Intermediate Thiopurine Methyltransferase Research Articles

Commentary: individual vs. weight‐based dosing of azathioprine in Crohn's disease

This study illustrates the difficulties of running a clinical study. There were several attempts to change the protocol, but low enrolment numbers resulted in an underpowered negative study. The primary end point of this study was clinical remission at 16 weeks. There was no difference in remission rates at 16 weeks, although the per-protocol analysis showed a trend towards better results in the individualised group (clinical remission in 3/12 in weight-based vs. 9/15 in the individualised group). These remission rates were below predicted levels, partly because a high proportion of patients had steroid dependency or steroid-refractory disease. The secondary outcomes were remission at 28 and 52 weeks, but only 4 patients were still in the study at week 52. This study shows that dose escalation does not always achieve the target concentration. For the individualised group, the mean 6-tioguanine (thioguanine) (6TGN) concentrations among normal thiopurine methyltransferase (TPMT) metabolisers was 198 pmol/8 9 10 red blood cells (significantly below the therapeutic target of 250, and not different from the mean 6TGN for the weight-based arm). These disappointingly low 6TGN levels may also explain the low remission rates. This study adds to the data showing the clinical benefit of achieving higher 6TGN levels and is consistent with a meta-analysis of 6TGN and IBD activity. There was a significant difference in 6TGN between patients achieving remission and those not achieving remission (216 vs. 149), although a wide range and significant overlap in values were observed. The study showed a modest difference in dose between individualised and weight-based groups (3.4 compared with 2.3 mg/kg for normal TPMT and 1.5 compared with 2.4 mg/kg for intermediate levels), but the trial was not long enough to observe a more significant divergence. A study by Gardiner et al. showed a two-fold divergence in dosing after 9 months of monitoring and dose change, although they predicted that a three-fold difference was likely to be more appropriate. The most useful test may be the initial TMPT activity with reduction of starting dose to 1.0 – 1.5 mg/kg for patients with intermediate TPMT (in this study, the numbers with intermediate TMPT were low and it is difficult to reach any conclusions). The only similar study is a randomised, open-label study where dose adjustment was based on 6TGN alone. Recruitment was also difficult, and the numbers in each group were small. Similar to this study, mean concentrations of 6TGN were below target in the dose-adjusted group and not different from the weightbased group. It is difficult to see this study being repeated. The concept of dose optimisation of azathioprine dose is now well accepted, and there will be reluctance among clinicians to enrol patients into a weight-based arm. There is a strong body of data, including studies with azathioprine/allopurinol combination, supporting this approach. Dose optimisation may be a slow process and not easy to replicate in a clinical trial. However, the disappointing remission rates in this study should not discourage use of azathioprine for an extended period with dose optimisation.

Randomised clinical trial: individualised vs. weight‐based dosing of azathioprine in Crohn's disease

Azathioprine (AZA), a pro-drug metabolised to the active metabolites 6-tioguanine nucleotides (6TGN), is a steroid-sparing therapy for Crohn's disease (CD). To investigate whether AZA therapy is optimised by individualised dosing based on thiopurine methyltransferase (TPMT) activity and 6TGN concentrations. This multicentre, double-blind, randomised controlled trial compared the efficacy and safety of weight-based vs. individualised AZA dosing in inducing and maintaining remission in adults and children with steroid-treated CD. The primary outcome was clinical remission (CR) at 16 weeks. In the weight-based arm, subjects received 2.5 mg/kg/day. In the individualised dosing arm, the initial AZA dose was 1.0 mg/kg/day (if intermediate TPMT) or 2.5 mg/kg/day (if normal TPMT). Starting at week 5, the dose was adjusted to target 6TGN concentrations of 250-400 pmol/8 × 10(8) red blood cells (RBC), or to a maximal dose of 4 mg/kg/day. After randomising 50 subjects, the trial was stopped prematurely due to insufficient enrolment. In intention-to-treat analysis, CR rates at week 16 were 40% in the individualised arm vs. 16% in the weight-based arm (P = 0.11). In per-protocol (PP) analysis, week 16 CR rates were 60% in the individualised arm and 25% in the weight-based arm (P = 0.12). At week 16, median 6TGN concentrations in PP remitters and nonremitters were 216 and 149 pmol/8 × 10(8) RBC respectively (P = 0.07). Despite trends favouring individualised over weight-based AZA dosing, there were no statistically significant differences in efficacy, likely due to low statistical power and inability to achieve the target 6TGN concentrations in the individualised arm. [Clinicaltrials.Gov Identifier Nct00113503].

