6-TGN Levels Research Articles

Thiopurines with low-dose allopurinol (ThiLDA)—a prospective clinical one-way crossover trial

Many patients with Crohn's disease (CD) and ulcerative colitis (UC) who have a high 6-methylmercaptopurine/6-thioguanine (6-MMP/6-TGN) ratio receive allopurinol 100mg in addition to thiopurines to optimize metabolite concentrations. However, some patients do not tolerate allopurinol at this dosage. The aim of this study was to determine the intra-patient effect of reducing the allopurinol dosage from 100 to 50mg, in terms of metabolite concentrations, enzyme activities, efficacy, and tolerability. A prospective non-inferiority one-way crossover study was performed. CD and UC patients with stable disease using a thiopurine and allopurinol 100mg were switched to 50mg for 1month. Primary outcomes were thiopurine metabolite concentrations. Secondary outcomes were enzyme activities of xanthine oxidase, thiopurine methyltransferase and hypoxanthine-guanine phosphoribosyltransferase, disease activity, and tolerability. Twenty-two patients were included. Treatment with allopurinol 50mg compared with 100mg resulted in a significant decrease in mean 6-TGN levels (761 to 625pmol/8 × 108 RBC; p = 0.005) and a significant increase in mean 6-MMP levels (451 to 665pmol/8 × 108 RBC; p = 0.01). However, the mean metabolite concentrations were still therapeutic. Enzyme activities, disease activity scores, and patient experiences did not alter significantly. Generally, UC patients were more positive about their improved treatment than CD patients. Combination therapy with 50mg allopurinol led to a decrease of 6-TGN levels compared with 100mg allopurinol. Disease activity, side effects, and patient experience, however, were similar between allopurinol 100 and 50mg. UC patients seem to benefit and prefer lower doses whereas the contrary is seen in CD patients. EudraCT trial registry - number 2016-001638-84.

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.

Systematic review with meta-analysis: risk factors for thiopurine-induced leukopenia in IBD.

Thiopurine-induced leukopenia, a frequently observed and potentially life-threatening adverse event, complicates the clinical management of IBD patients. To assess risk factors for thiopurine-induced leukopenia in IBD. MEDLINE, EMBASE, BIOSIS and Cochrane library were searched for studies reporting at least one risk factor for thiopurine-induced leukopenia. Pooled odds ratio (OR) was calculated for each potential risk factor using a random effects model. Studies that were not eligible for meta-analysis were described qualitatively. Seventy articles were included, 34 (11229 patients) were included in meta-analyses. A significantly higher thiopurine-induced leukopenia risk was found for TPMT (OR 3.9, 95% [CI] 2.5-6.1) and for NUDT15 R139C (OR 6.9, 95% CI 5.2-9.1), G52A (OR 3.2, 95% CI 1.3-7.9) and 36_37ins/delGGAGTC variant carriers (OR 5.6, 95% CI 2.8-11.4). A potential association between high 6-thioguanine nucleotides (6-TGN) or 6-methylmercaptopurine (6-MMP) levels and leukopenia was observed, since most studies reported higher metabolite levels in leukopenic patients (6-TGN: 204-308 (Lennard method) and 397 (Dervieux method), 6-MMP: 4020-10450pmol/8 x 108 RBC) compared to controls (6-TGN: 170-212 (Lennard method) and 269 (Dervieux method), 6-MMP: 1025-4550pmol/8 x 108 RBC). TPMT and NUDT15 variants predict thiopurine-induced leukopenia. High 6-TGN and 6-MMP levels might induce leukopenia, although exact cut-off values remain unclear. Potential preventive measures to reduce the risk of thiopurine-induced leukopenia include pre-treatment TPMT and NUDT15 genotyping. Routine thiopurine metabolite measurement might be efficient, yet cut-off levels must be validated in advance.

Thiopurine Methyltransferase Genetic Polymorphisms and Activity and Metabolic Products of Azathioprine in Patients with Inflammatory Bowel Disease.

