopurine levels decreased from an average of 503 pmol/8×108 RBC on single agent treatment to just 16 on combination therapy. Conclusion Combination treatment with reduced-dose thiopurine and allopurinol circumvents predominant methylation, optimising metabolite profiles, avoiding hepatotoxicity and improving clinical response. Using combination treatment as first line should lead to a faster remission in those with high TPMT activity. Allopurinol co-prescription may also overcome other side effects. Whether combination therapy should be used first-line in those with high TPMT activity, or more extensively, warrants further investigation in a prospective trial.

Monitorización de la tiopurina metiltransferasa y de los metabolitos tiopurínicos para optimizar el tratamiento con azatioprina en la enfermedad inflamatoria intestinal

Determination of the activity of thiopurine methyltransferase (TPMT) and of thiopurine metabolites (6-thioguanine and 6-methylmercaptopurine nucleotides) could be useful for individualized monitoring of azathioprine (AZA) and 6-mercaptopurine (6-MP) doses. TPMT activity in the general population follows a trimodal distribution, in which approximately 0.3% of the population is homozygotic for the low-activity allele. A notable correlation has been observed between the low TPMP activity genotype or phenotype and the risk of myelotoxicity. Patients with a high TPMT activity genotype or homozygous phenotype should receive immunosuppressive doses that have clearly been demonstrated to be effective. In contrast, in patients with a low TPMT activity genotype or homozygous phenotype, the use of AZA/6-MP should be contraindicated or only very small doses should be administered. Importantly, TPMP deficiency explains only some cases of myelotoxicity and consequently periodic laboratory testing should be performed in patients receiving AZA/6-MP, even though TPMP function may be normal. Currently, the utility of routine thiopurine metabolite determinations in patients undergoing AZA/6-MP therapy has not been established and this practice should be limited to specific situations such as lack of response to thiopurine therapy or the occurrence of thiopurine-related adverse effects. Randomized trials comparing the routine strategy of AZA/6-MP dosing (based exclusively on the patient's weight) versus individualized monitoring (based on quantification of TPMP activity and/or thiopurine metabolites) are required before definitive conclusions on the most effective alternative can be drawn.

Thiopurine Methyltransferase (TPMT) Activity and Adverse Effects of Azathioprine in Inflammatory Bowel Disease: Long-Term Follow-Up Study of 394 Patients

To prospectively evaluate whether a relationship between thiopurine methyltransferase (TPMT) activity and incidence of adverse effects (especially myelotoxicity) exists, in a long-term follow-up study of a large group of patients with inflammatory bowel disease treated with azathioprine. TPMT activity in red blood cells (RBC) was measured by a radiochemical method in 394 consecutive patients with Crohn's disease (238) or ulcerative colitis (156) starting treatment with azathioprine. The relationship among several variables and TPMT values was assessed, and the correlation between such levels and the incidence of adverse effects was evaluated. Mean TPMT value was 18.6 +/- 4 U/mL RBCs (range 9.4-33.7). No patient had low levels (<5), 7.1% had intermediate levels (5-13.7), and 92.9% had high levels (>13.8). Differences (P < 0.001) were demonstrated in TPMT activity depending on the type of inflammatory bowel disease, but not on the remaining variables (including treatment with 5-aminosalycilates). Adverse effects were reported in 74 patients (18.8%), the most frequent being gastrointestinal intolerance (9.1%) and myelotoxicity (4.3%). No patient having adverse effects had low TPMT levels. However, mean TPMT activity was lower in those with adverse effects (16.6 +/- 3 vs 19.1 +/- 4 U/mL, P < 0.001). Moreover, the probability of suffering myelotoxicity in the high TPMT group was only 3.5%, compared with 14.3% in the TPMT intermediate group (95% CI = 1.37-14.9; OR = 4.5). The strategy of determining TPMT activity in all patients prior to initiating treatment with azathioprine could help to minimize the risk of myelotoxicity, as patients with intermediate TPMT activity had fourfold more risk than high TPMT activity patients.