The Utility of Thiopurine Methyltransferase Enzyme Testing in Inflammatory Bowel Disease

To assess the levels of red blood cell thiopurine methyltransferase (TPMT) in subjects with inflammatory bowel disease (IBD) and to determine how these levels impacted thiopurine dosing and leukopenia over the first six months of therapy. A retrospective chart review was performed on all adult IBD patients (n=423, 88.2% Caucasian) who had TPMT levels measured by 11 participating gastroenterologists in Manitoba between 2008 and 2010. In addition to descriptive data, white blood cell count, dose and reason for discontinuation were analyzed for the first six months of therapy. Patients receiving ≥2.0 mg/kg of azathioprine (AZA) or ≥1.0 mg/kg of 6-mercapatopurine were considered to be 'substantially' dosed. Of the 423 patients, 8.3% had intermediate levels and 93.4% had normal levels of TPMT. Only one subject had a low level. A total of 216 patients had sufficient data to be included for full analysis. Patients with intermediate TPMT levels were generally started at lower doses of thiopurine than patients with normal TPMT levels (mean [± SD] 1.0±0.6 mg/kg versus 1.8±0.5 mg/kg). Of the subjects with normal TPMT levels, only 37.8% were dosed with ≥2.0 mg/kg of AZA. Each month, approximately 5% of subjects were leukopenic. These subjects received a mean overall AZA dose of 1.9±0.3 mg/kg and had a mean white blood cell count of 3.8±0.4×10(9)/L. Normal TPMT levels did not prevent the development of leukopenia, although life-threatening leukopenia was rare. Physicians are not using TPMT levels to substantially dose thiopurines at the outset, which may limit the speed at which adequate doses are reached to facilitate remission.

High TPMT enzyme activity does not explain drug resistance due to preferential 6‐methylmercaptopurine production in patients on thiopurine treatment

Up to 20% of patients on thiopurine therapy fail to achieve adequate drug response. Many of these patients preferentially produce the toxic 6-methylmercaptopurine metabolites (6-MMP) rather than the active 6-thioguanine nucleotides (6-TGN) resulting in a high 6-MMP/6-TGN ratio (>20) and increased risk of hepatotoxicity. To determine the prevalence of preferential 6-MMP producers and define the relationships between 6-TGN, 6-MMP and thiopurine methyltransferase (TPMT). The database of 6-TGN, 6-MMP and TPMT measurements from patients throughout New Zealand was used to calculate patients' 6-MMP/6-TGN ratios and identify those with high (>20) or normal ratio (≤20).The TPMT enzyme activity was compared amongst the groups. Of 1879 patients with TPMT, 6-TGN and 6-MMP results, 349 (19%) had a 6-MMP/6-TGN ratio >20. The mean TPMT enzyme activity was slightly lower for those with a 6-MMP/6-TGN ratio ≤20 vs. >20, which achieved statistical significance (12.2 vs. 13.2; P < 0.001). However, the distributions of TPMT enzyme activity were similar, with 97% of TPMT results falling between 5.0 and 17.6 IU/mL for both groups. In all, 17% of those with 6-MMP/6-TGN ratio ≤20 were intermediate TPMT metabolisers (TPMT 5.0-9.2 IU/mL) vs. 7% in those with a ratio >20. In this patient population with measured 6-MMP/6-TGN ratios, 19% of patients were preferential 6-MMP producers. The results show that high TPMT enzyme activity is not the major reason for preferential 6-MMP production in most patients with a high metabolite ratio. This suggests that there are one or more important alternative mechanisms for preferentially producing 6-MMP.

Abstract CN04-02: Interrogating the heritable genome to achieve the goal of personalized cancer therapeutics.