Thiopurine S-methyltransferase (TPMT) is a cytoplasmic enzyme that catalyzes thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. There is a correlation between thiopurine drug metabolism, response, and toxicity and genetic polymorphism of TPMT. The aim of this study is to assess TPMT genetic polymorphisms activity and metabolic products of AZA in patients with IBD. Blood samples were obtained from 50 IBD unrelated patients from a private laboratory. We used polymerase chain reaction-restriction length polymorphism (PCR-RFLP) and allele-specific PCRbased assays to determine the TPMT gene for the different variants. A high-performance liquid chromatography system (HPLC) was carried out to determine the whole blood 6-TGN concentration. Determination of serum TMPT activity was done by ELISA kit. In IBD patients, 46/50 (92%) subjects were homozygous for the wild-type allele (TPMT*1/*1). Mutant TPMT*1/*2 and TPMT*1/*3C alleles were found in 4/46 (8%) and 3/47 (6%) of IBD patients, respectively. TPMT*1/*3B variant was not detected in any of the IBD patients. TPMT enzyme activity was higher in wild-type than that mutant variants TPMT*1/*2 and TPMT*1/*3C, suggesting that there are statistically significant differences between 6-TG levels and polymorphisms of TMPT enzyme. 6-TG levels significantly increased in IBD patients mutant variants TPMT*1/*2 and TPMT*1/*3C. Our results showed that TPMT polymorphisms are associated with 6-TGN levels in patients using AZA. This study suggests that AZA dosage may be determined according to the high or low prevalence of a TPMT genotype. Moreover, the results present the determination of metabolite for assessing possible safe effective dosage of the drug.

Azathioprine dosing and metabolite measurement in pediatric inflammatory bowel disease: does one size fit all?

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

Low 6-thioguanine nucleotide level: Effective in maintaining remission in Chinese patients with Crohn's disease.

Measuring 6-thioguanine nucleotide (6-TGN) level is useful in optimizing dose of azathioprine (AZA) and monitoring for toxicity. Lower dose of AZA was suggested for maintenance of clinical remission in Asian patients than Caucasian patients with Crohn's disease (CD). However, the optimal 6-TGN threshold required in Asian patients is undetermined. Therefore, the aim of the current study is to explore the optimal 6-TGN threshold required in Asian patients with CD for maintenance of clinical remission. A retrospective cohort study in a tertiary referral center recruited 252 CD patients. The primary endpoint was disease relapse. The levels of 6-TGN and AZA dose were compared in remission group and relapse group. Remission rate was compared across the increased 6-TGN level and dose range. Patients with 6-TGN range of 0-180.94pmol/8×108 red blood cells (RBC) had lower remission rate compared with those with 180.94-255.50pmol/8×108 RBC (P=0.020). Quartile analysis showed that increasing 6-TGN level beyond 180pmol/8×108 RBC produced negligible gain in rate of remission. Frequency of adverse events significantly increased in patients with 6-TGN level>355pmol/8×108 RBC (8.0% with 6-TGN>355pmol/8×108 RBC vs 2.7% with 6-TGN<355pmol/8×108 RBC, P=0.035). Our study suggested that optimal 6-TGN threshold required to maintain clinical remission in Chinese patients was 180-355pmol/8×108 RBC.

Higher Thioguanine Nucleotide Metabolite Levels are Associated With Better Long-term Outcomes in Patients With Inflammatory Bowel Diseases.

The aim of this study was to assess whether sustained 6-thioguaninenucleotide (6-TGN) levels were associated with improved long-term outcomes in patients with inflammatory bowel diseases (IBD). Cross-sectional data have shown that thiopurine metabolites are correlated with clinical efficacy in patient receiving thiopurines for IBD but the role for serial measurements through treatment course is unclear. We conducted a retrospective cohort study including patients with IBD on thiopurine monotherapy and had serial 6-TGN levels measured. Predictive variables included demographics, disease phenotype, 6-TGN levels (nadir, median, and peak levels). The primary outcome was the development of a disease relapse. The secondary outcome was the need for IBD surgery. Two hundred eighteen 6-TGN samples from 87 patients were analyzed. Nadir and median 6-TGN levels were significantly higher in patients who did not relapse [185 and 233 pmol per 8×10 red blood cells (RBCs)] as compared with levels in patients who did relapse (150 and 167 pmol per 8×10 RBCs, P=0.025) but there was no significant difference in peak 6-TGN level. When adjusted for confounding factors, a nadir 6-TGN level ≥161 and a median 6-TGN level ≥264 were associated with a significant decrease in the rate of disease exacerbation (hazard ratio: 0.5; 95% confidence interval, 0.26-0.87; P=0.016 and hazard ratio: 0.4; 95% confidence interval, 0.2-0.82; P=0.14). Serial thiopurine metabolite level assessments and dose adjustment aiming to maintain higher 6-TGN levels may be helpful to improve long-term outcomes in patients with IBD.