Thiopurine S-methyltransferase as a target for drug interactions

The present study was undertaken to investigate the possible effects of various agents on thiopurine methyltransferase (TPMT) activity in red blood cells (RBCs) from patients with chronic inflammatory bowel disease (IBD). In three groups of patients with very high, normal and intermediate TPMT activity (each n=6), the inhibitory potential of furosemide, piretanide, azathioprine (AZA) and testosterone was assessed by ex vivo measurements of TPMT activity in RBCs. From individual concentration-response curves, IC50 values have been determined. Independent of the basal TPMT activity, lowest IC50 values were calculated for furosemide (15-19 microM), followed by testosterone (30-72 microM), piretanide (300-313 microM) and AZA (430-532 microM). Compared with reported plasma concentration achieved during treatment, only furosemide would have the potential to inhibit TPMT also in vivo, whereas the IC50 values of the other agents are far above the corresponding plasma levels. Our ex vivo study revealed that only furosemide has the potential to inhibit TPMT activity in patients with IBD. This possibility should be taken into consideration if the diuretic and AZA or 6-mercaptopurine are coadministered. However, the extrapolation to the clinical setting remains open.

THIOPURINE METHYLTRANSFERASE ACTIVITY IS CORRELATED WITH AZATHIOPRINE METABOLITE LEVELS IN INFLAMMATORY BOWEL DISEASE PATIENTS IN CLINICAL GASTROENTEROLOGY PRACTICE

Purpose: Genetic polymorphisms in thiopurine methyltransferase (TPMT) influence the metabolism of azathioprine (AZA) and 6-mercaptopurine (6-MP) in patients with inflammatory bowel disease (IBD). It has been suggested in studies using weight-based dosing that higher TPMT activity is associated with lower levels of 6-thioguanine (6-TG), the metabolite thought to provide therapeutic benefit, and higher levels of 6-methylmercaptopurine (6-MMP), the metabolite associated with hepatotoxicity. Some studies suggest that higher TPMT activity correlates with lower rates of clinical response. The aim of this study is to assess the relationship between TPMT activity and 6-TG and 6-MMP levels in patients with IBD in clinical practice. Methods: We analyzed the TPMT activity, the 6-TG level, and the 6-MMP level in all patients who had at least one TPMT assessment and at least two metabolite profiles sent from a gastroenterologist's office to Prometheus Laboratories (San Diego, CA) from June 2000 to February 2004. The use of AZA or 6-MP and the dosing of the drug was at the discretion of the patient's gastroenterologist. Linear regression was used to analyze the relationships and a Pearson correlation was calculated. Results: A total of 1,020 patients were identified. Linear regression showed a significant inverse relationship between TPMT activity and 6-TG level with a Pearson correlation of-0.162 (p <0.01), a significant direct relationship between TPMT activity and 6-MMP level with a Pearson correlation of 0.172 (p <0.01), and a significant direct relationship between TPMT activity and 6-MMP/6-TG ratio with a Pearson correlation of 0.141 (p <0.01). Conclusions: Higher TPMT activity is associated with lower 6-TG levels and higher 6-MMP levels in IBD patients treated with AZA or 6-MP in clinical practice with drug dosing at the discretion of the primary gastroenterologist. This suggests a possible role for TPMT activity assays in patients being considered for treatment with these agents.

Thiopurine Methyltransferase Activity in Red Blood Cells of Dogs

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6‐mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9‐fold range (7.9–71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9–20 U of RBC per milliliter; median, 13.1;P&lt; .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7–71.8 U of RBC per milliliter; median, 36.0;P&lt; .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.

Thiopurine methyltransferase activity in red blood cells of dogs.

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9-71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9-20 U of RBC per milliliter; median, 13.1; P < .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7-71.8 U of RBC per milliliter; median, 36.0; P < .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.

Thiopurine methyltransferase activity and the use of azathioprine in inflammatory bowel disease.

Azathioprine therapy is discontinued in one-third of patients with inflammatory bowel disease because of toxicity or a lack of clinical response. Patients with thiopurine methyltransferase (TPMT) deficiency are intolerant to azathioprine, whilst carriers are at increased risk of side-effects. To evaluate the importance of TPMT activity in the management of azathioprine therapy in inflammatory bowel disease. Clinical response, adverse effects and haematological parameters were determined and correlated with TPMT enzyme activity and genotype in 106 patients with inflammatory bowel disease. Ninety-six patients had high TPMT activity, and 10 had intermediate activity. Nineteen patients (18%) were intolerant to azathioprine. Fifteen (16%) of those with high TPMT activity were intolerant, compared with five (50%) with intermediate activity [odds ratio (OR), 5.4; 95% confidence interval (CI), 1.5-19.8]. Complete remission was achieved in 63% of cases, and complete or partial remission in 79%. Interestingly, very high TPMT activity (> 14 units/mL red blood cells) was significantly associated with non-response, irrespective of the time on azathioprine (OR, 0.21; 95% CI, 0.07-0.68). TPMT gene mutations correlated with TPMT activity. Inflammatory bowel disease patients with intermediate TPMT activity have an increased risk of azathioprine toxicity. Conversely, very high TPMT activity predicts treatment failure. TPMT genotype predicted TPMT phenotype in this study.