Abstract CN04-02: Interrogating the heritable genome to achieve the goal of personalized cancer therapeutics.

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.

The value of tpmt measurement in the routine clinical management of patients with IBD

IntroductionThe measurement of thiopurine methyl transferase (TPMT) can be used to identify patients at risk of marrow suppression from azathioprine. Across the medical specialities, there is no clear consensus or...

Influences of Thiopurine Methyltransferase Genotype and Activity on Thiopurine-induced Leukopenia in Korean Patients With Inflammatory Bowel Disease

Myelotoxicity has been shown to be very common in Korean patients with inflammatory bowel disease (IBD) during azathioprine (AZA) or 6-mercaptopurine (6-MP) treatment. The purpose of this study was to investigate the relative risk of the thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) genotypes and TPMT activity for the development of leukopenia in Korean IBD patients during AZA/6-MP treatment. We retrospectively analyzed 286 Korean patients with IBD who had been treated with AZA/6-MP for at least 6 months between June 1996 and September 2006. Common TPMT mutations, including TPMT*1, *2, *3A, *3B, and *3C, and ITPA mutations, including 94C>A and IVS2+21A>C, were determined using a high-performance liquid chromatography method. TPMT activity was measured using liquid chromatography with coupled mass spectrometry/mass spectrometry. Leukopenia occurred in 118 cases (41.3%). TPMT *1/*3C was detected in 7 cases (2.4%), and ITPA 94 C>A was detected in 66 cases (23.1%), including 63 heterozygotes (22.1%) and 3 homozygotes (1.0%). The median TPMT activity was 9.3 U/mL (interquartile range 10.4, range 2.1 to 76.2). Cox regression analysis revealed that patients with heterozygous *3C type TPMT had a higher probability of leukopenia than those with wild type TPMT (P=0.02). Patients with intermediate TPMT activity had a lower probability of leukopenia than those with low activity (P=0.01). However, the ITPA genotype did not affect the risk of leukopenia. Our data showed that it could be helpful to examine TPMT genotypes and to measure TPMT activity in Korean patients taking AZA/6-MP to predict the development of leukopenia.

Black Americans are More Likely to have Intermediate TPMT Enzyme Levels than White Americans

Purpose: Thiopurine methyltransferase (TPMT) metabolizes azathioprine (AZA) and mercaptopurine (6MP) into active and inactive metabolites. Optimal therapeutic effect is achieved at 6-thioguanine levels (6TG) between 230-400. Achieving therapeutic levels of 6TG is limited by the concurrent production of the inactive hepatotoxic metabolite 6-methylmercaptopurine (6MMP). A patient's TPMT enzyme activity is related to the ratio of production of these metabolites. Black Americans and Caucasians have differing frequencies of mutant alleles resulting in similar phenotypes of low, intermediate and normal levels. It is unclear whether there are racial differences in TPMT levels. Such differences could affect both medication choices and dosage regimens in the management of patients with inflammatory bowel disease. We sought to investigate ethnic variations in TMPT levels. Methods: We performed a retrospective chart review of patients who underwent TPMT level or genetic testing at our institution from June 2004 to June 2009. TPMT testing was performed at Prometheus Laboratories (San Diego, CA). Results: We identified 272 patients who had TPMT levels drawn (96 males, 176 females). Of the patients with quantitative TPMT levels available for review, 80 were self-described black American, 133 Caucasian, and 18 other. These levels are categorized into low, intermediate and normal (with less than 6.0 EU being low, 6.0- 21.0 EU intermediate and greater than 21.0 EU normal). The mean TPMT level for black Americans was 23.2 EU (SD 8.1), for Caucasians 27.7 EU (SD 9.4) and for other patients 25.7 EU (SD 8.7) (p=0.002). The mean levels are all within the normal range. Categorization of these levels into low, intermediate and normal showed that 0/80 (0.0%) of black Americans, 1/133 (0.8%) of Caucasians and 0/18 (0.0%) of others had low enzyme levels. 31/80 (38.8%) of black Americans, 26/133 (19.6%) of Caucasians and 5/18 (27.3%) of others had intermediate levels. 49/80 (61.3%) of black Americans, 106/133 (79.7%) of Caucasians and 13/18 (72.2%) of others had normal levels. The difference among the three categories is statistically significant (Fisher exact test p-value=0.017). Conclusion: Significant racial differences exist in mean TPMT enzyme levels among different American ethnic groups. While mean levels fall within the normal enzyme range, there was a statistically significant difference in the distribution of patients who fall into the intermediate versus the normal enzyme level between black and white Americans with a higher percentage of black Americans in the intermediate range and higher percentage of Caucasians in the normal range. Such differences may have important clinical implications insofar as ability to achieve therapeutic levels of 6TG.