The Impact of Thiopurine Metabolite Monitoring on the Durability of Thiopurine Monotherapy in Pediatric IBD.

Thiopurine metabolite monitoring to proactively dose optimize to achieve therapeutic levels has not been used consistently, and it is unclear if this would lead to better outcomes. We aimed to compare 6-month outcomes between standard and optimized dosing strategies and define long-term predictors of thiopurine durability. Two hundred sixteen pediatric IBD patients with at least 2 6-thioguanine nucleotide (6-TGN) levels were grouped for analysis by start dose: >2.5 mg/kg/day AZA (group 1) or <2.5 mg/kg/day (group 2) and further subgrouped depending on whether dosing was optimized to achieve 6-TGN >235 pmol/8 × 108 RBC. The metabolites, 6TGN and 6MMP, were univariate and multivariate analyses tested associations among metabolite levels, laboratory data, and the primary outcome of 6-month steroid-free clinical remission (SFR) (HBI ≤4 for CD; partial Mayo Score [pMS] ≤2 for UC). Thiopurine durability was measured using Kaplan Maier survival analysis. 6-MP, azathioprine, pediatrics, therapeutic drug monitoring, pediatrics were measured a median 59 (43-76) days after initiation of thiopurine. Both dosing strategies led to similar initial 6-TGN levels (group 1 = median 209 [IQR: 155-272] with 25% of patients >235; group 2 = 196 [139-274] with 29% >235). Steroid-free clinical remission was achieved in 74% of the 180 still on thiopurines at 6 months. Start dose was not associated with 6-month SFR-73% in group 1 and 77% in group 2 within those on thiopurines at 6 months (P = 0.61). Fixed- and optimized-dosing subgroups had similar 6-month 6-TGN levels, SFR rate, and percentage 6-TGN > 235. Only 6-TGN level >235 at 6 months predicted thiopurine durability (3 years [1.7-7.7] vs 2.5 years [0.83-5]; log-rank P < 0.001), and this did not retain significant in a multivariate model. Initial dosing strategy, first 6-TGN level, 6-month SFR, 6MMP:6TGN ratio, and delta-MCV did not predict durability. The rate of adverse events was 22%. Steroid-free clinical remission and 6-TGN levels at 6 months were no different between a standardized, fixed dosing strategy and a metabolite-driven, optimized dosing strategy.

6-Thioguanine Nucleotide Levels Are Associated With Mucosal Healing in Patients With Crohn's Disease.

Level of 6-thioguanine nucleotides (6-TGN) has been reported to be associated with clinical remission in patients with Crohn's disease (CD) receiving maintenance treatment with thiopurines. Whether 6-TGN levels are associated with mucosal healing (MH) has seldom been investigated. We aimed to assess the correlation between 6-TGN levels and MH in patients with CD. This was a retrospective, cross-sectional, observational, multicenter study of 119 patients with CD treated with thiopurines in 3 inflammatory bowel disease referral centers (France, Australia, and China) between June 2012 and April 2016. Established CD patients who underwent ileocolonoscopy during thiopurine treatment were included. MH was defined as simple endoscopic score-CD <3. Univariate and multivariable regression analyses were used to evaluate variables associated with MH. The mean concentration of 6-TGN in the MH group was higher compared with that in the non-MH group (359.0 ± 226.7 pmol/8 × 108 red blood cell count [RBC] vs 277.1 ± 170.5 pmol/8 × 108 RBC; P = 0.017). The cutoff 6-TGN concentration of 397.3 pmol/8 × 108 RBC was 86.7% specific to MH, with a sensitivity of 35.3% and area under curve (AUC) of 0.631 (P = 0.010). On multivariable analysis, 6-TGN levels were associated with MH (odds ratio [OR], 3.287; 95% confidence interval [CI], 1.348-8.017; P = 0.009) whereas late initiation of AZA (longer duration from disease onset) was inversely associated with MH (OR, 0.972; 95% CI, 0.954-0.991; P = 0.004). Higher 6-TGN levels are independently associated with a reduced rate of endoscopically active disease and a higher rate of mucosal healing in CD patients. Prospective studies of adequate sample size are required to confirm these findings.