Phenotype Determination of Thiopurine Methyltransferase in Erythrocytes by HPLC

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S -methylation of thiopurine drugs, which are used in cancer chemotherapy and as immunosuppressive agents (1). TPMT activity is controlled by a common genetic polymorphism that contributes to interindividual variability in drug response and, consequently, to implications for thiopurine therapeutic efficacy and toxicity (2). Severe myelosuppression has been reported for TPMT-deficient patients treated with standard doses of thiopurines (3)(4)(5), and high TPMT activity has been associated with the rejection of transplanted organs (6). Because of the clinical significance of the TPMT genetic polymorphism, determination of the TPMT phenotype in red blood cells is routinely performed to optimize and individualize thiopurine treatment (5). Variant alleles of the TPMT gene have been characterized and associated with low TPMT activity (7)(8). Recently, Spire-Vayron de la Moureyre et al. (9) reported that genotypic analysis of TPMT allows the correct determination of metabolic capacity for 87% of individuals. A lower correlation was found for individuals with TPMT activity that was close to the antimode value. Thus, phenotypic analysis may be useful and could be performed concomitantly with genotyping tests. TPMT activity has classically been measured using a radiochemical assay (10). Few HPLC methods using nonradiolabeled calibrators with liquid-liquid extraction (11)(12) or solid-phase extraction (13) have been reported. Here, we report a reversed-phase HPLC method that uses a simple and rapid treatment procedure for the determination …

Screening for azathioprine toxicity: A pharmacoeconomic analysis based on a target case

The risk of azathioprine-induced myelosuppression can be predicted by detecting patients with intermediate or low thiopurine methyltransferase (TPMT) activity. Population studies have shown that 89% of whites have high TPMT activity, 11% have intermediate TPMT activity, and 0.3% have low TPMT activity. Three specific mutations in the TPMT gene that cause decreased TPMT activity have recently been identified. Patients homozygous for the TPMT mutations have low TPMT activity, and patients heterozygous for TPMT mutations have intermediate TPMT activity. This has led to the development of a technique for TPMT genotype analysis that will identify patients at risk of azathioprine-induced myelosuppression. We report a case of a patient with bullous pemphigoid who experienced azathioprine-induced myelosuppression and who was later found to be homozygous for TPMT mutant alleles. Using the cost of treatment required for this patient and the estimated population prevalence of TPMT mutations, we examined the cost impact of screening for TPMT mutations in all patients being considered for azathioprine therapy. We calculated that screening is cost-neutral assuming patients homozygous for TPMT mutations experience myelosuppression, and that it is cost-beneficial assuming patients heterozygous for TPMT mutations also experience myelosuppression while receiving azathioprine. Screening patients for TPMT mutations will reduce the risk of azathioprine-induced myelosuppression, and our study suggests that it may also be a cost-attractive strategy. (J Am Acad Dermatol 2000;42:628-32.)

Thiopurine methyltransferase activity and its relationship to the occurrence of rejection episodes in paediatric renal transplant recipients treated with azathioprine.