The Increasing Complexity of Mercaptopurine Pharmacogenomics

Thiopurine methyltransferase (TPMT) activity shows significant interindividual variation, with approximately 90% of individuals having high (wild-type) activity, 10% with intermediate activity, and 0.3% with low activity. Low and intermediate TPMT activity leads to toxicity from mercaptopurine and the need for dose reduction. Common variants in the TPMT gene have a strong association with mercaptopurine toxicity. However, recent research has shown that genetic contribution to mercaptopurine toxicity is more complex, possibly involving other genes, in particular ITPA, which encodes inosine triphosphate pyrophosphatase.

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

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. To confirm the hypothesis that allopurinol may alleviate thiopurine hepatotoxicity by similar mechanisms as proposed in our renal study. 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. 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 (stealosis on biopsy). Leucopenia occurred in two cases and resolved with minor dose reductions. 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.

Thiopurine S-methyltransferase activity in three major Asian populations: a population-based study in Singapore

The distribution of thiopurine methyltransferase (TPMT) activity in Asian populations has not been well documented. We studied the TPMT phenotype in three major Asian ethnic groups in Singapore, namely the Chinese (Ch), Malays (Mal) and Indians (Ind), with the aim of carrying out a comprehensive survey of the distribution of TPMT activity in Asians. A radiochemical assay was used to measure the enzymatic activity of TPMT in the red blood cells (RBCs) of 479 healthy adults (Ch=153, Mal=163 and Ind=163). Cut-off points for intermediate TPMT activity were validated using a receiver operating curve (ROC) analysis. PCR-based methods were used to screen for the TPMT*3C, TPMT*3A and TPMT*6 variants. The histogram of the combined population cohort showed a bimodal distribution of TPMT activity, with no subject having low TPMT activity (<5 units). In total, TPMT variants were detected in 14 subjects (*1/*3C in 13 subjects; *1/*3A in one subject). We observed significant inter-ethnic differences in terms of TPMT activity (p<0.001), with the Malays showing a higher median activity than the Chinese or Indians (17.8 units vs 16.4 units). The Malays also showed a higher methylation rate--with a cut-off point for intermediate TPMT activity of 11.3 units--than the Chinese (9.9 units) or Indians (9.4 units). A high phenotype-genotype correlation of >97% was observed in all three races. We also genotyped 418 childhood leukaemias. The combined analysis of subjects participating in this and a previous study--1585 subjects--showed that 4.7% of Chinese (n=30/644), 4.4% of Malays (n=24/540) and 2.7% of Indians (n=11/401) were heterozygous at the TPMT gene locus. This is the first comprehensive TPMT phenotype and genotype study in Asian populations, particularly in the Malays and Indians.

Assessment of thiopurine methyltransferase enzyme activity is superior to genotype in predicting myelosuppression following azathioprine therapy in patients with inflammatory bowel disease

Myelosuppression occurs in 2-7% of inflammatory bowel disease (IBD) patients treated with azathioprine, and can be associated with reduced activity of thiopurine methyltransferase (TPMT) in some patients. It has been proposed that pretreatment assessment of TPMT status reduces the incidence of toxicity and is cost-effective. To determine if screening for TPMT status predicts side-effects to azathioprine in patients with IBD and to ascertain whether screening by TPMT enzyme activity or genotype is superior. Sequential IBD patients were identified and azathioprine tolerance recorded. Blood was collected for measurement of TPMT activity and TPMT*3C, TPMT*3A and TPMT*2 genotypes. Of 130 patients, 25% stopped azathioprine because of toxicity. Four patients experienced severe myelosuppression (WCC < 2). Eleven of 17 patients with reduced TPMT activity were heterozygotes, including one patient with marked TPMT deficiency who experienced severe myelosuppression. There was no association between intermediate TPMT deficiency and any side-effect. Moderate reduction of TPMT activity in heterozygotes was not associated with toxicity, but very low TPMT activity caused severe myelosuppression in one patient. This would have been predicted by measuring TPMT activity but not by genotyping. Measurement of TPMT activity may therefore be superior to genotype in predicting severe myelosuppression.