Randomised clinical trial: efficacy, safety and dosage of adjunctive allopurinol in azathioprine/mercaptopurine nonresponders (AAA Study).

Thiopurine hypermethylation towards 6-methylmercaptopurine (6MMP) instead of 6-thioguanine nucleotides (6TGN) is associated with inefficacy in patients with IBD. Allopurinol reverses such hypermethylation. To prospectively determine efficacy of allopurinol-thiopurine combination and to compare 2 doses of allopurinol. In a multicentre, double-blind trial, patients with clinically active or steroid-dependent IBD and thiopurine shunting were randomised to 50 or 100mg/d allopurinol and 25% of their screening thiopurine dose, which was subsequently optimised, aiming for 6TGN of 260-500pmol/8x108 RBCs. The primary endpoint was steroid-free clinical remission at 24weeks. Of 73 patients, 39 (53% [95% CI 42-65]) achieved steroid-free remission, (54% with 50mg/d and 53% with 100mg/d). 81% were able to discontinue steroids. Therapeutic 6TGN levels were achieved in both groups. Final thiopurine doses were lower with 100mg/d allopurinol (P<0.005). 6MMP: 6TGN ratio decreased from mean 64 to 4 (P<0.001), being higher with 50mg/d (6±1.83) than for 100mg/d ([1±0.16], P=0.003). Three patients on 50mg/d failed to sustain low ratios at 24weeks. Toxicity was minimal; three patients on 50mg/d allopurinol developed transient leukopenia. Alanine aminotransferase concentrations decreased (P<0.001) similarly in both arms. Faecal calprotectin levels at study end were lower in patients who achieved the primary endpoint (median 171 [85-541] vs 821[110-5892] ug/g, P=0.03). Low-dose allopurinol-thiopurine combination safely reverses shunting and optimises 6TGN with associated improvement in disease activity. 100mg/d allopurinol is preferable due to greater metabolite profile stability and lower thiopurine dose without additional toxicity.

P551 6-Thioguanine nucleotide levels are associated with mucosal healing in patients with Crohn’s disease: A multi-centre, international study

The level of 6-thioguanine nucleotides (6-TGN) has been reported to be associated with clinical remission in patients with Crohn’s disease (CD) receiving maintenance treatment with thiopurines. Whether 6-TGN levels are associated with mucosal healing (MH) has seldom been investigated. We aimed to assess the correlation between 6-TGN levels and MH in patients with CD. This was a retrospective cross-sectional observational multi-centre study of 119 patients with CD treated with thiopurines in three IBD referral centres (France, Australia and China) between June 2012 and April 2016. Established CD patients who underwent ileocolonoscopy during thiopurine treatment were included. MH was defined as simple endoscopic score-CD <3. Univariate and multivariable regression analyses were used to evaluate variables associated with MH. The mean concentration of 6-TGN in MH group was higher compared with that in non-MH group (359.0 ± 226.7 pmol/8 × 108 RBC vs. 277.1 ± 170.5 pmol/8 × 108 RBC) (p = 0.017). The cut-off 6-TGN concentration of 397.3 pmol/8x108RBC was 86.7% specific for MH, with a sensitivity of 35.3% and area under curve (AUC) of 0.631 (p = 0.010). On multivariate analysis, 6-TGN levels were associated with MH (odds ratio [OR] 3.287, 95% confidence interval [CI] 1.348–8.017; p = 0.009) while late initiation of AZA (longer duration from disease onset) was inversely associated with MH (OR 0.972, 95% CI 0.954–0.991; p = 0.004). Higher 6-TGN levels are independently associated with a reduced rate of endoscopically active disease and a higher rate of mucosal healing in CD patients. Prospective studies of adequate sample size are required to confirm these findings.