Azathioprine is a prodrug commonly used in combination therapy to prevent allograft rejection after renal transplantation. After conversion to 6-mercaptopurine, the drug is metabolized into 6-thioguanine nucleotides (6-TGN) and catabolized by thiopurine methyltransferase (TPMT), an enzyme under monogenic control. The aim of this study was to evaluate the inter- and intraindividual variability of red blood cell thiopurine methyltransferase and 6-TGN concentrations and their relationship to the clinical effects of azathioprine in paediatric patients. In the present study, the inter- and intraindividual variations in red blood cell TPMT activity and 6-TGN concentrations and their relationship to the actions of azathioprine were evaluated during the first year after renal transplantation in 22 paediatric patients. 6-TGN concentration reached steady-state values after 6 months and correlated negatively with TPMT activity (P=0.004). Initial TPMT activity (median: 20.8 nmol h-1 ml-1, range 7.8-34.6) and 6-TGN concentration at steady-state (median: 80 pmol 8 x 10(8-1) cells, range not detected to 366) were not related to the occurrence of rejection episodes during the period of the study. In contrast, TPMT activity and the percentage difference in TPMT activity from the day of transplantation determined at month 1 were higher in the patients with rejection episodes by comparison with those that did not reject during the first 3 months or the first year following transplantation (P<0.005). We report a relationship between TPMT activity and occurrence of rejection in paediatric kidney transplant patients undergoing azathioprine therapy. These data suggest a link between high red blood cell TPMT activity and poor clinical outcome probably caused by rapid azathioprine catabolism.

Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia.

The role of 6-mercaptopurine (6MP) in the treatment of childhood acute lymphoblastic leukaemia (ALL) is well established. However, the efficacy of 6MP is significantly influenced by inactivation by the polymorphic enzyme thiopurine methyltransferase (TPMT). In the general population 89-94% have high TPMT activity, 6-11% have intermediate activity, and approximately 0.3% have low activity. Individuals with low-activity experience severe or fatal toxicity with standard 6MP doses. Prospective identification of this group of patients might prevent this problem. Recent identification of the molecular basis for low TPMT activity enabled rapid assessment of altered 6MP metabolism by PCR methods. This study evaluated the frequency of mutant TPMT alleles in 147 children with ALL. One patient was homozygous mutant (0.7%), and 16 patients were heterozygous for variant TPMT alleles (10.9%). The majority of mutant alleles were TPMT*3A. Both the allele frequency and the pattern of TPMT mutations were similar to that observed in an adult British population. The number of weeks when 6MP therapy was administered at full dose was determined in patients on MRC UKALL X and XI. The 94 patients spent a median 11% of the maintenance period receiving no therapy as a result of haematological toxicity. There was no significant difference in the number of weeks when no therapy could be administered among patients with a wild-type or heterozygous genotype. However, the one patient with a homozygous mutant genotype had severe haematological toxicity and no therapy could be administered for 53% of the maintenance period. This study demonstrates that 11.6% of the children had variant TPMT alleles. Prospective identification of TPMT genotype may be a promising tool for decreasing excessive haematological toxicity in individuals with low activity.

The frequency and distribution of thiopurine methyltransferase alleles in Caucasian and Asian populations.

Thiopurine methyltransferase metabolizes 6-mercaptopurine, thioguanine and azathioprine, thereby regulating cytotoxicity and clinical response to these thiopurine drugs. In healthy Caucasian populations, 89-94% of individuals have high thiopurine methyltransferase activity, 6-11% intermediate and 0.3% low, resulting from genetic polymorphism. Four variant thiopurine methyltransferase alleles were detected in over 80% of individuals with low or intermediate thiopurine methyltransferase activity. The wild-type allele is defined as TPMT*1 and the mutant alleles are TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3B (A719G). The frequency of these alleles in different ethnic groups is not well defined. In this study, DNA from 199 British Caucasian, 99 British South West Asian and 192 Chinese individuals was analysed for the presence of these variant alleles using polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction based assays. The frequency of individuals with a variant thiopurine methyltransferase genotype was: Caucasians 10.1% (20/199), South West Asians 2.0% (2/99) and Chinese 4.7% (9/192). Two TPMT*2 heterozygotes were identified in the Caucasian population, but this allele was not found in the two Asian populations. TPMT*3A was the only mutant allele found in the South West Asians (two heterozygotes). This was also the most common mutant allele in the Caucasians (16 heterozygotes and one homozygote) but was not found in the Chinese. All mutant alleles identified in the Chinese population were TPMT*3C (nine heterozygotes). This allele was found at a low frequency in the Caucasians (one heterozygote). This suggests that A719G is the oldest mutation, with G460A being acquired later to form the TPMT*3A allele in the Caucasian and South West Asian populations. TPMT*2 appears to be a more recent allele, which has only been detected in Caucasians to date. These ethnic differences may be important in the clinical use of thiopurine drugs.