Relationship Between 6-Mercaptopurine Dose and 6-Thioguanine Nucleotide Levels in Patients With Inflammatory Bowel Disease

6-Thioguanine nucleotides (6-TGN) are active metabolites of azathioprine (AZA) and 6-mercaptopurine (6MP). Higher remission rates have been observed in patients with higher 6-TGN levels. However, many physicians prescribe AZA/6MP using milligrams per kilogram (mg/kg) dosing regimens without measuring 6-TGN levels. The aim of this study was to examine the association between 6MP dose and 6-TGN levels. We conducted a cross-sectional study of patients treated for inflammatory bowel disease (IBD) with AZA or 6MP, whose 6-TGN levels were measured. Patients with low or intermediate thiopurine methyl transferase (TPMT) activity were excluded. AZA dose was converted to 6MP equivalents. The relationship between dose and 6-TGN levels was assessed with the Spearman correlation coefficient. We used logistic regression to assess the relationship between dose and 6-TGN levels of >230 pmol/8 x 10(8). In this study, 155 patients met our inclusion criteria (median dose, 1.01 +/- 0.40; range, 0.61-1.41 mg/kg). There was a weak correlation between 6-TGN levels and the absolute dose (rho = 0.18, P = 0.04) and the dose in mg/kg (rho = 0.19, P = 0.03). The correlation between mg/kg dosage and 6-TGN levels was slightly stronger in those using concomitant 5-aminosalicylate (5-ASA) medications (rho = 0.24, P = 0.02). Compared with <1.0 mg/kg per day, doses of > or =1.5 mg/kg per day were strongly associated with 6-TGN levels of >230 pmol/8 x 10(8) RBC (adjusted odds ratio [OR] = 3.7, 95% confidence interval [CI] = 1.1-11.8). However, only 37% of patients receiving > or =1.0 mg/kg per day had 6-TGN levels of >230 pmol/8 x 10(8) RBC. 6MP dose is weakly associated with 6-TGN levels. The use of standard mg/kg dosing regimens will result in low 6-TGN levels in most patients.

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.

Assessment of Thiopurine Methyltransferase and Metabolite Formation During Thiopurine Therapy

This study examined thiopurine methyltransferase (TPMT) and the relationship to thioguanine nucleotides (TGN) and methylthioinosine monophosphate (meTIMP) in a large Swedish patient population. The current hypothesis is that the cytotoxic effects of thiopurine drugs are mediated by the incorporation of TGN into DNA. The authors assayed the TPMT activity in red blood cells from 1151 subjects and the concentrations of TGN (n = 602) and meTIMP (n = 593) from patients treated with thiopurine drugs. The TPMT frequency distribution in both adults and children showed some differences from what had been found in unselected general populations. Children had lower median TPMT activity than adults (12.0 versus 12.9 U/mL RBC; P < 0.001). Relative differences in both TGN formation [medians: normal TPMT, 1.3; intermediate TPMT, 3.3; low TPMT, 47.9 pmol/8 x 10(8) RBC per mg azathioprine (AZA); P < 0.001] and meTIMP formation (medians: normal TPMT, 13; intermediate TPMT, 7.3; low TPMT, 0 pmol/8 x 10(8) RBC per mg AZA; P = 0.001) per 1 mg administered drug were noted among the 3 TPMT activity groups. Women formed higher concentrations of both TGN (1.5 versus 1.3 pmol/8 x 10(8) RBC per mg AZA; P = 0.01) and meTIMP (14.4 versus 10.7 pmol/8 x 10(8) RBC per mg AZA; P = 0.01) than men did. There was a significant correlation between the AZA dose and the meTIMP concentrations (r = 0.45; P < 0.001). Furthermore, dose alterations made in subjects with normal TPMT (n = 84) and intermediate TPMT (n = 22) activity resulted in more pronounced increases in TGN concentrations (170 versus 30 pmol/8 x 10(8) RBC; P < 0.001) in intermediate TPMT activity, whereas in normal TPMT activity changes in meTIMP concentrations were more pronounced (1.3 versus 0 nmol/8 x 10(8) RBC; P < 0.001). In normal TPMT activity both metabolites increased in a dose-dependent fashion, whereas in intermediate TPMT activity only TGN concentrations increased. The results of this study demonstrate the dynamic nature of thiopurine metabolism and its importance for thiopurine dosing.