P677 Azathioprine dosing and metabolite measurement in paediatric inflammatory bowel disease—does one size fit all?

Azathioprine (AZA) is an effective immunomodulatory maintenance therapy in children with inflammatory bowel disease (IBD). Current recommended dosing is 2–2.5 mg/kg/dose in the presence of normal thiopurine methyltransferase (TPMT) level. Elevated levels of the AZA metabolites 6-TGN and 6-MMP have been associated with increased risk of myelotoxicity and hepatotoxicity, however evidence suggests that monitoring metabolites can help optimise effectiveness and safety of thiopurines. We aim to assess association between AZA weight-based dosing, thiopurine metabolites and disease activity index in a cohort of paediatric IBD patients. Retrospective data review (2015–2017) from the paediatric gastroenterology department at Evelina London Children’s Hospital. Children with IBD who were taking AZA and had regular thiopurine metabolites measurement were included. PCDAI and PUCAI were used to assess disease activity. A total of 41 patients, 38% female, 62% male with mean (±SD) age of diagnosis 12.2 (±3.4) years, were included. Twelve patients had ulcerative colitis (UC), 27 Crohn’s disease (CD) and 2 very early onset IBD (VEOIBD). Mean AZA dose, in mg/kg (±SD), 1.3 (±0.08). Mean 6TGN value (±SEM) 260 (±25.2), and mean MMT value (±SEM) 1023 (±168). Thiopurine metabolites were measured at (mean ± SEM) after (75 ± 13.5) days after starting AZA, 5 patients had elevated 6TGN levels >450. Mean lymphocyte count at time of metabolite measurement (±SD) 1.7 (±0.8). Activity index at the start of thiopurine were (±SD) 22.5 (±16.3) and when metabolites were measured 8.1 (±12). We have identified that the majority of our patients maintain clinical remission (defined by an activity index—PUCAI/PCDAI—of less than 10) on lower AZA doses than currently recommended. We have also identified a number of patients with elevated 6TGN levels without signs of myelosuppression. Higher doses of AZA are associated with higher metabolite levels, and we propose that regular routine measurement of AZA metabolites will help optimise therapy, minimise side effects and identify children at risk of hepato- and myelotoxicity.

P170 Azathioprine metabolite (6-TGN) levels within a defined therapeutic range are associated with lower faecal calprotectin in Crohn’s disease

P170 Azathioprine metabolite (6-TGN) levels within a defined therapeutic range are associated with lower faecal calprotectin in Crohn’s disease

Measurements of 6-thioguanine nucleotide levels with TPMT and NUDT15 genotyping in patients with Crohn's disease.

The association between the 6-thioguanine nucleotide (6-TGN) level and clinical remission in Crohn’s disease (CD) remains controversial. Thiopurine-induced leukopenia is a life-threatening complication of CD in Asians that was recently shown to strongly correlate with NUDT15 genetic variants. This study aimed to determine the relationship between thiopurine metabolite levels and therapeutic response, and to investigate the association of NUDT15, TPMT, and thiopurine metabolites with leukopenia in patients with CD. We enrolled 165 adult patients with CD undergoing thiopurine treatment. Clinical evaluation and laboratory examinations were carried out every 2–3 months. We measured thiopurine metabolites levels and genotyped NUDT15 and TPMT. During the median 12-month observational period, 95 (67.9%) patients exhibited clinical response and 45 (32.1%) did not respond to the treatment. The median 6-TGN level was significantly higher in responders than in non-responders (P < 0.001). The odds ratio of patients with a 6-TGN level ≥230 pmol/8 × 108 red blood cells for showing a clinical response was 4.63 (95% CI 1.62–11.9). NUDT15 variant types were strongly associated with developing leukopenia. Patients with NUDT15 homozygous variant genotype developed severe early leukopenia with an average reduction of 88.2% (range, 84–94%) from the baseline white blood cell count at 4 weeks. Our findings support the role of therapeutic drug monitoring in thiopurine maintenance treatment to optimize thiopurine therapy, especially, for non-responding CD patients. Thiopurine treatment should not be recommended to patients with NUDT15 homozygous variant genotype due to severe early leukopenia.

A Role for Thiopurine Metabolites in the Synergism Between Thiopurines and Infliximab in Inflammatory Bowel Disease.

Interactions between principal cytotoxic thiopurine metabolites, that is 6-thioguanine nucleotides [6-TGN], and infliximab [IFX] and anti-IFX antibodies [Abs] may contribute to higher effectiveness of IFX-thiopurine combination therapy than monotherapies in inflammatory bowel disease. To examine if thiopurine metabolites influenced trough IFX and anti-IFX Abs, 89 patients previously assessed for anti-IFX Abs were included. To assess if IFX influenced thiopurine metabolites, eight patients who had responded to 12 weeks of intensified IFX at a constant thiopurine dosing were included. In the first cohort, IFX-thiopurine combination therapy reduced anti-IFX Ab detection [8/40; 20%] as compared with IFX monotherapy [22/49; 45%], odds ratio [OR] 0.31 [0.12-0.80], p < 0.05. 6-TGN was significantly lower in anti-IFX Ab-positive patients (50 pmol/8 × 108 red blood cells [RBC] vs 105, p < 0.01). All anti-IFX Ab-positive patients had 6-TGN < 117 pmol/8 × 108 RBC (sensitivity 100% [63-100], specificity 47% [29-65], area under the curveROC = 0.82, p < 0.01). Trough IFX was similar between anti-IFX Ab-negative patients in IFX monotherapy and IFX-thiopurine combination therapy [5.1 μg/mL vs 4.9, p = 0.76]. 6-TGN and IFX did not correlate [rP = 0.04, p = 0.83; rS = 0.02, p = 0.89, respectively]. In the second cohort, trough IFX increased during IFX intensification [ΔIFX median 6.5 μg/mL, p = 0.02], but 6-TGN was stable [6-TGN at Weeks 0, 4, 8, 12: 90 pmol/8 × 108 RBC, 93, 101, 90; p > 0.05]. Methylated mercaptopurine metabolite associations were consistently negative. Superior effect of IFX-thiopurine combination therapy over monotherapies partly relates to decrease in anti-IFX Abs, which associates with 6-TGN levels and has a lower therapeutic threshold than during thiopurine monotherapy. Additional benefit likely ascribes to synergy between different anti-inflammatory modes of action rather than direct drug interactions.

Allopurinol in combination with thiopurine induces mucosal healing and improves clinical and metabolic outcomes in IBD.

Background:Thiopurines, azathioprine (AZA) and 6-mercaptopurine (6-MP) are common maintenance medications for inflammatory bowel disease (IBD). Excessive methylation via thiopurine methyltransferase (TPMT) frequently causes therapeutic failure. Allopurinol reduces excessive 6-methyl-mercaptopurine (6-MMP) while enhancing 6-thioguanine (6-TGN) levels. The aim of this study was to evaluate clinical, metabolic and endoscopic impact of allopurinol in combination with low-dose thiopurine in IBD.Methods:Retrospective review of consecutive cases treated with allopurinol. Metabolites and their ratios (6-MMP/6-TGN) were compared pre- and post-allopurinol. Clinical and endoscopic remission were assessed.Results:Allopurinol (n = 66) reduced mean dose of AZA by 70% (p < 0.01). Baseline levels (SD) 6-TGN, 6-MMP and 6-MMP/6-TGN were 165 (64), 9388 (5234) and 59.8 (30.3), respectively. These values improved on allopurinol to 297 (102), 896 (1031) and 3.4 (4.0), respectively (p < 0.0001). Therapeutic 6-TGN level (>235) was achieved in 49/58 cases on allopurinol combination therapy, versus 9/58 monotherapy (p = 0.0001). Among the thiopurine failure group (40 patients), clinical remission or response was observed in 65% and 22% of patients, respectively. In the asymptomatic group with excessive 6-MMP, 11/14 achieved sustained remission on allopurinol. Repeat colonoscopy (n = 28) showed mostly endoscopic remission (67.9%) or improvement (17.8%). Few had unimproved lesions (14.3%). Importantly, 46% of cases had complete mucosal healing. Two patients had cancer on combination therapy (de novo pancreatic cancer and fatal recurrence of metastatic testicular cancer). Elevated transaminases were reduced on allopurinol (48.2 versus 6.9%) (p < 0.001); no change in leukopenic or infectious events occurred.Conclusion:Allopurinol in combination with low-dose thiopurine corrected excessive 6-MMP levels, resulting in clinical remission and mucosal healing in the majority of cases. The potential cancer risk of allopurinol and thiopurine combination therapy needs further research.