Relationship between Red Blood Cell Thiopurine Methyltransferase Activity and Myelotoxicity in Dogs Receiving Azathioprine

Thiopurine methyltransferase (TPMT) produces inactive metabolites of azathioprine and, in humans, has a variable amount of activity. Humans with low TPMT activity commonly develop myelotoxicity when receiving azathioprine. Our study sought to characterize the distribution of TPMT activity in a population of dogs and to determine whether the pretreatment knowledge of red blood cell (RBC) TPMT activity could predict myelotoxicity in dogs receiving azathioprine. RBC TPMT activity was measured in 299 healthy dogs, and 9 dogs that represented a wide range of enzyme activity received azathioprine at a standard therapeutic dose for 30 days. TPMT activity in healthy dogs was log normally distributed and varied over an approximately 7‐fold range. Geometric mean, minimum, and maximum RBC TPMT activities were 37.1, 16.3, and 115 nmol per gram of hemoglobin (gHb) per hour, respectively. TPMT deficiency was not identified. Two populations of TPMT activity in dogs were detected by statistical modeling (commingling analysis). Dogs with intermediate TPMT activity (14–38 nmol/gHb/h) receiving azathioprine had significantly lower neutrophil counts during week 4 than during weeks 0–3, whereas those with high activity (&gt;39 nmol/gHb/h) did not have a significant change in neutrophil count. An analysis of TPMT activity in 6 dogs with a history of azathioprine‐associated myelotoxicity in a clinical setting revealed either intermediate or high TPMT enzyme activity in all dogs, suggesting that TPMT activity, as measured in RBCs, is not the sole cause of severe azathioprine‐associated myelosuppression in dogs.

Relationship between red blood cell thiopurine methyltransferase activity and myelotoxicity in dogs receiving azathioprine.

Thiopurine methyltransferase (TPMT) produces inactive metabolites of azathioprine and, in humans, has a variable amount of activity. Humans with low TPMT activity commonly develop myelotoxicity when receiving azathioprine. Our study sought to characterize the distribution of TPMT activity in a population of dogs and to determine whether the pretreatment knowledge of red blood cell (RBC) TPMT activity could predict myelotoxicity in dogs receiving azathioprine. RBC TPMT activity was measured in 299 healthy dogs, and 9 dogs that represented a wide range of enzyme activity received azathioprine at a standard therapeutic dose for 30 days. TPMT activity in healthy dogs was log normally distributed and varied over an approximately 7-fold range. Geometric mean, minimum, and maximum RBC TPMT activities were 37.1, 16.3, and 115 nmol per gram of hemoglobin (gHb) per hour, respectively. TPMT deficiency was not identified. Two populations of TPMT activity in dogs were detected by statistical modeling (commingling analysis). Dogs with intermediate TPMT activity (14-38 nmol/gHb/h) receiving azathioprine had significantly lower neutrophil counts during week 4 than during weeks 0-3, whereas those with high activity (>39 nmol/gHb/h) did not have a significant change in neutrophil count. An analysis of TPMT activity in 6 dogs with a history of azathioprine-associated myelotoxicity in a clinical setting revealed either intermediate or high TPMT enzyme activity in all dogs, suggesting that TPMT activity, as measured in RBCs, is not the sole cause of severe azathioprine-associated myelosuppression in dogs.