Pharmacokinetic Effect of Anti-tumor Necrosis Factor-alpha Agents on Thiopurine Drug Metabolism in Inflammatory Bowel Diseases

Introduction: Thiopurines and anti-tumor necrosis factor-alpha (anti-TNF) drugs are commonly used in treatment of inflammatory bowel diseases (IBD). Studies have established that thiopurines, when used in combination with anti-TNF drugs, affect the metabolism of anti-TNF drugs, resulting in higher serum drug concentrations. However, the data on the effect of anti-TNF drugs on thiopurine metabolism is limited. Methods: All adult IBD patients followed at our IBD center who had thiopurine metabolite concentrations measured between 2000-2016 were eligible for inclusion if they had at least 1 level measured while on thiopurine monotherapy, and 1 level measured while on combination therapy with an anti-TNF and thiopurine drug with the same thiopurine drug dose. Thiopurine metabolite concentration, white blood cell (WBC) count, mean corpuscular volume (MCV) and serum alanine aminotransferase (ALT) levels were compared on monotherapy and combination therapy. Paired t-test and Wilcoxon rank sum test were used for analysis of variables with normal and non-normal distribution, respectively. Results: 30 patients met the inclusion criteria (Table 1). Mean 6TGN level (in pmol/8 x 108 RBCs) on monotherapy and combination therapy were 201 and 267 respectively (% increase 33%; 95% CI 30.1-105.8; p 0.0004). There was a corresponding decrease in mean WBC count (in x1000/mL) from 7.6 to 6.1 (% decrease 20%; 95% CI 0.49-2.85; p 0.01) (Figure 1). There was no significant difference in 6-MMP levels, MCV and ALT levels on monotherapy and combination therapy (Table 2). On subgroup analysis, patients with therapeutic 6TGN levels >230 (n=13) had a significant increase in 6TGN levels (301 vs 369; p 0.03), and a corresponding decrease in WBC count (8.0 vs 5.7; p 0.07) on combination therapy. One patient developed leukopenia (WBC 2.8) after starting infliximab with an increase in 6TGN levels from 425 to 516.Figure: Thiopurine metabolite concentration and WBC count on monotherapy and combination therapy.Table: Table. Baseline CharacteristicsTable: Table. Lab parameters on monotherapy and combination therapyConclusion: In patients with IBD on thiopurine monotherapy, the addition of an anti-TNF agent can lead to an increase in 6-TGN concentration, thereby increasing the risk of leukopenia. Hence, elective thiopurine dose reduction should be considered prior to starting an anti-TNF agent, particularly in patients with therapeutic 6TGN concentration on thiopurine monotherapy. Our findings also suggest that benefits of combination therapy may be, in part, due to increase in thiopurine metabolite concentration with addition of an anti-TNF agent.

Thiopurine metabolite levels in patients with atopic dermatitis and/or chronic hand/foot eczema treated with azathioprine

Background: Azathioprine is frequently used in severe eczema. It is converted in the liver into active metabolites, including 6-thioguanine nucleotide (6-TGN) and methylated 6-methylmercaptopurine (6-MMP). In the past, the therapeutic potential of azathioprine may have not been fully utilized. Recent investigations on inflammatory bowel disease have led to a better understanding of azathioprine metabolism and optimizing treatment.Objective: To investigate whether measuring thiopurine metabolites in circulation can improve the effectiveness and safety of azathioprine treatment in patients with atopic dermatitis and/or chronic hand/foot eczema.Methods: Azathioprine metabolite levels were measured in eczema patients during maintenance treatment (Part I) and dose escalation (Part II). Clinical effectiveness, hepatotoxicity, and bone marrow suppression were analyzed and TPMT genotype was assessed.Results: A wide variation in metabolite levels in all dose groups was observed. In Part I (32 patients), there were no significant differences in 6-TGN levels between clinical responders and non-responders (p = .806). No hepatoxicity or myelotoxicity was observed. In Part II, all 6-TGN and 6-MMP levels increased during dose escalation. Hypermethylation was observed in 2/8 patients.Conclusion: For individual eczema patients treated with azathioprine, routinely measuring 6-TGN and 6-MMP can be helpful in optimizing azathioprine dose, improving clinical effectiveness, and preventing side effects.

NUDT15 Variants Cause Hematopoietic Toxicity with Low 6-TGN Levels in Children with Acute Lymphoblastic Leukemia.

Purpose We aimed to identify the impact of NUDT15 variants on thiopurine intolerance and 6-thioguanine nucleotide (6-TGN) levels in Korean children with acute lymphoblastic leukemia (ALL).Materials and Methods Genotyping of NUDT15 was tested in 258 patients with ALL registered at Samsung Medical Center. Patients were classified into normal-activity (wild-type), intermediate-activity (heterozygous variant), and low-activity groups (homozygous or compound heterozygous variant). Clinical and laboratory features during the first year of maintenance therapy were investigated. ResultsA total of 182 patients were included in the final analysis. There were five (2.7%), 46 (25.3%), and 131 (72.0%) patients in low-, intermediate-, and normal-activity groups, respectively. The lowest 6-mercaptopurine (6-MP) dose (mg/m2/day) was administered to the low-activity group (low-activity group 7.5 vs. intermediate-activity group 24.4 vs. normalactivity group 31.1, p < 0.01) from three months to a year after beginning maintenance therapy. The low-activity group experienced the longest duration of therapy interruption during the first year (low-activity group 169 days vs. intermediate-activity group 30 days vs. normal-activity group 16 days, p < 0.01). They also showed the lowest blood cell counts and had a longer duration of leukopenia (low-activity group 131 days vs. intermediate-activity group 92 days vs. normal-activity group 59 days, p < 0.01). 6-TGN level and its ratio to 6-MP dose were lowest in the low-activity group.ConclusionNUDT15 variants cause hematopoietic toxicity with low 6-TGN levels. NUDT15 genotyping should be conducted before administering thiopurine, and dose adjustments require caution regardless of 6-TGN levels.

Significance of measurement of serum trough level and anti-drug antibody of adalimumab as personalised pharmacokinetics in patients with Crohn's disease: asubanalysis of the DIAMOND trial.

SummaryBackgroundSignificance of monitoring adalimumab trough levels and anti‐adalimumab antibodies (AAA) for disease outcome in Crohn's disease (CD) patients remained unclear.AimTo evaluate the association of adalimumab trough levels and AAA at week 26 with clinical remission at week 52, the effect of azathiopurine on AAA and factors influencing trough levels in CD patients in the DIAMOND trial.MethodsWe performed this study using adalimumab trough levels, AAA at week 26 and 6‐thioguanine nucleotide (TGN) in red blood cells at week 12. A multiple regression model and receiver operating analysis was performed to identify factors influencing adalimumab trough levels and AAA, and adalimumab thresholds for predicting disease activity.ResultsThere was a significant difference of adalimumab trough level at week 26 between patients with disease remission and without at week 52 (7.7 ± 3.3 μg/mL vs 5.4 ± 4.3 μg/mL: P <.001). Adalimumab trough level of 5.0 μg/mL yielded optimal sensitivity and specificity for remission prediction (80.2% and 55.6%, respectively). AAA development at week 26 significantly affected remission at week 52 (P = .021), which was strongly associated with adalimumab trough levels. Female gender and increasing body weight were independently associated with low adalimumab trough levels, and female gender was associated with AAA development. A cut‐off 6TGN level of >222.5 p mol/8 ×108 RBCs yielded sensitivity (100%) and specificity (60.6%) for AAA negativity.ConclusionAdalimumab trough levels and AAA occurrence were significantly associated with clinical remission. Higher 6TGN affected AAA negativity. The combination therapy is beneficial in some relevant aspects for CD patients. (UMIN Registration No. 000005146)